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Publications

    2025

    • Deep-learning triage of 3D pathology datasets for comprehensive and efficient pathologist assessments.

      Gao, G., Yan, R., Song, A. H., Hsieh, H. C., Barner, L. A., Wang, F., Brenes, D., Chow, S. S., Wang, R., Bishop, K. W., Liu, Y., Farre, X., Divatia, M., Downes, M. R., Vakar-Lopez, F., Lal, P., Burke, W., Madabhushi, A., True, L. D., Reddi, D. M., Grady, W. M., Mahmood, F., Liu, J. T.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Traumatic brain injury (TBI) often induces blood leakage into brain tissues, which causes further tissue loss after the initial injury. To mitigate this secondary injury, we hypothesized that delivery of a fibrin-binding hemostatic polymer, PolySTAT, would act as a molecular patch and ameliorate brain vessel damage following TBI. We developed a three-dimensional (3D) pathology and analysis workflow to quantify the effects of PolySTAT versus a control polymer, PolySCRM, on neurovascular networks and neural tissue in whole mouse brains. Using a panel of fluorescent probes, our 3D pathology pipeline revealed that PolySTAT treatment preserves neurovascular density and function, reduces hypoxia and blood extravasation, and reduces brain tissue loss after TBI. To further corroborate the 3D microscopy-based findings, gene expression analyses show that PolySTAT attenuates the expression of inflammation and reactive gliosis biomarkers. These findings support future translational investigation of intravenous PolySTAT as an early post-injury therapy to mitigate neural tissue loss after TBI.
    • 3D pathology-guided microdissection.

      Hsieh, H. C., Gao, G., Han, Q., Brenes, D., Baraznenok, E., Yan, R., Serafin, R., Bishop, K. W., Wang, R., Konnick, E. Q., Pritchard, C. C., Figiel, S., Hamdy, F. C., Mills, I. G., Reder, N. P., Reddi, D. M., Paulson, T. G., Grady, W. M., Valk, J. E., True, L. D., Haffner, M. C., Rao, S. R., Woodcock, D. J., Liu, J. T.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Standard-of-care slide-based 2D histopathology severely undersamples spatially heterogeneous tissue specimens, with each thin 2D section representing <1% of the entire tissue volume (in the case of a biopsy). Recent advances in non-destructive 3D pathology, such as open-top light-sheet microscopy (OTLS), enable comprehensive high-resolution imaging of large clinical specimens. While fully automated computational analyses of such 3D pathology datasets are being explored, a potential low-risk route for accelerated clinical adoption would be to continue to rely upon pathologists to provide final diagnoses. Since manual review of these massive and complex 3D datasets is infeasible for routine clinical practice, we present CARP3D, a deep learning triage framework that identifies high-risk 2D cross sections within large 3D pathology datasets to enable time-efficient pathologist evaluation. CARP3D assigns risk scores to all 2D levels within a tissue volume by leveraging context from a subset of neighboring depth levels, outperforming models in which predictions are based on isolated 2D levels. In two use cases - risk stratification based on prostate cancer biopsies and screening for dysplasia/cancer in endoscopic biopsies of Barrett's esophagus - AI-triaged 3D pathology, enabled by CARP3D, demonstrates the potential to improve the detection of high-risk diseases in comparison to slide-based 2D histopathology while optimizing pathologist workloads.
    • AI-driven Multi-cohort Analysis of 3D Prostate Gland-Skeleton Morphology for Prognosis of Biochemical Recurrence

      Salguero, J., Medina, S., Serafin, R., Corredor, G., Manandhar, S., Mutha, P., Dhamdhere, R., Wang, R., Chow, S., Romero, E., Bishop, K., Daniel, R., Tokuyama, N., Pathak, T., Mirtti, T., True, L., Lal, P., Liu, J., Madabhushi, A.

      ABSTRACT
      Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.
    • Imaging 3D cell cultures with optical microscopy

      Hsieh, H., Han, Q., Brenes, D., Bishop, K. W., Wang, R., Wang, Y., Poudel, C., Glaser, A. K., Freedman, B. S., Vaughan, J. C., Allbritton, N. L., Liu, J. T. C.

      NATURE METHODS

      ABSTRACT
      Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.
    • Delivery of a fibrin-binding hemostatic polymer ameliorates neurovascular damage and neural tissue loss after traumatic brain injury.

      Han, Q., Brenes, D., Bishop, K. W., Pichon, T. J., Ling, M., McPheron, G. D., White, N. J., Pun, S. H., Liu, J. T., Sellers, D. L.

      Science advances

      ABSTRACT
      Three-dimensional (3D) cell cultures have gained popularity in recent years due to their ability to represent complex tissues or organs more faithfully than conventional two-dimensional (2D) cell culture. This article reviews the application of both 2D and 3D microscopy approaches for monitoring and studying 3D cell cultures. We first summarize the most popular optical microscopy methods that have been used with 3D cell cultures. We then discuss the general advantages and disadvantages of various microscopy techniques for several broad categories of investigation involving 3D cell cultures. Finally, we provide perspectives on key areas of technical need in which there are clear opportunities for innovation. Our goal is to guide microscope engineers and biomedical end users toward optimal imaging methods for specific investigational scenarios and to identify use cases in which additional innovations in high-resolution imaging could be helpful.

    2024

    • Analysis of 3D pathology samples using weakly supervised AI

      Song, A. H., Williams, M., Williamson, D. F. K., Chow, S. S. L., Jaume, G., Gao, G., Zhang, A., Chen, B., Baras, A. S., Serafin, R., Colling, R., Downes, M. R., Farre, X., Humphrey, P., Verrill, C., True, L. D., Parwani, A. V., Liu, J. T. C., Mahmood, F.

      CELL

      ABSTRACT
      Recent advances in 3D pathology offer the ability to image orders of magnitude more tissue than conventional pathology methods while also providing a volumetric context that is not achievable with 2D tissue sections, and all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis, however, is not trivial and requires careful attention to a series of details during tissue preparation, imaging and initial data processing, as well as iterative optimization of the entire process. Here, we provide an end-to-end procedure covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. Although 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol focuses on the use of a fluorescent analog of hematoxylin and eosin, which remains the most common stain used for gold-standard pathological reports. We present our guidelines for a broad range of end users (e.g., biologists, clinical researchers and engineers) in a simple format. The end-to-end workflow requires 3-6 d to complete, bearing in mind that data analysis may take longer.
    • Axially swept open-top light-sheet microscopy for densely labeled clinical specimens

      Bishop, K. W., Barner, L., Baraznenok, E., Lan, L., Poudel, C., Brenes, D., Serafin, R. B., True, L. D., Vaughan, J. C., Glaser, A. K., Liu, J. T. C.

      OPTICS LETTERS

      ABSTRACT
      In recent years, we and others have developed non-destructive methods to obtain three-dimensional (3D) pathology datasets of clinical biopsies and surgical specimens. For prostate cancer risk stratification (prognostication), standard-of-care Gleason grading is based on examining the morphology of prostate glands in thin 2D sections. This motivates us to perform 3D segmentation of prostate glands in our 3D pathology datasets for the purposes of computational analysis of 3D glandular features that could offer improved prognostic performance.To facilitate prostate cancer risk assessment, we developed a computationally efficient and accurate deep learning model for 3D gland segmentation based on open-top light-sheet microscopy datasets of human prostate biopsies stained with a fluorescent analog of hematoxylin and eosin (H&E).For 3D gland segmentation based on our H&E-analog 3D pathology datasets, we previously developed a hybrid deep learning and computer vision-based pipeline, called image translation-assisted segmentation in 3D (ITAS3D), which required a complex two-stage procedure and tedious manual optimization of parameters. To simplify this procedure, we use the 3D gland-segmentation masks previously generated by ITAS3D as training datasets for a direct end-to-end deep learning-based segmentation model, nnU-Net. The inputs to this model are 3D pathology datasets of prostate biopsies rapidly stained with an inexpensive fluorescent analog of H&E and the outputs are 3D semantic segmentation masks of the gland epithelium, gland lumen, and surrounding stromal compartments within the tissue.nnU-Net demonstrates remarkable accuracy in 3D gland segmentations even with limited training data. Moreover, compared with the previous ITAS3D pipeline, nnU-Net operation is simpler and faster, and it can maintain good accuracy even with lower-resolution inputs.Our trained DL-based 3D segmentation model will facilitate future studies to demonstrate the value of computational 3D pathology for guiding critical treatment decisions for patients with prostate cancer.
    • LiverMap pipeline for 3D imaging of human liver reveals volumetric spatial dysregulation of cirrhotic vasculobiliary architecture.

      Fabyan, W. B., Fortin, C. L., Kenerson, H. L., Simmonds, S. P., Liu, J. T., Yeh, M. M., Carr, R. M., Yeung, R. S., Stevens, K. R.

      bioRxiv : the preprint server for biology

      ABSTRACT
      Human tissue, which is inherently three-dimensional (3D), is traditionally examined through standard-of-care histopathology as limited two-dimensional (2D) cross-sections that can insufficiently represent the tissue due to sampling bias. To holistically characterize histomorphology, 3D imaging modalities have been developed, but clinical translation is hampered by complex manual evaluation and lack of computational platforms to distill clinical insights from large, high-resolution datasets. We present TriPath, a deep-learning platform for processing tissue volumes and efficiently predicting clinical outcomes based on 3D morphological features. Recurrence risk-stratification models were trained on prostate cancer specimens imaged with open-top light-sheet microscopy or microcomputed tomography. By comprehensively capturing 3D morphologies, 3D volume-based prognostication achieves superior performance to traditional 2D slice-based approaches, including clinical/histopathological baselines from six certified genitourinary pathologists. Incorporating greater tissue volume improves prognostic performance and mitigates risk prediction variability from sampling bias, further emphasizing the value of capturing larger extents of heterogeneous morphology.
    • Automated, Scalable, and Comprehensive Three-Dimensional Analysis of Glomeruli and Whole Nephrons in Kidney Tissues

      Poudel, C., Brenes, D., Sandoval, R. M., Martinez-Irizarry, M. M., Dunn, K., Dagher, P. C., Liu, J. T. C., Vaughan, J. C.

      ABSTRACT
      Open-top light-sheet (OTLS) microscopy offers rapid 3D imaging of large optically cleared specimens. This enables nondestructive 3D pathology, which provides key advantages over conventional slide-based histology including comprehensive sampling without tissue sectioning/destruction and visualization of diagnostically important 3D structures. With 3D pathology, clinical specimens are often labeled with small-molecule stains that broadly target nucleic acids and proteins, mimicking conventional hematoxylin and eosin (H&E) dyes. Tight optical sectioning helps to minimize out-of-focus fluorescence for high-contrast imaging in these densely labeled tissues but has been challenging to achieve in OTLS systems due to trade-offs between optical sectioning and field of view. Here we present an OTLS microscope with voice-coil-based axial sweeping to circumvent this trade-off, achieving 2 µm axial resolution over a 750 × 375 µm field of view. We implement our design in a non-orthogonal dual-objective (NODO) architecture, which enables a 10-mm working distance with minimal sensitivity to refractive index mismatches, for high-contrast 3D imaging of clinical specimens.
    • Triage of 3D pathology data via 2.5D multiple-instance learning to guide pathologist assessments

      Gao, G., Song, A. H., Wang, F., Brenes, D., Wang, R., Chow, S. S. L., Bishop, K. W., True, L. D., Mahmood, F., Liu, J. T. C., IEEE

      ABSTRACT
      Prostate cancer prognostication largely relies on visual assessment of a few thinly sectioned biopsy specimens under a microscope to assign a Gleason grade group (GG). Unfortunately, the assigned GG is not always associated with a patient's outcome in part because of the limited sampling of spatially heterogeneous tumors achieved by 2-dimensional histopathology. In this study, open-top light-sheet microscopy was used to obtain 3-dimensional pathology data sets that were assessed by 4 human readers. Intrabiopsy variability was assessed by asking readers to perform Gleason grading of 5 different levels per biopsy for a total of 20 core needle biopsies (ie, 100 total images). Intrabiopsy variability (Cohen κ) was calculated as the worst pairwise agreement in GG between individual levels within each biopsy and found to be 0.34, 0.34, 0.38, and 0.43 for the 4 pathologists. These preliminary results reveal that even within a 1-mm-diameter needle core, GG based on 2-dimensional images can vary dramatically depending on the location within a biopsy being analyzed. We believe that morphologic assessment of whole biopsies in 3 dimension has the potential to enable more reliable and consistent tumor grading.
    • An end-to-end workflow for nondestructive 3D pathology

      Bishop, K. W., Erion Barner, L. A., Han, Q., Baraznenok, E., Lan, L., Poudel, C., Gao, G., Serafin, R. B., Chow, S. S. L., Glaser, A. K., Janowczyk, A., Brenes, D., Huang, H., Miyasato, D., True, L. D., Kang, S., Vaughan, J. C., Liu, J. T. C.

      NATURE PROTOCOLS

      ABSTRACT
      Early detection of esophageal neoplasia via evaluation of endoscopic surveillance biopsies is the key to maximizing survival for patients with Barrett's esophagus, but it is hampered by the sampling limitations of conventional slide-based histopathology. Comprehensive evaluation of whole biopsies with 3-dimensional (3D) pathology may improve early detection of malignancies, but large 3D pathology data sets are tedious for pathologists to analyze. Here, we present a deep learning-based method to automatically identify the most critical 2-dimensional (2D) image sections within 3D pathology data sets for pathologists to review. Our method first generates a 3D heatmap of neoplastic risk for each biopsy, then classifies all 2D image sections within the 3D data set in order of neoplastic risk. In a clinical validation study, we diagnose esophageal biopsies with artificial intelligence-triaged 3D pathology (3 images per biopsy) vs standard slide-based histopathology (16 images per biopsy) and show that our method improves detection sensitivity while reducing pathologist workloads.
    • Direct three-dimensional segmentation of prostate glands with nnU-Net

      Wang, R., Chow, S. S. L., Serafin, R. B., Xie, W., Han, Q., Baraznenok, E., Lan, L., Bishop, K. W., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      Early detection of esophageal neoplasia via evaluation of endoscopic surveillance biopsies is the key to maximizing survival for patients with Barrett's esophagus, but it is hampered by the sampling limitations of conventional slide-based histopathology. Comprehensive evaluation of whole biopsies with 3-dimensional (3D) pathology may improve early detection of malignancies, but large 3D pathology data sets are tedious for pathologists to analyze. Here, we present a deep learning-based method to automatically identify the most critical 2-dimensional (2D) image sections within 3D pathology data sets for pathologists to review. Our method first generates a 3D heatmap of neoplastic risk for each biopsy, then classifies all 2D image sections within the 3D data set in order of neoplastic risk. In a clinical validation study, we diagnose esophageal biopsies with artificial intelligence-triaged 3D pathology (3 images per biopsy) vs standard slide-based histopathology (16 images per biopsy) and show that our method improves detection sensitivity while reducing pathologist workloads.

    2023

    • Miniature line-scanned dual-axis confocal microscope for versatile clinical use

      Bishop, K., Hu, B., Yawhare, R., Yang, Z., Liang, D., Gao, G., Baraznenok, E., Han, Q., Lan, L., Chow, S., Sanai, N., Liu, J.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      Recent advances in 3D pathology offer the ability to image orders-of-magnitude more tissue than conventional pathology while providing a volumetric context that is lacking with 2D tissue sections, all without requiring destructive tissue sectioning. Generating high-quality 3D pathology datasets on a consistent basis is non-trivial, requiring careful attention to many details regarding tissue preparation, imaging, and data/image processing in an iterative process. Here we provide an end-to-end protocol covering all aspects of a 3D pathology workflow (using light-sheet microscopy as an illustrative imaging platform) with sufficient detail to perform well-controlled preclinical and clinical studies. While 3D pathology is compatible with diverse staining protocols and computationally generated color palettes for visual analysis, this protocol will focus on a fluorescent analog of hematoxylin and eosin (H&E), which remains the most common stain for gold-standard diagnostic determinations. We present our guidelines for a broad range of end-users (e.g., biologists, clinical researchers, and engineers) in a simple tutorial format.
    • Comprehensive Surface Histology of Fresh Resection Margins With Rapid Open-Top Light-Sheet (OTLS) Microscopy

      Gao, G., Miyasato, D., Barner, L. A., Serafin, R., Bishop, K. W., Xie, W., Glaser, A. K., Rosenthal, E. L., True, L. D., Liu, J. T. C.

      IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING

      ABSTRACT
      Prostate cancer treatment decisions rely heavily on subjective visual interpretation [assigning Gleason patterns or International Society of Urological Pathology (ISUP) grade groups] of limited numbers of two-dimensional (2D) histology sections. Under this paradigm, interobserver variance is high, with ISUP grades not correlating well with outcome for individual patients, and this contributes to the over- and undertreatment of patients. Recent studies have demonstrated improved prognostication of prostate cancer outcomes based on computational analyses of glands and nuclei within 2D whole slide images. Our group has also shown that the computational analysis of three-dimensional (3D) glandular features, extracted from 3D pathology datasets of whole intact biopsies, can allow for improved recurrence prediction compared to corresponding 2D features. Here we seek to expand on these prior studies by exploring the prognostic value of 3D shape-based nuclear features in prostate cancer (e.g. nuclear size, sphericity). 3D pathology datasets were generated using open-top light-sheet (OTLS) microscopy of 102 cancer-containing biopsies extracted ex vivo from the prostatectomy specimens of 46 patients. A deep learning-based workflow was developed for 3D nuclear segmentation within the glandular epithelium versus stromal regions of the biopsies. 3D shape-based nuclear features were extracted, and a nested cross-validation scheme was used to train a supervised machine classifier based on 5-year biochemical recurrence (BCR) outcomes. Nuclear features of the glandular epithelium were found to be more prognostic than stromal cell nuclear features (area under the ROC curve [AUC] = 0.72 versus 0.63). 3D shape-based nuclear features of the glandular epithelium were also more strongly associated with the risk of BCR than analogous 2D features (AUC = 0.72 versus 0.62). The results of this preliminary investigation suggest that 3D shape-based nuclear features are associated with prostate cancer aggressiveness and could be of value for the development of decision-support tools. © 2023 The Pathological Society of Great Britain and Ireland.
    • Tracking the 3D Architecture of Hundreds of Nephrons and Peritubular Capillaries in Health and Disease Using Light Sheet Microscopy and Deep Learning

      Poudel, C., Sandoval, R. M., Wong, M. K., Liu, J. T., Vaughan, J. C., Vaughan Lab

      ABSTRACT
      Anatomic pathologists render diagnosis on tissue samples sectioned onto glass slides and viewed under a bright-field microscope. This approach is destructive to the sample, which can limit its use for ancillary assays that can inform patient management. Furthermore, the subjective interpretation of a relatively small number of 2D tissue sections per sample contributes to low interobserver agreement among pathologists for the assessment (diagnosis and grading) of various lesions.To evaluate 3D pathology data sets of thick formalin-fixed Barrett esophagus specimens imaged nondestructively with open-top light-sheet (OTLS) microscopy.Formalin-fixed, paraffin-embedded Barrett esophagus samples (N = 15) were deparaffinized, stained with a fluorescent analog of hematoxylin-eosin, optically cleared, and imaged nondestructively with OTLS microscopy. The OTLS microscopy images were subsequently compared with archived hematoxylin-eosin histology sections from each sample.Barrett esophagus samples, both small endoscopic forceps biopsies and endoscopic mucosal resections, exhibited similar resolvable structures between OTLS microscopy and conventional light microscopy with up to a ×20 objective (×200 overall magnification). The 3D histologic images generated by OTLS microscopy can enable improved discrimination of cribriform and well-formed gland morphologies. In addition, a much larger amount of tissue is visualized with OTLS microscopy, which enables improved assessment of clinical specimens exhibiting high spatial heterogeneity.In esophageal specimens, OTLS microscopy can generate images comparable in quality to conventional light microscopy, with the advantages of providing 3D information for enhanced evaluation of glandular morphologies and enabling much more of the tissue specimen to be visualized nondestructively.
    • Weakly Supervised AI for Efficient Analysis of 3D Pathology Samples.

      Song, A. H., Williams, M., Williamson, D. F., Jaume, G., Zhang, A., Chen, B., Serafin, R., Liu, J. T., Baras, A., Parwani, A. V., Mahmood, F.

      ArXiv

      ABSTRACT
      Prostate cancer treatment decisions rely heavily on subjective visual interpretation [assigning Gleason patterns or International Society of Urological Pathology (ISUP) grade groups] of limited numbers of two-dimensional (2D) histology sections. Under this paradigm, interobserver variance is high, with ISUP grades not correlating well with outcome for individual patients, and this contributes to the over- and undertreatment of patients. Recent studies have demonstrated improved prognostication of prostate cancer outcomes based on computational analyses of glands and nuclei within 2D whole slide images. Our group has also shown that the computational analysis of three-dimensional (3D) glandular features, extracted from 3D pathology datasets of whole intact biopsies, can allow for improved recurrence prediction compared to corresponding 2D features. Here we seek to expand on these prior studies by exploring the prognostic value of 3D shape-based nuclear features in prostate cancer (e.g. nuclear size, sphericity). 3D pathology datasets were generated using open-top light-sheet (OTLS) microscopy of 102 cancer-containing biopsies extracted ex vivo from the prostatectomy specimens of 46 patients. A deep learning-based workflow was developed for 3D nuclear segmentation within the glandular epithelium versus stromal regions of the biopsies. 3D shape-based nuclear features were extracted, and a nested cross-validation scheme was used to train a supervised machine classifier based on 5-year biochemical recurrence (BCR) outcomes. Nuclear features of the glandular epithelium were found to be more prognostic than stromal cell nuclear features (area under the ROC curve [AUC] = 0.72 versus 0.63). 3D shape-based nuclear features of the glandular epithelium were also more strongly associated with the risk of BCR than analogous 2D features (AUC = 0.72 versus 0.62). The results of this preliminary investigation suggest that 3D shape-based nuclear features are associated with prostate cancer aggressiveness and could be of value for the development of decision-support tools. © 2023 The Pathological Society of Great Britain and Ireland.
    • Engineering the future of 3D pathology

      Liu, J. T. C., Chow, S. S. L., Colling, R., Downes, M. R., Farre, X., Humphrey, P., Janowczyk, A., Mirtti, T., Verrill, C., Zlobec, I., True, L. D.

      JOURNAL OF PATHOLOGY CLINICAL RESEARCH

      ABSTRACT
      A miniature optical-sectioning fluorescence microscope with high sensitivity and resolution would enable non-invasive and real-time tissue inspection, with potential use cases including early disease detection and intraoperative guidance. Previously, we developed a miniature MEMS-based dual-axis confocal (DAC) microscope that enabled video-rate optically sectioned in vivo microscopy of human tissues. However, the device's clinical utility was limited due to a small field of view, a non-adjustable working distance, and a lack of a sterilization strategy. In our latest design, we have made improvements to achieve a 2x increase in the field of view (600 × 300 µm) and an adjustable working distance range of 150 µm over a wide range of excitation/emission wavelengths (488-750 nm), all while maintaining a high frame rate of 15 frames per second (fps). Furthermore, the device is designed to image through a disposable sterile plastic drape for convenient clinical use. We rigorously characterize the performance of the device and show example images of ex vivo tissues to demonstrate the optical performance of our new design, including fixed mouse skin and human prostate, as well as fresh mouse kidney, mouse intestine, and human head and neck surgical specimens with corresponding H&E histology. These improvements will facilitate clinical testing and translation.
    • Nondestructive 3D Pathology with Light-Sheet Fluorescence Microscopy for Translational Research and Clinical Assays

      Liu, J. T. C., Glaser, A. K., Poudel, C., Vaughan, J. C.

      ANNUAL REVIEW OF ANALYTICAL CHEMISTRY

      ABSTRACT
      There have been numerous academic and commercial efforts to develop high-resolution in vivo microscopes for a variety of clinical use cases, including early disease detection and surgical guidance. While many high-profile studies, commercialized products, and publications have resulted from these efforts, mainstream clinical adoption has been relatively slow other than for a few clinical applications (e.g., dermatology).Here, our goals are threefold: (1) to introduce and motivate the need for in vivo microscopy (IVM) as an adjunctive tool for clinical detection, diagnosis, and treatment, (2) to discuss the key translational challenges facing the field, and (3) to propose best practices and recommendations to facilitate clinical adoption.We will provide concrete examples from various clinical domains, such as dermatology, oral/gastrointestinal oncology, and neurosurgery, to reinforce our observations and recommendations.While the incremental improvement and optimization of IVM technologies should and will continue to occur, future translational efforts would benefit from the following: (1) integrating clinical and industry partners upfront to define and maintain a compelling value proposition, (2) identifying multimodal/multiscale imaging workflows, which are necessary for success in most clinical scenarios, and (3) developing effective artificial intelligence tools for clinical decision support, tempered by a realization that complete adoption of such tools will be slow.The convergence of imaging modalities, academic-industry-clinician partnerships, and new computational capabilities has the potential to catalyze rapid progress and adoption of IVM in the next few decades.
    • Visual Assessment of 2-Dimensional Levels Within 3-Dimensional Pathology Data Sets of Prostate Needle Biopsies Reveals Substantial Spatial Heterogeneity

      Koyuncu, C., Janowczyk, A., Farre, X., Pathak, T., Mirtti, T., Fernandez, P. L., Pons, L., Reder, N. P., Sera, R., Chow, S. S. L., Viswanathan, V. S., Glaser, A. K., True, L. D., Liu, J. T. C., Madabhushi, A.

      LABORATORY INVESTIGATION

      ABSTRACT
      A miniature optical-sectioning fluorescence microscope with high sensitivity and resolution would enable non-invasive and real-time tissue inspection, with potential use cases including early disease detection and intraoperative guidance. Previously, we developed a miniature MEMS-based dual-axis confocal (DAC) microscope that enabled video-rate optically sectioned in vivo microscopy of human tissues. However, the device's clinical utility was limited due to a small field of view, a non-adjustable working distance, and a lack of a sterilization strategy. In our latest design, we have made improvements to achieve a 2x increase in the field of view (600 × 300 µm) and an adjustable working distance range of 150 µm over a wide range of excitation/emission wavelengths (488-750 nm), all while maintaining a high frame rate of 15 frames per second (fps). Furthermore, the device is designed to image through a disposable sterile plastic drape for convenient clinical use. We rigorously characterize the performance of the device and show example images of ex vivo tissues to demonstrate the optical performance of our new design, including fixed mouse skin and human prostate, as well as fresh mouse kidney, mouse intestine, and human head and neck surgical specimens with corresponding H&E histology. These improvements will facilitate clinical testing and translation.
    • Artificial Intelligence-Triaged 3-Dimensional Pathology to Improve Detection of Esophageal Neoplasia While Reducing Pathologist Workloads.

      Erion Barner, L. A., Gao, G., Reddi, D. M., Lan, L., Burke, W., Mahmood, F., Grady, W. M., Liu, J. T.

      Modern pathology : an official journal of the United States and Canadian Academy of Pathology, Inc

      ABSTRACT
      In recent years, technological advances in tissue preparation, high-throughput volumetric microscopy, and computational infrastructure have enabled rapid developments in nondestructive 3D pathology, in which high-resolution histologic datasets are obtained from thick tissue specimens, such as whole biopsies, without the need for physical sectioning onto glass slides. While 3D pathology generates massive datasets that are attractive for automated computational analysis, there is also a desire to use 3D pathology to improve the visual assessment of tissue histology. In this perspective, we discuss and provide examples of potential advantages of 3D pathology for the visual assessment of clinical specimens and the challenges of dealing with large 3D datasets (of individual or multiple specimens) that pathologists have not been trained to interpret. We discuss the need for artificial intelligence triaging algorithms and explainable analysis methods to assist pathologists or other domain experts in the interpretation of these novel, often complex, large datasets.
    • Introduction to the Biophotonics Congress 2022 feature issue

      Liu, J. T. C., Bale, G., Choe, R., Elson, D. S., Oldenburg, A., Ian, L., Tkaczyk, E. R.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      Light-sheet microscopy has emerged as the preferred means for high-throughput volumetric imaging of cleared tissues. However, there is a need for a flexible system that can address imaging applications with varied requirements in terms of resolution, sample size, tissue-clearing protocol, and transparent sample-holder material. Here, we present a 'hybrid' system that combines a unique non-orthogonal dual-objective and conventional (orthogonal) open-top light-sheet (OTLS) architecture for versatile multi-scale volumetric imaging. We demonstrate efficient screening and targeted sub-micrometer imaging of sparse axons within an intact, cleared mouse brain. The same system enables high-throughput automated imaging of multiple specimens, as spotlighted by a quantitative multi-scale analysis of brain metastases. Compared with existing academic and commercial light-sheet microscopy systems, our hybrid OTLS system provides a unique combination of versatility and performance necessary to satisfy the diverse requirements of a growing number of cleared-tissue imaging applications.
    • Innovations in Remote Teaching of Engineering Design Teams.

      Kang, S., Blakeney, E. A., Yasuhara, K., Kearney, K. E., Payne, S., Seibel, E., Liu, J. T., Reinhall, P., Posner, J.

      Annual Conference & Exposition : final program and proceedings. American Society for Engineering Education

      ABSTRACT
      In recent years, there has been a revived appreciation for the importance of spatial context and morphological phenotypes for both understanding disease progression and guiding treatment decisions. Compared with conventional 2D histopathology, which is the current gold standard of medical diagnostics, nondestructive 3D pathology offers researchers and clinicians the ability to visualize orders of magnitude more tissue within their natural volumetric context. This has been enabled by rapid advances in tissue-preparation methods, high-throughput 3D microscopy instrumentation, and computational tools for processing these massive feature-rich data sets. Here, we provide a brief overview of many of these technical advances along with remaining challenges to be overcome. We also speculate on the future of 3D pathology as applied in translational investigations, preclinical drug development, and clinical decision-support assays.
    • An end-to-end workflow for non-destructive 3D pathology.

      Bishop, K. W., Barner, L. A., Han, Q., Baraznenok, E., Lan, L., Poudel, C., Gao, G., Serafin, R. B., Chow, S. S., Glaser, A. K., Janowczyk, A., Brenes, D., Huang, H., Miyasato, D., True, L. D., Kang, S., Vaughan, J. C., Liu, J. T.

      bioRxiv : the preprint server for biology

      ABSTRACT
      For tumor resections, margin status typically correlates with patient survival but positive margin rates are generally high (up to 45% for head and neck cancer). Frozen section analysis (FSA) is often used to intraoperatively assess the margins of excised tissue, but suffers from severe under-sampling of the actual margin surface, inferior image quality, slow turnaround, and tissue destructiveness.Here, we have developed an imaging workflow to generate en face histologic images of freshly excised surgical margin surfaces based on open-top light-sheet (OTLS) microscopy. Key innovations include (1) the ability to generate false-colored H&E-mimicking images of tissue surfaces stained for < 1 min with a single fluorophore, (2) rapid OTLS surface imaging at a rate of 15 min/cm2 followed by real-time post-processing of datasets within RAM at a rate of 5 min/cm2, and (3) rapid digital surface extraction to account for topological irregularities at the tissue surface.In addition to the performance metrics listed above, we show that the image quality generated by our rapid surface-histology method approaches that of gold-standard archival histology.OTLS microscopy has the feasibility to provide intraoperative guidance of surgical oncology procedures.The reported methods can potentially improve tumor-resection procedures, thereby improving patient outcomes and quality of life.
    • Nondestructive 3D pathology with analysis of nuclear features for prostate cancer risk assessment

      Serafin, R., Koyuncu, C., Xie, W., Huang, H., Glaser, A. K., Reder, N. P., Janowczyk, A., True, L. D., Madabhushi, A., Liu, J. T. C.

      JOURNAL OF PATHOLOGY

      ABSTRACT
      A feature issue is being presented by a team of guest editors containing papers based on studies presented at the Optica Biophotonics Congress: Biomedical Optics held on April 24-27, 2022 in Fort Lauderdale, Florida, USA.
    • Nondestructive 3D Pathology Image Atlas of Barrett Esophagus With Open-Top Light-Sheet Microscopy

      Reddi, D. M., Barner, L. A., Burke, W., Gao, G., Grady, W. M., Liu, J. T. C.

      ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE

      ABSTRACT
      Human tissue consists of complex structures that display a diversity of morphologies, forming a tissue microenvironment that is, by nature, three-dimensional (3D). However, the current standard-of-care involves slicing 3D tissue specimens into two-dimensional (2D) sections and selecting a few for microscopic evaluation1,2, with concomitant risks of sampling bias and misdiagnosis3-6. To this end, there have been intense efforts to capture 3D tissue morphology and transition to 3D pathology, with the development of multiple high-resolution 3D imaging modalities7-18. However, these tools have had little translation to clinical practice as manual evaluation of such large data by pathologists is impractical and there is a lack of computational platforms that can efficiently process the 3D images and provide patient-level clinical insights. Here we present Modality-Agnostic Multiple instance learning for volumetric Block Analysis (MAMBA), a deep-learning-based platform for processing 3D tissue images from diverse imaging modalities and predicting patient outcomes. Archived prostate cancer specimens were imaged with open-top light-sheet microscopy12-14 or microcomputed tomography15,16 and the resulting 3D datasets were used to train risk-stratification networks based on 5-year biochemical recurrence outcomes via MAMBA. With the 3D block-based approach, MAMBA achieves an area under the receiver operating characteristic curve (AUC) of 0.86 and 0.74, superior to 2D traditional single-slice-based prognostication (AUC of 0.79 and 0.57), suggesting superior prognostication with 3D morphological features. Further analyses reveal that the incorporation of greater tissue volume improves prognostic performance and mitigates risk prediction variability from sampling bias, suggesting that there is value in capturing larger extents of spatially heterogeneous 3D morphology. With the rapid growth and adoption of 3D spatial biology and pathology techniques by researchers and clinicians, MAMBA provides a general and efficient framework for 3D weakly supervised learning for clinical decision support and can help to reveal novel 3D morphological biomarkers for prognosis and therapeutic response.
    • 3D open-top light-sheet microscopy and 3D microdissection of neuoadjuvant-treated primary prostate cancer reveals latent subclonal mutations.

      Reder, N., Pritchard, C. C., Konnick, E. Q., Huang, H., Lerma, A., Glaser, A. K., True, L. D., Liu, J. T. C., Schweizer, M.

      ABSTRACT
      The University of Washington's Engineering Innovation in Health program is a yearlong engineering design course sequence where senior undergraduate and graduate engineering students across different disciplines work in teams with health professionals to address their unmet needs. With the onset of the COVID-19 pandemic, these team- and project-based courses shifted from an in-person to remote course environment. Here, we share innovative teaching strategies for a team-based, remote course environment. We show how this shift affected productivity by comparing survey results from before (in person) and during (remote) the pandemic. Preliminary results show that overall project outcomes and productivity were as high or, in some cases, higher during the pandemic than prior to the pandemic. These findings suggest that the innovative remote teaching strategies implemented by the teaching team provided effective options in the absence of certain hands-on experiences that are considered critical to engineering capstone design courses. A discussion on these teaching strategies in the context beyond the pandemic are considered in the discussion.

    2022

    • 3D Light-Sheet Microscopy and Microdissection of Primary Prostate Cancer Reveals Important Latent Sub-clonal Mutations

      Lerma, L., Pritchard, C., Konnick, E., Haffner, M., Schweizer, M., Huang, H., Glaser, A., True, L., Liu, J., Reder, N.

      ABSTRACT
      Fluorescence microscopy is a vital tool in biomedical research but faces considerable challenges in achieving uniform or bright labeling. For instance, fluorescent proteins are limited to model organisms, and antibody conjugates can be inconsistent and difficult to use with thick specimens. To partly address these challenges, we developed a labeling protocol that can rapidly visualize many well-contrasted key features and landmarks on biological specimens in both thin and thick tissues or cultured cells. This approach uses established reactive fluorophores to label a variety of biological specimens for cleared-tissue microscopy or expansion super-resolution microscopy and is termed FLARE (fluorescent labeling of abundant reactive entities). These fluorophores target chemical groups and reveal their distribution on the specimens; amine-reactive fluorophores such as hydroxysuccinimidyl esters target accessible amines on proteins, while hydrazide fluorophores target oxidized carbohydrates. The resulting stains provide signals analogous to traditional general histology stains such as H&E or periodic acid-Schiff but use fluorescent probes that are compatible with volumetric imaging. In general, the stains for FLARE are performed in the order of carbohydrates, amine and DNA, and the incubation time for the stains varies from 1 h to 1 d depending on the combination of stains and the type and thickness of the biological specimens. FLARE is powerful, robust and easy to implement in laboratories that already routinely do fluorescence microscopy.
    • <i>In vivo</i> microscopy as an adjunctive tool to guide detection, diagnosis, and treatment

      Bishop, K. W., Maitland, K. C., Rajadhyaksha, M., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      For breast cancer patients, the extent of regional lymph node (LN) metastasis influences the decision to remove all axillary LNs. Metastases are currently identified and classified with visual analysis of a few thin tissue sections with conventional histology that may underrepresent the extent of metastases.We sought to enable nondestructive three-dimensional (3D) pathology of human axillary LNs and to develop a practical workflow for LN staging with our method. We also sought to evaluate whether 3D pathology improves staging accuracy in comparison to two-dimensional (2D) histology.We developed a method to fluorescently stain and optically clear LN specimens for comprehensive imaging with multiresolution open-top light-sheet microscopy. We present an efficient imaging and data-processing workflow for rapid evaluation of H&E-like datasets in 3D, with low-resolution screening to identify potential metastases followed by high-resolution localized imaging to confirm malignancy.We simulate LN staging with 3D and 2D pathology datasets from 10 metastatic nodes, showing that 2D pathology consistently underestimates metastasis size, including instances in which 3D pathology would lead to upstaging of the metastasis with important implications on clinical treatment.Our 3D pathology method may improve clinical management for breast cancer patients by improving staging accuracy of LN metastases.
    • Prostate Cancer Risk Stratification via Nondestructive 3D Pathology with Deep Learning-Assisted Gland Analysis

      Xie, W., Reder, N. P., Koyuncu, C., Leo, P., Hawley, S., Huang, H., Mao, C., Postupna, N., Kang, S., Serafin, R., Gao, G., Han, Q., Bishop, K. W., Barner, L. A., Fu, P., Wright, J. L., Keene, C., Vaughan, J. C., Janowczyk, A., Glaser, A. K., Madabhushi, A., True, L. D., Liu, J. T. C.

      CANCER RESEARCH

      ABSTRACT
      The rate of positive margins in head and neck cancers has remained stagnant over the past three decades and is consistently associated with poor overall survival. This suggests that significant improvements must be made intraoperatively to ensure negative margins. We discuss the important role of fluorescence imaging to guide surgical oncology in head and neck cancer. This review includes a general overview of the principles of fluorescence, available fluorophores used for fluorescence imaging, and specific clinical applications of fluorescence-guided surgery, as well as challenges and future directions in head and neck surgical oncology. Laryngoscope, 131:529-534, 2021.
    • A hybrid open-top light-sheet microscope for versatile multi-scale imaging of cleared tissues

      Glaser, A. K., Bishop, K. W., Barner, L. A., Susaki, E. A., Kubota, S. I., Gao, G., Serafin, R. B., Balaram, P., Turschak, E., Nicovich, P. R., Lai, H., Lucas, L. A. G., Yi, Y., Nichols, E. K., Huang, H., Reder, N. P., Wilson, J. J., Sivakumar, R., Shamskhou, E., Stoltzfus, C. R., Wei, X., Hempton, A. K., Pende, M., Murawala, P., Dodt, H., Imaizumi, T., Shendure, J., Beliveau, B. J., Gerner, M. Y., Xin, L., Zhao, H., True, L. D., Reid, R., Chandrashekar, J., Ueda, H. R., Svoboda, K., Liu, J. T. C.

      NATURE METHODS

      ABSTRACT
      For breast cancer patients, the extent of regional lymph node (LN) metastasis influences the decision to remove all axillary LNs. Metastases are currently identified and classified with visual analysis of a few thin tissue sections with conventional histology that may underrepresent the extent of metastases.We sought to enable nondestructive three-dimensional (3D) pathology of human axillary LNs and to develop a practical workflow for LN staging with our method. We also sought to evaluate whether 3D pathology improves staging accuracy in comparison to two-dimensional (2D) histology.We developed a method to fluorescently stain and optically clear LN specimens for comprehensive imaging with multiresolution open-top light-sheet microscopy. We present an efficient imaging and data-processing workflow for rapid evaluation of H&E-like datasets in 3D, with low-resolution screening to identify potential metastases followed by high-resolution localized imaging to confirm malignancy.We simulate LN staging with 3D and 2D pathology datasets from 10 metastatic nodes, showing that 2D pathology consistently underestimates metastasis size, including instances in which 3D pathology would lead to upstaging of the metastasis with important implications on clinical treatment.Our 3D pathology method may improve clinical management for breast cancer patients by improving staging accuracy of LN metastases.
    • Fluorescent labeling of abundant reactive entities (FLARE) for cleared-tissue and super-resolution microscopy

      Lee, M., Mao, C., Glaser, A. K., Woodworth, M. A., Halpern, A. R., Ali, A., Liu, J. T. C., Vaughan, J. C.

      NATURE PROTOCOLS

      ABSTRACT
      Interdisciplinary academic teams perform better when competent in teamwork; however, there is a lack of best practices of how to introduce and facilitate the development of effective learning and functioning within these teams in academic environments.To close this gap, we tailored, implemented, and evaluated team science training in the year-long Engineering Innovation in Health (EIH) program at the University of Washington (UW), a project-based course in which engineering students across several disciplines partner with health professionals to develop technical solutions to clinical and translational health challenges. EIH faculty from the UW College of Engineering and the Institute of Translational Health Sciences' (ITHS) Team Science Core codeveloped and delivered team science training sessions and evaluated their impact with biannual surveys. A student cohort was surveyed prior to the implementation of the team science trainings, which served as a baseline.Survey responses were compared within and between both cohorts (approximately 55 students each Fall Quarter and 30 students each Spring Quarter). Statistically significant improvements in measures of self-efficacy and interpersonal team climate (i.e., psychological safety) were observed within and between teams.Tailored team science training provided to student-professional teams resulted in measurable improvements in self-efficacy and interpersonal climate both of which are crucial for teamwork and intellectual risk taking. Future research is needed to determine long-term impacts of course participation on individual and team outcomes (e.g., patents, start-ups). Additionally, adaptability of this model to clinical and translational research teams in alternate formats and settings should be tested.
    • Multiresolution nondestructive 3D pathology of whole lymph nodes for breast cancer staging

      Barner, L. A., Glaser, A. K., Mao, C., Susaki, E. A., Vaughan, J. C., Dintzis, S. M., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      Prostate cancer treatment planning is largely dependent upon examination of core-needle biopsies. The microscopic architecture of the prostate glands forms the basis for prognostic grading by pathologists. Interpretation of these convoluted three-dimensional (3D) glandular structures via visual inspection of a limited number of two-dimensional (2D) histology sections is often unreliable, which contributes to the under- and overtreatment of patients. To improve risk assessment and treatment decisions, we have developed a workflow for nondestructive 3D pathology and computational analysis of whole prostate biopsies labeled with a rapid and inexpensive fluorescent analogue of standard hematoxylin and eosin (H&E) staining. This analysis is based on interpretable glandular features and is facilitated by the development of image translation-assisted segmentation in 3D (ITAS3D). ITAS3D is a generalizable deep learning-based strategy that enables tissue microstructures to be volumetrically segmented in an annotation-free and objective (biomarker-based) manner without requiring immunolabeling. As a preliminary demonstration of the translational value of a computational 3D versus a computational 2D pathology approach, we imaged 300 ex vivo biopsies extracted from 50 archived radical prostatectomy specimens, of which, 118 biopsies contained cancer. The 3D glandular features in cancer biopsies were superior to corresponding 2D features for risk stratification of patients with low- to intermediate-risk prostate cancer based on their clinical biochemical recurrence outcomes. The results of this study support the use of computational 3D pathology for guiding the clinical management of prostate cancer. SIGNIFICANCE: An end-to-end pipeline for deep learning-assisted computational 3D histology analysis of whole prostate biopsies shows that nondestructive 3D pathology has the potential to enable superior prognostic stratification of patients with prostate cancer.

    2021

    • Implementation and evaluation of team science training for interdisciplinary teams in an engineering design program

      Blakeney, E., Kang, S., Henrikson, K., Liu, J. T. C., Seibel, E. J., Sprecher, J., Summerside, N., Vogel, M. T., Zierler, B. K., Posner, J. D.

      JOURNAL OF CLINICAL AND TRANSLATIONAL SCIENCE

      ABSTRACT
      High-throughput methods for slide-free three-dimensional (3D) pathological analyses of whole biopsies and surgical specimens offer the promise of modernizing traditional histology workflows and delivering improvements in diagnostic performance. Advanced optical methods now enable the interrogation of orders of magnitude more tissue than previously possible, where volumetric imaging allows for enhanced quantitative analyses of cell distributions and tissue structures that are prognostic and predictive. Non-destructive imaging processes can simplify laboratory workflows, potentially reducing costs, and can ensure that samples are available for subsequent molecular assays. However, the large size of the feature-rich datasets that they generate poses challenges for data management and computer-aided analysis. In this Perspective, we provide an overview of the imaging technologies that enable 3D pathology, and the computational tools-machine learning, in particular-for image processing and interpretation. We also discuss the integration of various other diagnostic modalities with 3D pathology, along with the challenges and opportunities for clinical adoption and regulatory approval.
    • Three-dimensional histo-morphometric features from light-sheet microscopy images result in improved discrimination of benign from malignant glands in prostate cancer

      Koyuncu, C. F., Janowczyk, A., Lu, C., Leo, P., Alilou, M., Glaser, A. K., Reder, N. P., Liu, J. T. C., Madabhushi, A.

      ABSTRACT
      Open-top light-sheet (OTLS) microscopes have been developed for user-friendly and versatile high-throughput 3D microscopy of thick specimens. As with all imaging modalities, spatial resolution trades off with imaging and analysis times. A hierarchical multi-scale imaging workflow would therefore be of value for many volumetric microscopy applications. We describe a compact multi-resolution OTLS microscope, enabled by a novel solid immersion meniscus lens (SIMlens), which allows users to rapidly transition between air-based objectives for low- and high-resolution 3D imaging. We demonstrate the utility of this system by showcasing an efficient 3D analysis workflow for a diagnostic pathology application.
    • Microdissected "cuboids" for microfluidic drug testing of intact tissues

      Horowitz, L. F., Rodriguez, A. D., Au-Yeung, A., Bishop, K. W., Barner, L. A., Mishra, G., Raman, A., Delgado, P., Liu, J. T. C., Gujral, T. S., Mehrabi, M., Yang, M., Pierce, R. H., Folch, A.

      LAB ON A CHIP

      ABSTRACT
      Processing and diagnosing a set of 12 prostate biopsies using conventional histology methods typically take at least one day. A rapid and accurate process performed while the patient is still on-site could significantly improve the patient's quality of life.We develop and assess the feasibility of a one-hour-to-diagnosis (1Hr2Dx) method for processing and providing a preliminary diagnosis of a set of 12 prostate biopsies.We developed a fluorescence staining, optical clearing, and 3D open-top light-sheet microscopy workflow to enable 12 prostate needle core biopsies to be processed and diagnosed within an hour of receipt. We analyzed 44 biopsies by the 1Hr2Dx method, which does not consume tissue. The biopsies were then processed for routine, slide-based 2D histology. Three pathologists independently evaluated the 3D 1Hr2Dx and 2D slide-based datasets in a blinded, randomized fashion. Turnaround times were recorded, and the accuracy of our method was compared with gold-standard slide-based histology.The average turnaround time for tissue processing, imaging, and diagnosis was 44.5 min. The sensitivity and specificity of 1Hr2Dx in diagnosing cancer were both >90  %  .The 1Hr2Dx method has the potential to improve patient care by providing an accurate preliminary diagnosis within an hour of biopsy.
    • Harnessing non-destructive 3D pathology

      Liu, J. T. C., Glaser, A. K., Bera, K., True, L. D., Reder, N. P., Eliceiri, K. W., Madabhushi, A.

      NATURE BIOMEDICAL ENGINEERING

      ABSTRACT
      Processing and diagnosing a set of 12 prostate biopsies using conventional histology methods typically take at least one day. A rapid and accurate process performed while the patient is still on-site could significantly improve the patient's quality of life.We develop and assess the feasibility of a one-hour-to-diagnosis (1Hr2Dx) method for processing and providing a preliminary diagnosis of a set of 12 prostate biopsies.We developed a fluorescence staining, optical clearing, and 3D open-top light-sheet microscopy workflow to enable 12 prostate needle core biopsies to be processed and diagnosed within an hour of receipt. We analyzed 44 biopsies by the 1Hr2Dx method, which does not consume tissue. The biopsies were then processed for routine, slide-based 2D histology. Three pathologists independently evaluated the 3D 1Hr2Dx and 2D slide-based datasets in a blinded, randomized fashion. Turnaround times were recorded, and the accuracy of our method was compared with gold-standard slide-based histology.The average turnaround time for tissue processing, imaging, and diagnosis was 44.5 min. The sensitivity and specificity of 1Hr2Dx in diagnosing cancer were both >90  %  .The 1Hr2Dx method has the potential to improve patient care by providing an accurate preliminary diagnosis within an hour of biopsy.
    • Intraoperative Fluorescence-Guided Surgery in Head and Neck Squamous Cell Carcinoma.

      Lee, Y. J., Krishnan, G., Nishio, N., van den Berg, N. S., Lu, G., Martin, B. A., van Keulen, S., Colevas, A. D., Kapoor, S., Liu, J. T., Rosenthal, E. L.

      The Laryngoscope

      ABSTRACT
      As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

    2020

    • Light-Sheet Microscopy for 3D Pathology of a Variety of Human Tissues

      Glaser, A., Reder, N., True, L., Liu, J.

    • Real-time video mosaicking to guide handheld in vivo microscopy

      Yin, C., Wei, L., Kose, K., Glaser, A. K., Peterson, G., Rajadhyaksha, M., Liu, J. T. C.

      JOURNAL OF BIOPHOTONICS

    • Multi-resolution open-top light-sheet microscopy to enable efficient 3D pathology workflows

      Barner, L. A., Glaser, A. K., Huang, H., True, L. D., Liu, J. T. C.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      Intraoperative confocal microscopy can enable high-resolution cross-sectional imaging of intact tissues as a non-invasive real-time alternative to gold-standard histology. However, all current means of intraoperative confocal microscopy are hindered by a limited field of view (FOV), presenting a challenge for evaluating gliomas, which are highly heterogeneous.This study explored the use of image mosaicking with handheld dual-axis confocal (DAC) microscopy of fresh human glioma specimens.In this preliminary technical feasibility study, fresh human glioma specimens from 6 patients were labeled with a fast-acting topical stain (acridine orange) and imaged using a newly developed DAC microscope prototype.In comparison to individual image frames with small fields of view, mosaicked images from a DAC microscope correlate better with gold-standard H&E-stained histology images, including the ability to visualize gradual transitions from areas of dense cellularity to sparse cellularity within the tumor.LS-DAC microscopy provides high-resolution, high-contrast images of glioma tissues that agree with corresponding H&E histology. Compared with individual image frames, mosaicked images provide more accurate representations of the overall cytoarchitecture of heterogeneous glioma tissues. Further investigation is needed to evaluate the ability of high-resolution mosaicked microscopy to improve the extent of glioma resection and patient outcomes.
    • Feature-rich covalent stains for super-resolution and cleared tissue fluorescence microscopy

      Mao, C., Lee, M., Jhan, J., Halpern, A. R., Woodworth, M. A., Glaser, A. K., Chozinski, T. J., Shin, L., Pippin, J. W., Shankland, S. J., Liu, J., Vaughan, J. C.

      SCIENCE ADVANCES

    • Diagnosing 12 prostate needle cores within an hour of biopsy via open-top light-sheet microscopy

      Xie, W., Glaser, A. K., Vakar-Lopez, F., Wright, J. L., Reder, N. P., Liu, J. T. C., True, L. D.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      Slide-free digital pathology techniques, including nondestructive 3D microscopy, are gaining interest as alternatives to traditional slide-based histology. In order to facilitate clinical adoption of these fluorescence-based techniques, software methods have been developed to convert grayscale fluorescence images into color images that mimic the appearance of standard absorptive chromogens such as hematoxylin and eosin (H&E). However, these false-coloring algorithms often require manual and iterative adjustment of parameters, with results that can be inconsistent in the presence of intensity nonuniformities within an image and/or between specimens (intra- and inter-specimen variability). Here, we present an open-source (Python-based) rapid intensity-leveling and digital-staining package that is specifically designed to render two-channel fluorescence images (i.e. a fluorescent analog of H&E) to the traditional H&E color space for 2D and 3D microscopy datasets. However, this method can be easily tailored for other false-coloring needs. Our package offers (1) automated and uniform false coloring in spite of uneven staining within a large thick specimen, (2) consistent color-space representations that are robust to variations in staining and imaging conditions between different specimens, and (3) GPU-accelerated data processing to allow these methods to scale to large datasets. We demonstrate this platform by generating H&E-like images from cleared tissues that are fluorescently imaged in 3D with open-top light-sheet (OTLS) microscopy, and quantitatively characterizing the results in comparison to traditional slide-based H&E histology.
    • Performance tradeoffs for single- and dual-objective open-top light-sheet microscope designs: a simulation-based analysis

      Bishop, K. W., Glaser, A. K., Liu, J. T. C.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      Handheld and endoscopic optical-sectioning microscopes are being developed for noninvasive screening and intraoperative consultation. Imaging a large extent of tissue is often desired, but miniature in vivo microscopes tend to suffer from limited fields of view. To extend the imaging field during clinical use, we have developed a real-time video mosaicking method, which allows users to efficiently survey larger areas of tissue. Here, we modified a previous post-processing mosaicking method so that real-time mosaicking is possible at >30 frames/second when using a device that outputs images that are 400 × 400 pixels in size. Unlike other real-time mosaicking methods, our strategy can accommodate image rotations and deformations that often occur during clinical use of a handheld microscope. We perform a feasibility study to demonstrate that the use of real-time mosaicking is necessary to enable efficient sampling of a desired imaging field when using a handheld dual-axis confocal microscope.
    • Video-Mosaicked Handheld Dual-Axis Confocal Microscopy of Gliomas: An<i>ex vivo</i>Feasibility Study in Humans

      Fujita, Y., Wei, L., Cimino, P. J., Liu, J. T. C., Sanai, N.

      FRONTIERS IN ONCOLOGY

      ABSTRACT
      Fluorescence microscopy is a workhorse tool in biomedical imaging but often poses substantial challenges to practitioners in achieving bright or uniform labeling. In addition, while antibodies are effective specific labels, their reproducibility is often inconsistent, and they are difficult to use when staining thick specimens. We report the use of conventional, commercially available fluorescent dyes for rapid and intense covalent labeling of proteins and carbohydrates in super-resolution (expansion) microscopy and cleared tissue microscopy. This approach, which we refer to as Fluorescent Labeling of Abundant Reactive Entities (FLARE), produces simple and robust stains that are modern equivalents of classic small-molecule histology stains. It efficiently reveals a wealth of key landmarks in cells and tissues under different fixation or sample processing conditions and is compatible with immunolabeling of proteins and in situ hybridization labeling of nucleic acids.
    • Multi-immersion open-top light-sheet microscopy

      Glaser, A. K., Liu, J. T. C.

      ABSTRACT
      Open-top light-sheet (OTLS) microscopy has been developed for rapid volumetric imaging of large pathology specimens. A challenge with OTLS microscopy is the transmission of oblique illumination and detection beams through a horizontal sample plate without introducing aberrations. Previous solutions prevented vertical sample movement, constrained the refractive index of the sample, and/or hindered multi-resolution imaging. Here we describe a solid immersion meniscus lens, a wavefront-matching element that suppresses aberrations when illumination and detection beam transition between air and various high-index immersion media, thereby enabling multi-resolution OTLS microscopy of specimens cleared with diverse protocols.
    • FalseColor-Python: A rapid intensity-leveling and digital-staining package for fluorescence-based slide-free digital pathology

      Serafin, R., Xie, W., Glaser, A. K., Liu, J. T. C.

      PLOS ONE

      ABSTRACT
      Light-sheet microscopy (LSM) has emerged as a powerful tool for high-speed volumetric imaging of live model organisms and large optically cleared specimens. When designing cleared-tissue LSM systems with certain desired imaging specifications (e.g. resolution, contrast, and working distance), various design parameters must be taken into consideration. In order to elucidate some of the key design tradeoffs for LSM systems, we present a diffraction-based analysis of single- and dual-objective LSM configurations using simulations of LSM point spread functions. We assume Gaussian illumination is utilized. Specifically, we analyze the effects of the illumination and collection numerical aperture (NA), as well as their crossing angle, on spatial resolution and contrast. Assuming an open-top light-sheet (OTLS) architecture, we constrain these parameters based on fundamental geometric considerations as well as those imposed by currently available microscope objectives. In addition to revealing the performance tradeoffs of various single- and dual-objective LSM configurations, our analysis showcases the potential advantages of a novel, non-orthogonal dual-objective (NODO) architecture, especially for moderate-resolution imaging applications (collection NA of 0.5 to 0.8).
    • 3D Open-Top Light-Sheet (OTLS) Fluorescence Microscopy of Entire Prostate Core Biopsies Improves Diagnostic Accuracy

      Malleis, J., Glaser, A., Kang, S., Pang, K., True, L., Liu, J., Reder, N.

      ABSTRACT
      Open-top light-sheet (OTLS) microscopy has been developed for rapid volumetric imaging of large pathology specimens. A challenge with OTLS microscopy is the transmission of oblique illumination and detection beams through a horizontal sample plate without introducing aberrations. Previous solutions prevented vertical sample movement, constrained the refractive index of the sample, and/or hindered multi-resolution imaging. Here we describe a solid immersion meniscus lens, a wavefront-matching element that suppresses aberrations when illumination and detection beam transition between air and various high-index immersion media, thereby enabling multi-resolution OTLS microscopy of specimens cleared with diverse protocols.
    • Diagnosis of All 12 Needle Cores within an Hour of a Prostate Biopsy Procedure

      Xie, W., Reder, N., Vakar-Lopez, F., Liu, J., Glaser, A., True, L.

      ABSTRACT
      Open-top light-sheet (OTLS) microscopy has been developed for rapid volumetric imaging of large pathology specimens. A challenge with OTLS microscopy is the transmission of oblique illumination and detection beams through a horizontal sample plate without introducing aberrations. Previous solutions prevented vertical sample movement, constrained the refractive index of the sample, and/or hindered multi-resolution imaging. Here we describe a solid immersion meniscus lens, a wavefront-matching element that suppresses aberrations when illumination and detection beam transition between air and various high-index immersion media, thereby enabling multi-resolution OTLS microscopy of specimens cleared with diverse protocols.

    2019

    • Microscopy with ultraviolet surface excitation for wide-area pathology of breast surgical margins

      Xie, W., Chen, Y., Wang, Y., Wei, L., Yin, C., Glaser, A. K., Fauver, M. E., Seibel, E. J., Dintzis, S. M., Vaughan, J. C., Reder, N. P., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      5-ALA-based fluorescence-guided surgery has been shown to be a safe and effective method to improve intraoperative visualization and resection of malignant gliomas. However, it remains ineffective in guiding the resection of lower-grade, non-enhancing, and deep-seated tumors, mainly because these tumors do not produce detectable fluorescence with conventional visualization technologies, namely, wide-field (WF) surgical microscopy.We describe some of the main factors that limit the sensitivity and accuracy of conventional WF surgical microscopy, and then provide a survey of commercial and research prototypes being developed to address these challenges, along with their principles, advantages and disadvantages, as well as the current status of clinical translation for each technology. We also provide a neurosurgical perspective on how these visualization technologies might best be implemented for guiding glioma surgeries in the future.Detection of PpIX expression in low-grade gliomas and at the infiltrative margins of all gliomas has been achieved with high-sensitivity probe-based visualization techniques. Deep-tissue PpIX imaging of up to 5 mm has also been achieved using red-light illumination techniques. Spectroscopic approaches have enabled more accurate quantification of PpIX expression.Advancements in visualization technologies have extended the sensitivity and accuracy of conventional WF surgical microscopy. These technologies will continue to be refined to further improve the extent of resection in glioma patients using 5-ALA-induced fluorescence.
    • Dual-Axis Confocal Microscopy for Point-of-Care Pathology

      Wei, L., Yin, C., Liu, J. T. C.

      IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS

      ABSTRACT
      Light-sheet fluorescence microscopy (LSFM) has emerged as a powerful method for rapid and optically efficient 3D microscopy. Initial LSFM designs utilized a static sheet of light, termed selective plane illumination microscopy (SPIM), which exhibited shadowing artifacts and deteriorated contrast due to light scattering. These issues have been addressed, in part, by multidirectional selective plane illumination microscopy (mSPIM), in which rotation of the light sheet is used to mitigate shadowing artifacts, and digital scanned light-sheet microscopy (DSLM), in which confocal line detection is used to reject scattered light. Here we present a simple and passive multidirectional digital scanned light-sheet microscopy (mDSLM) architecture that combines the benefits of mSPIM and DSLM. By utilizing an elliptical Gaussian beam with increased angular diversity in the imaging plane, mDSLM provides mitigation of shadowing artifacts and contrast-enhanced imaging of fluorescently labeled samples.
    • Rapid pathology of lumpectomy margins with open open-top light-sheet (OTLS) microscopy

      Chen, Y., Xie, W., Glaser, A. K., Reder, N. P., Mao, C., Dintzis, S. M., Vaughan, J. C., Liu, J. T. C.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      Nanoparticle (NP) contrast agents targeted to cancer biomarkers are increasingly being engineered for the early detection of cancer, guidance of therapy, and monitoring of response. There have been recent efforts to topically apply biomarker-targeted NPs on tissue surfaces to image the expression of cell-surface receptors over large surface areas as a means of evaluating tumor margins to guide wide local excision surgeries. However, diffusion and nonspecific binding of the NPs present challenges for relating NP retention on the tissue surface with the expression of cancer cell receptors. Paired-agent methods that employ a secondary 'control' NP to account for these nonspecific effects can improve cancer detection. Yet these paired-agent methods introduce multidimensional complexity (with tissue staining, rinsing, imaging, and data analysis protocols all being subject to alteration), and could be greatly simplified with accurate, predictive in silico models of NP binding and diffusion. Here, we outline and validate such a model to predict the diffusion, as well as specific and nonspecific binding, of targeted and control NPs topically applied on tissue surfaces. In order to inform the model, in vitro experiments were performed to determine relevant NP diffusion and binding rate constants in tissues. The predictive capacity of the model was validated by comparing simulated distributions of various sizes of NPs in comparison with experimental results. The regression of predicted and experimentally measured concentration-depth profiles yielded  <15% error (compared to ~70% error obtained using a previous model of NP diffusion and binding).
    • Handheld line-scanned dual-axis confocal microscope with pistoned MEMS actuation for flat-field fluorescence imaging

      Wei, L., Yin, C., Fujita, Y., Sanai, N., Liu, J. T. C.

      OPTICS LETTERS

      ABSTRACT
      Raman spectroscopic imaging has shown great promise for improved cancer detection and localization with the use of tumor targeting surface enhanced Raman scattering (SERS) nanoparticles. With the ultrasensitive detection and multiplexing capabilities that SERS imaging has to offer, scientists have been investigating several clinical applications that could benefit from this unique imaging strategy. Recently, there has been a push to develop new image-guidance tools for surgical resection to help surgeons sensitively and specifically identify tumor margins in real time. We hypothesized that SERS nanoparticles (NPs) topically applied to breast cancer resection margins have the potential to provide real-time feedback on the presence of residual cancer in the resection margins during lumpectomy. Here, we explore the ability of SERS nanoparticles conjugated with a cluster of differentiation-47 (CD47) antibody to target breast cancer. CD47 is a cell surface receptor that has recently been shown to be overexpressed on several solid tumor types. The binding potential of our CD47-labeled SERS nanoparticles was assessed using fluorescence assisted cell sorting (FACS) on seven different human breast cancer cell lines, some of which were triple negative (negative expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2)). Xenograft mouse models were also used to assess the ability of our Raman imaging system to identify tumor from normal tissue. A ratiometric imaging strategy was used to quantify specific vs. nonspecific probe binding, resulting in improved tumor-to-background ratios. FACS analysis showed that CD47-labeled SERS nanoparticles bound to seven different breast cancer cell lines at levels 12-fold to 70-fold higher than isotype control-labeled nanoparticles (p < 0.01), suggesting that our CD47-targeted nanoparticles actively bind to CD47 on breast cancer cells. In a mouse xenograft model of human breast cancer, topical application of CD47-targeted nanoparticles to excised normal and cancer tissue revealed increased binding of CD47-targeted nanoparticles on tumor relative to normal adjacent tissue. The findings of this study support further investigation and suggest that SERS nanoparticles topically applied to breast cancer could guide more complete surgical resection during lumpectomy.
    • Label-free <i>in vivo</i> pathology of human epithelia with a high-speed handheld dual-axis confocal microscope

      Yin, C., Wei, L., Abeytunge, S., Peterson, G., Rajadhyaksha, M., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      Intraoperative assessment of breast surgical margins will be of value for reducing the rate of re-excision surgeries for lumpectomy patients. While frozen-section histology is used for intraoperative guidance of certain cancers, it provides limited sampling of the margin surface (typically <1  %   of the margin) and is inferior to gold-standard histology, especially for fatty tissues that do not freeze well, such as breast specimens. Microscopy with ultraviolet surface excitation (MUSE) is a nondestructive superficial optical-sectioning technique that has the potential to enable rapid, high-resolution examination of excised margin surfaces. Here, a MUSE system is developed with fully automated sample translation to image fresh tissue surfaces over large areas and at multiple levels of defocus, at a rate of ∼5  min  /  cm2. Surface extraction is used to improve the comprehensiveness of surface imaging, and 3-D deconvolution is used to improve resolution and contrast. In addition, an improved fluorescent analog of conventional H&E staining is developed to label fresh tissues within ∼5  min for MUSE imaging. We compare the image quality of our MUSE system with both frozen-section and conventional H&E histology, demonstrating the feasibility to provide microscopic visualization of breast margin surfaces at speeds that are relevant for intraoperative use.
    • Trends and Challenges for the Clinical Adoption of Fluorescence-Guided Surgery

      Liu, J. T. C., Sanai, N.

      JOURNAL OF NUCLEAR MEDICINE

      ABSTRACT
      Open-top light-sheet microscopy is a technique that can potentially enable rapid ex vivo inspection of large tissue surfaces and volumes. Here, we have optimized an open-top light-sheet (OTLS) microscope and image-processing workflow for the comprehensive examination of surgical margin surfaces, and have also developed a novel fluorescent analog of H&E staining that is robust for staining fresh unfixed tissues. Our tissue-staining method can be achieved within 2.5 minutes followed by OTLS microscopy of lumpectomy surfaces at a rate of up to 1.5 cm2/minute. An image atlas is presented to show that OTLS image quality surpasses that of intraoperative frozen sectioning and can approximate that of gold-standard H&E histology of formalin-fixed paraffin-embedded (FFPE) tissues. Qualitative evidence indicates that these intraoperative methods do not interfere with downstream post-operative H&E histology and immunohistochemistry. These results should facilitate the translation of OTLS microscopy for intraoperative guidance of lumpectomy and other surgical oncology procedures.
    • INTRAOPERATIVE HAND-HELD LINE-SCANNED DUAL-AXIS CONFOCAL MICROSCOPY FOR VISUALIZING LOW-GRADE GLIOMAS

      Fujita, Y., Wei, L., Liu, J. T. C., Sanai, N.

      ABSTRACT
      Ex vivo microscopy encompasses a range of techniques to examine fresh or fixed tissue with microscopic resolution, eliminating the need to embed the tissue in paraffin or produce a glass slide. One such technique is light-sheet microscopy, which enables rapid 3D imaging. Our pathology-engineering collaboration has resulted in an open-top light-sheet (OTLS) microscope that is specifically tailored to the needs of pathology practice.To present an image atlas of OTLS images of prostate core needle biopsies.Core needle biopsies (N = 9) were obtained from fresh radical prostatectomy specimens. Each biopsy was fixed in formalin, dehydrated in ethanol, stained with TO-PRO3 and eosin, optically cleared, and imaged using OTLS microscopy. The biopsies were then processed, paraffin embedded, and sectioned. Hematoxylin-eosin and immunohistochemical staining for cytokeratin 5 and cytokeratin 8 was performed.Benign and neoplastic histologic structures showed high fidelity between OTLS and traditional light microscopy. OTLS microscopy had no discernible effect on hematoxylin-eosin or immunohistochemical staining in this pilot study. The 3D histology information obtained using OTLS microscopy enabled new structural insights, including the observation of cribriform and well-formed gland morphologies within the same contiguous glandular structures, as well as the continuity of poorly formed glands with well-formed glands.Three-dimensional OTLS microscopy images have a similar appearance to traditional hematoxylin-eosin histology images, with the added benefit of useful 3D structural information. Further studies are needed to continue to document the OTLS appearance of a wide range of tissues and to better understand 3D histologic structures.
    • Toward Quantitative Neurosurgical Guidance With High-Resolution Microscopy of 5-Aminolevulinic Acid-Induced Protoporphyrin IX

      Wei, L., Fujita, Y., Sanai, N., Liu, J. T. C.

      FRONTIERS IN ONCOLOGY

      ABSTRACT
      There would be clinical value in a miniature optical-sectioning microscope to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology for early disease detection and surgical guidance. To address this need, a reflectance-based handheld line-scanned dual-axis confocal microscope was developed and fully packaged for label-free imaging of human skin and oral mucosa. This device can collect images at >15  frames/s with an optical-sectioning thickness and lateral resolution of 1.7 and 1.1  μm, respectively. Incorporation of a sterile lens cap design enables pressure-sensitive adjustment of the imaging depth by the user during clinical use. In vivo human images and videos are obtained to demonstrate the capabilities of this high-speed optical-sectioning microscopy device.
    • Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

      Glaser, A. K., Reder, N. P., Chen, Y., Yin, C., Wei, L., Kang, S., Barner, L. A., Xie, W., McCarty, E. F., Mao, C., Halpern, A. R., Stoltzfus, C. R., Daniels, J. S., Gerner, M. Y., Nicovich, P. R., Vaughan, J. C., True, L. D., Liu, J. T. C.

      NATURE COMMUNICATIONS

      ABSTRACT
      There would be clinical value in a miniature optical-sectioning microscope to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology for early disease detection and surgical guidance. To address this need, a reflectance-based handheld line-scanned dual-axis confocal microscope was developed and fully packaged for label-free imaging of human skin and oral mucosa. This device can collect images at >15  frames/s with an optical-sectioning thickness and lateral resolution of 1.7 and 1.1  μm, respectively. Incorporation of a sterile lens cap design enables pressure-sensitive adjustment of the imaging depth by the user during clinical use. In vivo human images and videos are obtained to demonstrate the capabilities of this high-speed optical-sectioning microscopy device.
    • Visualization technologies for 5-ALA-based fluorescence-guided surgeries

      Wei, L., Roberts, D. W., Sanai, N., Liu, J. T. C.

      JOURNAL OF NEURO-ONCOLOGY

      ABSTRACT
      Dual-axis confocal (DAC) microscopy is an optical imaging modality that utilizes simple low-numerical aperture (NA) lenses to achieve effective optical sectioning and superior image contrast in biological tissues. The unique architecture of DAC microscopy also provides some advantages for miniaturization, facilitating the development of endoscopic and handheld DAC systems for in vivo imaging. This article reviews the principles of DAC microscopy, including its differences from conventional confocal microscopy, and surveys several variations of DAC microscopy that have been developed and investigated as non-invasive real-time alternatives to conventional biopsy and histopathology.
    • Open-Top Light-Sheet Microscopy Image Atlas of Prostate Core Needle Biopsies

      Reder, N. P., Glaser, A. K., McCarty, E. F., Chen, Y., True, L. D., Liu, J. T. C.

      ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE

      ABSTRACT
      Low-power fluorescence microscopy of 5-ALA-induced PpIX has emerged as a valuable intraoperative imaging technology for improving the resection of malignant gliomas. However, current fluorescence imaging tools are not highly sensitive nor quantitative, which limits their effectiveness for optimizing operative decisions near the surgical margins of gliomas, in particular non-enhancing low-grade gliomas. Intraoperative high-resolution optical-sectioning microscopy can potentially serve as a valuable complement to low-power fluorescence microscopy by providing reproducible quantification of tumor parameters at the infiltrative margins of diffuse gliomas. In this forward-looking perspective article, we provide a brief discussion of recent technical advancements, pilot clinical studies, and our vision of the future adoption of handheld optical-sectioning microscopy at the final stages of glioma surgeries to enhance the extent of resection. We list a number of challenges for clinical acceptance, as well as potential strategies to overcome such obstacles for the surgical implementation of these in vivo microscopy techniques.
    • Modeling the binding and diffusion of receptor-targeted nanoparticles topically applied on fresh tissue specimens

      Kang, S., Xu, X., Navarro, E., Wang, Y., Liu, J. T. C., Tichauer, K. M.

      PHYSICS IN MEDICINE AND BIOLOGY

      ABSTRACT
      A handheld line-scanned dual-axis confocal (LS-DAC) microscope has been developed for high-speed (16 frames/s) fluorescence imaging of tissues with sub-nuclear resolution. This is the first miniature fluorescence LS-DAC system that has been fully packaged for handheld clinical use on patients. A novel micro-electro-mechanical system scanning mechanism, with synchronized tilting and pistoning, is used to achieve flat-field en face imaging. We show that this facilitates video mosaicking to generate images that sample an extended lateral field of view.
    • Solid immersion meniscus lens (SIMlens) for open-top light-sheet microscopy

      Barner, L. A., Glaser, A. K., True, L. D., Reder, N. P., Liu, J. T. C.

      OPTICS LETTERS

      ABSTRACT
      Recent advances in optical clearing and light-sheet microscopy have provided unprecedented access to structural and molecular information from intact tissues. However, current light-sheet microscopes have imposed constraints on the size, shape, number of specimens, and compatibility with various clearing protocols. Here we present a multi-immersion open-top light-sheet microscope that enables simple mounting of multiple specimens processed with a variety of clearing protocols, which will facilitate wide adoption by preclinical researchers and clinical laboratories. In particular, the open-top geometry provides unsurpassed versatility to interface with a wide range of accessory technologies in the future.

    2018

    • A handheld MEMS-scanned in vivo optical-sectioning microscope for early detection and surgical guidance

      Yin, C., Wei, L., Abeytunge, S., Peterson, G., Glaser, A. K., Mandella, M. J., Rajadhyaksha, M., Liu, J. T. C.

      ABSTRACT
      Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.
    • Thick tissue diffusion model with binding to optimize topical staining in fluorescence breast cancer margin imaging

      Xu, X., Kang, S., Navarro-Comes, E., Wang, Y., Liu, J. T. C., Tichauer, K. M.

      ABSTRACT
      Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.
    • Identification of Tissue Invasive Fungi by 2D and 3D Light Sheet Microscopy

      Lieberman, J., Glaser, A. K., Reder, N., True, L. D., Bryan, A., Liu, J. T., Bourassa, L.

      ABSTRACT
      Intraoperative identification of carcinoma at lumpectomy margins would enable reduced re-excision rates, which are currently as high as 20% to 50%. Although imaging of disease-associated biomarkers can identify malignancies with high specificity, multiplexed imaging of such biomarkers is necessary to detect molecularly heterogeneous carcinomas with high sensitivity. We have developed a Raman-encoded molecular imaging (REMI) technique in which targeted nanoparticles are topically applied on excised tissues to enable rapid visualization of a multiplexed panel of cell surface biomarkers at surgical margin surfaces. A first-ever clinical study was performed in which 57 fresh specimens were imaged with REMI to simultaneously quantify the expression of four biomarkers HER2, ER, EGFR, and CD44. Combined detection of these biomarkers enabled REMI to achieve 89.3% sensitivity and 92.1% specificity for the detection of breast carcinoma. These results highlight the sensitivity and specificity of REMI to detect biomarkers in freshly resected tissue, which has the potential to reduce the rate of re-excision procedures in cancer patients. Cancer Res; 77(16); 4506-16. ©2017 AACR.
    • High-speed Raman-encoded molecular imaging of freshly excised tissue surfaces with topically applied SERRS nanoparticles

      Wang, Y., Yang, Q., Kang, S., Wall, M. A., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

    • MEASURING AND MITIGATING SPECKLE NOISE IN DUAL-AXIS CONFOCAL MICROSCOPY IMAGES

      Gigilashvili, D., Yin, C., Liu, J. T. C., Hardeberg, J., Pedersen, M.

      ABSTRACT
      For the 1.7 million patients per year in the U.S. who receive a new cancer diagnosis, treatment decisions are largely made after a histopathology exam. Unfortunately, the gold standard of slide-based microscopic pathology suffers from high inter-observer variability and limited prognostic value due to sampling limitations and the inability to visualize tissue structures and molecular targets in their native 3D context. Here, we show that an open-top light-sheet microscope optimized for non-destructive slide-free pathology of clinical specimens enables the rapid imaging of intact tissues at high resolution over large 2D and 3D fields of view, with the same level of detail as traditional pathology. We demonstrate the utility of this technology for various applications: wide-area surface microscopy to triage surgical specimens (with ~200 μm surface irregularities), rapid intraoperative assessment of tumour-margin surfaces (12.5 sec/cm2), and volumetric assessment of optically cleared core-needle biopsies (1 mm in diameter, 2 cm in length). Light-sheet microscopy can be a versatile tool for both rapid surface microscopy and deep volumetric microscopy of human specimens.
    • Microscopic investigation of" topically applied nanoparticles for molecular imaging of fresh tissue surfaces

      Kang, S., Wang, Y., Xu, X., Navarro, E., Tichauer, K. M., Liu, J. T. C.

      JOURNAL OF BIOPHOTONICS

    • Reaction-Driven Nucleation Theory

      Wall, M. A., Cossairt, B. M., Liu, J. T. C.

      JOURNAL OF PHYSICAL CHEMISTRY C

      ABSTRACT
      Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) are increasingly being engineered for a variety of disease-detection and treatment applications. For example, we have previously developed a fiber-optic Raman-encoded molecular imaging (REMI) system for spectral imaging of biomarker-targeted SERS NPs topically applied on tissue surfaces to identify residual tumors at surgical margins. Although accurate tumor detection was achieved, the commercial SERS NPs used in our previous studies lacked the signal strength to enable high-speed imaging with high pixel counts (large fields of view and/or high spatial resolution), which limits their use for certain time-constrained clinical applications. As a solution, we explored the use of surface-enhanced resonant Raman scattering (SERRS) NPs to enhance imaging speeds. The SERRS NPs were synthesized de novo, and then conjugated to HER2 antibodies to achieve high binding affinity, as validated by flow cytometry. Under identical tissue-staining and imaging conditions, the targeted SERRS NPs enabled reliable identification of HER2-overexpressed tumor xenografts with 50-fold-enhanced imaging speed compared with our standard targeted SERS NPs. This enables our REMI system to image tissue surfaces at a rate of 150  cm2 per minute at a spatial resolution of 0.5 mm.
    • Multidirectional digital scanned light-sheet microscopy enables uniform fluorescence excitation and contrast-enhanced imaging

      Glaser, A. K., Chen, Y., Yin, C., Wei, L., Barner, L. A., Reder, N. P., Liu, J. T. C.

      SCIENTIFIC REPORTS

      ABSTRACT
      Previous studies have shown that functionalized nanoparticles (NPs) topically applied on fresh tissues are able to rapidly target cell-surface protein biomarkers of cancer. Furthermore, studies have shown that a paired-agent approach, in which an untargeted NP is co-administered with a panel of targeted NPs, controls for the nonspecific behavior of the NPs, enabling quantitative imaging of biomarker expression. However, given the complexities in nonspecific accumulation, diffusion, and chemical binding of targeted NPs in tissues, studies are needed to better understand these processes at the microscopic scale. Here, fresh tissues were stained with a paired-agent approach, frozen, and sectioned to image the depth-dependent accumulation of targeted and untargeted NPs. The ratio of targeted-to-untargeted NP concentrations-a parameter used to distinguish between tumor and benign tissues-was found to diminish with increasing NP diffusion depths due to nonspecific accumulation and poor washout. It was then hypothesized and experimentally demonstrated that larger NPs would exhibit less diffusion below tissue surfaces, enabling higher targeted-to-untargeted NP ratios. In summary, these methods and investigations have enabled the design of NP agents with improved sensitivity and contrast for rapid molecular imaging of fresh tissues.
    • A Raman Imaging Approach Using CD47 Antibody-Labeled SERS Nanoparticles for Identifying Breast Cancer and Its Potential to Guide Surgical Resection.

      Davis, R. M., Campbell, J. L., Burkitt, S., Qiu, Z., Kang, S., Mehraein, M., Miyasato, D., Salinas, H., Liu, J. T., Zavaleta, C.

      Nanomaterials (Basel, Switzerland)

      ABSTRACT
      Previous studies have shown that functionalized nanoparticles (NPs) topically applied on fresh tissues are able to rapidly target cell-surface protein biomarkers of cancer. Furthermore, studies have shown that a paired-agent approach, in which an untargeted NP is co-administered with a panel of targeted NPs, controls for the nonspecific behavior of the NPs, enabling quantitative imaging of biomarker expression. However, given the complexities in nonspecific accumulation, diffusion, and chemical binding of targeted NPs in tissues, studies are needed to better understand these processes at the microscopic scale. Here, fresh tissues were stained with a paired-agent approach, frozen, and sectioned to image the depth-dependent accumulation of targeted and untargeted NPs. The ratio of targeted-to-untargeted NP concentrations-a parameter used to distinguish between tumor and benign tissues-was found to diminish with increasing NP diffusion depths due to nonspecific accumulation and poor washout. It was then hypothesized and experimentally demonstrated that larger NPs would exhibit less diffusion below tissue surfaces, enabling higher targeted-to-untargeted NP ratios. In summary, these methods and investigations have enabled the design of NP agents with improved sensitivity and contrast for rapid molecular imaging of fresh tissues.

    2017

    • Rinsing paired-agent model (RPAM) to quantify cell-surface receptor concentrations in topical staining applications of thick tissues

      Xu, X., Wang, Y., Xiang, J., Liu, J. T. C., Tichauer, K. M.

      PHYSICS IN MEDICINE AND BIOLOGY

    • Light-sheet microscopy for slide-free non-destructive pathology of large clinical specimens

      Glaser, A. K., Reder, N. P., Chen, Y., McCarty, E. F., Yin, C., Wei, L., Wang, Y., True, L. D., Liu, J. T. C.

      NATURE BIOMEDICAL ENGINEERING

      ABSTRACT
      Systemic delivery of 5-aminolevulinic acid leads to enhanced fluorescence image contrast in many tumors due to the increased accumulation of protoporphyrin IX (PpIX), a fluorescent porphyrin that is associated with tumor burden and proliferation. The value of PpIX-guided resection of malignant gliomas has been demonstrated in prospective randomized clinical studies in which a twofold greater extent of resection and improved progression-free survival have been observed. In low-grade gliomas and at the diffuse infiltrative margins of all gliomas, PpIX fluorescence is often too weak to be detected with current low-resolution surgical microscopes that are used in operating rooms. However, it has been demonstrated that high-resolution optical-sectioning microscopes are capable of detecting the sparse and punctate accumulations of PpIX that are undetectable via conventional low-power surgical fluorescence microscopes. To standardize the performance of high-resolution optical-sectioning devices for future clinical use, we have developed an imaging phantom and methods to ensure that the imaging of PpIX-expressing brain tissues can be performed reproducibly. Ex vivo imaging studies with a dual-axis confocal microscope demonstrate that these methods enable the acquisition of images from unsectioned human brain tissues that quantitatively and consistently correlate with images of histologically processed tissue sections.
    • Numerical modeling of illumination and detection methods for light-sheet microscopy of optically clear biological tissues

      Glaser, A. K., Liu, J. T. C., IEEE Photon Soc

      ABSTRACT
      One of the main challenges for laser-scanning microscopy of biological tissues with refractive heterogeneities is the degradation in spatial resolution that occurs as a result of beam steering and distortion. This challenge is particularly significant for dual-axis confocal (DAC) microscopy, which achieves improved spatial-filtering and optical-sectioning performance over traditional confocal microscopy through off-axis illumination and collection of light with low-numerical aperture (NA) beams that must intersect precisely at their foci within tissues. DAC microscope image quality is sensitive to positional changes and distortions of these illumination- and collection-beam foci. Previous studies have shown that Bessel beams display improved positional stability and beam quality than Gaussian beams when propagating through tissues with refractive heterogeneities, which suggests that Bessel-beam illumination may enhance DAC microscopy of such tissues. Here, we utilize both Gaussian and Bessel illumination in a point-scanned DAC microscope and quantify the resultant degradation in resolution when imaging within heterogeneous optical phantoms and fresh tissues. Results indicate that DAC microscopy with Bessel illumination exhibits reduced resolution degradation from microscopic tissue heterogeneities compared to DAC microscopy with conventional Gaussian illumination.
    • Raman-Encoded Molecular Imaging with Topically Applied SERS Nanoparticles for Intraoperative Guidance of Lumpectomy

      Wang, Y., Reder, N. P., Kang, S., Glaser, A. K., Yang, Q., Wall, M. A., Javid, S. H., Dintzis, S. M., Liu, J. T. C.

      CANCER RESEARCH

      ABSTRACT
      Conventional molecular assessment of tissue through histology, if adapted to fresh thicker samples, has the potential to enhance cancer detection in surgical margins and monitoring of 3D cell culture molecular environments. However, in thicker samples, substantial background staining is common despite repeated rinsing, which can significantly reduce image contrast. Recently, 'paired-agent' methods-which employ co-administration of a control (untargeted) imaging agent-have been applied to thick-sample staining applications to account for background staining. To date, these methods have included (1) a simple ratiometric method that is relatively insensitive to noise in the data but has accuracy that is dependent on the staining protocol and the characteristics of the sample; and (2) a complex paired-agent kinetic modeling method that is more accurate but is more noise-sensitive and requires a precise serial rinsing protocol. Here, a new simplified mathematical model-the rinsing paired-agent model (RPAM)-is derived and tested that offers a good balance between the previous models, is adaptable to arbitrary rinsing-imaging protocols, and does not require calibration of the imaging system. RPAM is evaluated against previous models and is validated by comparison to estimated concentrations of targeted biomarkers on the surface of 3D cell culture and tumor xenograft models. This work supports the use of RPAM as a preferable model to quantitatively analyze targeted biomarker concentrations in topically stained thick tissues, as it was found to match the accuracy of the complex paired-agent kinetic model while retaining the low noise-sensitivity characteristics of the ratiometric method.
    • A handheld MEMS-based line-scanned dual-axis confocal microscope for early cancer detection and surgical guidance (Conference Presentation)

      Chen, Y., Yin, C., Wei, L., Glaser, A. K., Abeytunge, S., Peterson, G., Mandella, M. J., Sanai, N., Rajadhyaksha, M., Liu, J. T.

      ABSTRACT
      In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.
    • Cost-Effective Triaging of Prostatectomy Specimens Using Light-Sheet Microscopy

      Reder, N. P., Glaser, A. K., McCarty, E., Chen, Y., Liu, J. T. C., True, L.

      ABSTRACT
      In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.
    • Bessel-beam illumination in dual-axis confocal microscopy mitigates resolution degradation caused by refractive heterogeneities

      Chen, Y., Glaser, A., Liu, J. T. C.

      JOURNAL OF BIOPHOTONICS

      ABSTRACT
      Modulated-alignment dual-axis (MAD) confocal microscopy combines the benefits of dual-axis confocal (DAC) microscopy and focal-modulation microscopy (FMM) for rejecting out-of-focus and multiply scattered light in tissues. The DAC architecture, which utilizes off-axis and separated beam paths for illumination and detection, has previously been shown to be superior to single-axis confocal (SAC) microscopy for the spatial filtering (rejection) of unwanted background light. With the MAD approach, a modulation of the alignment between the illumination and collection beam paths tags ballistic photons emanating from the focal volume with a characteristic radio frequency that can be extracted and separated from background signal using lock-in detection. We report here an optimized form of MAD confocal microscopy where we have fully mitigated tradeoffs in performance in an initial proof-of-concept system in order to recover the imaging speed of DAC microscopy while retaining contrast enhancement of 6 dB (signal-to-background ratio) with a secondary improvement in optical-sectioning and in-plane resolution. Validation is demonstrated with light-scattering tissue phantoms and freshly excised tissues.
    • 3D Light-Sheet Microscopy Improves the Accuracy of Grading Prostate Cancer by Distinguishing Pattern 3 Glands from Poorly Formed Pattern 4 Glands

      Reder, N. P., Glaser, A. K., McCarty, E., Chen, Y., Liu, J. T. C., True, L.

      ABSTRACT
      In recent decades, various classes of nanoparticles have been developed for optical imaging of cancers. Many of these nanoparticles are designed to specifically target tumor sites, and specific cancer biomarkers, to facilitate the visualization of tumors. However, one challenge for accurate detection of tumors is that the molecular profiles of most cancers vary greatly between patients as well as spatially and temporally within a single tumor mass. To overcome this challenge, certain nanoparticles and imaging systems have been developed to enable multiplexed imaging of large panels of cancer biomarkers. Multiplexed molecular imaging can potentially enable sensitive tumor detection, precise delineation of tumors during interventional procedures, and the prediction/monitoring of therapy response. In this review, we summarize recent advances in systems that have been developed for the imaging of optical nanoparticles that can be heavily multiplexed, which include surface-enhanced Raman-scattering nanoparticles (SERS NPs) and quantum dots (QDs). In addition to surveying the optical properties of these various types of nanoparticles, and the most-popular multiplexed imaging approaches that have been employed, representative preclinical and clinical imaging studies are also highlighted.
    • Multiplexed Optical Imaging of Tumor-Directed Nanoparticles: A Review of Imaging Systems and Approaches.

      Wang, Y. W., Reder, N. P., Kang, S., Glaser, A. K., Liu, J. T.

      Nanotheranostics

      ABSTRACT
      There is a need for intraoperative imaging technologies to guide breast-conserving surgeries and to reduce the high rates of re-excision for patients in which residual tumor is found at the surgical margins during postoperative pathology analyses. Feasibility studies have shown that utilizing topically applied surface-enhanced Raman scattering (SERS) nanoparticles (NPs), in conjunction with the ratiometric imaging of targeted versus untargeted NPs, enables the rapid visualization of multiple cell-surface biomarkers of cancer that are overexpressed at the surfaces of freshly excised breast tissues. In order to reliably and rapidly perform multiplexed Raman-encoded molecular imaging of large numbers of biomarkers (with five or more NP flavors), an enhanced staining method has been developed in which tissue surfaces are cyclically dipped into an NP-staining solution and subjected to high-frequency mechanical vibration. This dipping and mechanical vibration (DMV) method promotes the convection of the SERS NPs at fresh tissue surfaces, which accelerates their binding to their respective biomarker targets. By utilizing a custom-developed device for automated DMV staining, this study demonstrates the ability to simultaneously image four cell-surface biomarkers of cancer at the surfaces of fresh human breast tissues with a mixture of five flavors of SERS NPs (four targeted and one untargeted control) topically applied for 5 min and imaged at a spatial resolution of 0.5 mm and a raster-scanned imaging rate of >5 cm2 min-1 .
    • Clarification of Prostate Core Needle Biopsies with 2,2′ Thiodiethanol Rapidly and Efficiently Prepares Tissue for 3D Light-Sheet Microscopy

      McCarty, E., Reder, N. P., Glaser, A. K., Chen, Y., Liu, J. T. C., True, L.

    • Optical-sectioning microscopy of protoporphyrin IX fluorescence in human gliomas: standardization and quantitative comparison with histology

      Wei, L., Chen, Y., Yin, C., Borwege, S., Sanai, N., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

    2016

    • Fractal propagation method enables realistic optical microscopy simulations in biological tissues

      Glaser, A. K., Chen, Y., Liu, J. T. C.

      OPTICA

      ABSTRACT
      Earlier and more accurate detection of oral squamous cell carcinoma (OSCC) is essential to improve the prognosis of patients and to reduce the morbidity of surgical therapy. Here, we demonstrate that the nuclear enzyme Poly(ADP-ribose)Polymerase 1 (PARP1) is a promising target for optical imaging of OSCC with the fluorescent dye PARPi-FL. In patient-derived OSCC specimens, PARP1 expression was increased 7.8 ± 2.6-fold when compared to normal tissue. Intravenous injection of PARPi-FL allowed for high contrast in vivo imaging of human OSCC models in mice with a surgical fluorescence stereoscope and high-resolution imaging systems. The emitted signal was specific for PARP1 expression and, most importantly, PARPi-FL can be used as a topical imaging agent, spatially resolving the orthotopic tongue tumors in vivo. Collectively, our results suggest that PARP1 imaging with PARPi-FL can enhance the detection of oral cancer, serve as a screening tool and help to guide surgical resections.
    • Multiplexed Molecular Imaging of Biomarker-Targeted SERS Nanoparticles on Fresh Tissue Specimens with Channel-Compressed Spectrometry

      Kang, S., Wang, Y., Reder, N. P., Liu, J. T. C.

      PLOS ONE

      ABSTRACT
      The imaging of dysregulated cell-surface receptors (or biomarkers) is a potential means of identifying the presence of cancer with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Recently, surface-enhanced Raman-scattering (SERS) nanoparticles (NPs) have attracted wide interest due to their potential for sensitive and multiplexed biomarker detection. In this review, we focus on the most recent advances in tumor imaging using SERS-coded NPs. A brief introduction of the structure and optical properties of SERS NPs is provided, followed by a detailed discussion of key imaging issues such as the administration of NPs in tissue (topical versus systemic), the optical configuration and imaging approach of Raman imaging systems, spectral demultiplexing methods for quantifying NP concentrations, and the disambiguation of specific vs. nonspecific sources of contrast through ratiometric imaging of targeted and untargeted (control) NP pairs. Finally, future challenges and directions are briefly outlined.
    • Modulated-Alignment Dual-Axis (MAD) Confocal Microscopy Optimized for Speed and Contrast

      Leigh, S. Y., Chen, Y., Liu, J. T. C.

      IEEE TRANSACTIONS ON BIOMEDICAL ENGINEERING

      ABSTRACT
      Current simulation methods for light transport in biological media have limited efficiency and realism when applied to three-dimensional microscopic light transport in biological tissues with refractive heterogeneities. We describe here a technique which combines a beam propagation method valid for modeling light transport in media with weak variations in refractive index, with a fractal model of refractive index turbulence. In contrast to standard simulation methods, this fractal propagation method (FPM) is able to accurately and efficiently simulate the diffraction effects of focused beams, as well as the microscopic heterogeneities present in tissue that result in scattering, refractive beam steering, and the aberration of beam foci. We validate the technique and the relationship between the FPM model parameters and conventional optical parameters used to describe tissues, and also demonstrate the method's flexibility and robustness by examining the steering and distortion of Gaussian and Bessel beams in tissue with comparison to experimental data. We show that the FPM has utility for the accurate investigation and optimization of optical microscopy methods such as light-sheet, confocal, and nonlinear microscopy.
    • Multiplexed Molecular Imaging of Fresh Tissue Surfaces Enabled by Convection-Enhanced Topical Staining with SERS-Coded Nanoparticles

      Wang, Y. W., Doerksen, J. D., Kang, S., Walsh, D., Yang, Q., Hong, D., Liu, J. T. C.

      SMALL

      ABSTRACT
      Biomarker-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have been explored as a viable option for targeting and imaging multiple cell-surface protein biomarkers of cancer. While it has been demonstrated that this Raman-encoded molecular imaging (REMI) technology may potentially be used to guide tumor-resection procedures, the REMI strategy would benefit from further improvements in imaging speed. Previous implementations of REMI have utilized 1024 spectral channels (camera pixels) in a commercial spectroscopic CCD to detect the spectral signals from multiplexed SERS NPs, a strategy that enables accurate demultiplexing of the relative concentration of each NP "flavor" within a mixture. Here, we investigate the ability to significantly reduce the number of spectral-collection channels while maintaining accurate imaging and demultiplexing of up to five SERS NP flavors, a strategy that offers the potential for improved imaging speed and/or detection sensitivity in future systems. This strategy was optimized by analyzing the linearity of five multiplexed flavors of SERS NPs topically applied on tissues. The accuracy of this binning approach was then validated by staining tumor xenografts and human breast tumor specimens with a mixture of five NP flavors (four targeted NPs and one untargeted NP) and performing ratiometric imaging of specific vs. nonspecific NP accumulation. We demonstrate that with channel-compressed spectrometry using as few as 16 channels, it is possible to perform REMI with five NP flavors, with < 20% error, at low concentrations (< 10 pM) that are relevant for clinical applications.
    • Miniature in vivo MEMS-based line-scanned dual-axis confocal microscope for point-of-care pathology.

      Yin, C., Glaser, A. K., Leigh, S. Y., Chen, Y., Wei, L., Pillai, P. C., Rosenberg, M. C., Abeytunge, S., Peterson, G., Glazowski, C., Sanai, N., Mandella, M. J., Rajadhyaksha, M., Liu, J. T.

      Biomedical optics express

      ABSTRACT
      Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.
    • Quantitative molecular phenotyping with topically applied SERS nanoparticles for intraoperative guidance of breast cancer lumpectomy

      Wang, Y., Kang, S., Khan, A., Ruttner, G., Leigh, S. Y., Murray, M., Abeytunge, S., Peterson, G., Rajadhyaksha, M., Dintzis, S., Javid, S., Liu, J. T. C.

      SCIENTIFIC REPORTS

      ABSTRACT
      Video-rate optical-sectioning microscopy of living organisms would allow for the investigation of dynamic biological processes and would also reduce motion artifacts, especially for in vivo imaging applications. Previous feasibility studies, with a slow stage-scanned line-scanned dual-axis confocal (LS-DAC) microscope, have demonstrated that LS-DAC microscopy is capable of imaging tissues with subcellular resolution and high contrast at moderate depths of up to several hundred microns. However, the sensitivity and performance of a video-rate LS-DAC imaging system, with low-numerical aperture optics, have yet to be demonstrated. Here, we report on the construction and validation of a video-rate LS-DAC system that possesses sufficient sensitivity to visualize fluorescent contrast agents that are topically applied or systemically delivered in animal and human tissues. We present images of murine oral mucosa that are topically stained with methylene blue, and images of protoporphyrin IX-expressing brain tumor from glioma patients that have been administered 5-aminolevulinic acid prior to surgery. In addition, we demonstrate in vivo fluorescence imaging of red blood cells trafficking within the capillaries of a mouse ear, at frame rates of up to 30 fps. These results can serve as a benchmark for miniature in vivo microscopy devices under development.
    • Detection and delineation of oral cancer with a PARP1 targeted optical imaging agent

      Kossatz, S., Brand, C., Gutiontov, S., Liu, J. T. C., Lee, N. Y., Goenen, M., Weber, W. A., Reiner, T.

      SCIENTIFIC REPORTS

      ABSTRACT
      There is a need for miniature optical-sectioning microscopes to enable in vivo interrogation of tissues as a real-time and noninvasive alternative to gold-standard histopathology. Such devices could have a transformative impact for the early detection of cancer as well as for guiding tumor-resection procedures. Miniature confocal microscopes have been developed by various researchers and corporations to enable optical sectioning of highly scattering tissues, all of which have necessitated various trade-offs in size, speed, depth selectivity, field of view, resolution, image contrast, and sensitivity. In this study, a miniature line-scanned (LS) dual-axis confocal (DAC) microscope, with a 12-mm diameter distal tip, has been developed for clinical point-of-care pathology. The dual-axis architecture has demonstrated an advantage over the conventional single-axis confocal configuration for reducing background noise from out-of-focus and multiply scattered light. The use of line scanning enables fast frame rates (16 frames/sec is demonstrated here, but faster rates are possible), which mitigates motion artifacts of a hand-held device during clinical use. We have developed a method to actively align the illumination and collection beams in a DAC microscope through the use of a pair of rotatable alignment mirrors. Incorporation of a custom objective lens, with a small form factor for in vivo clinical use, enables our device to achieve an optical-sectioning thickness and lateral resolution of 2.0 and 1.1 microns respectively. Validation measurements with reflective targets, as well as in vivo and ex vivo images of tissues, demonstrate the clinical potential of this high-speed optical-sectioning microscopy device.
    • Surgical Guidance via Multiplexed Molecular Imaging of Fresh Tissues Labeled with SERS-Coded Nanoparticles.

      Wang, Y., Kang, S., Doerksen, J. D., Glaser, A. K., Liu, J. T.

      IEEE journal of selected topics in quantum electronics : a publication of the IEEE Lasers and Electro-optics Society

      ABSTRACT
      There is a need to image excised tissues during tumor-resection procedures in order to identify residual tumors at the margins and to guide their complete removal. The imaging of dysregulated cell-surface receptors is a potential means of identifying the presence of diseases with high sensitivity and specificity. However, due to heterogeneities in the expression of protein biomarkers in tumors, molecular-imaging technologies should ideally be capable of visualizing a multiplexed panel of cancer biomarkers. Here, we demonstrate that the topical application and quantification of a multiplexed cocktail of receptor-targeted surface-enhanced Raman scattering (SERS) nanoparticles (NPs) enables rapid quantitative molecular phenotyping (QMP) of the surface of freshly excised tissues to determine the presence of disease. In order to mitigate the ambiguity due to nonspecific sources of contrast such as off-target binding or uneven delivery, a ratiometric method is employed to quantify the specific vs. nonspecific binding of the multiplexed NPs. Validation experiments with human tumor cell lines, fresh human tumor xenografts in mice, and fresh human breast specimens demonstrate that QMP imaging of excised tissues agrees with flow cytometry and immunohistochemistry, and that this technique may be achieved in less than 15 minutes for potential intraoperative use in guiding breast-conserving surgeries.
    • Bessel beam illumination reduces resolution degradation due to micro-architectural heterogeneities for dual-axis confocal microscopy of tissues

      Chen, Y., Liu, J. T. C., IEEE

      ABSTRACT
      The biological investigation and detection of esophageal cancers could be facilitated with an endoscopic technology to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and investigation through the sensitive and multiplexed detection of cell-surface biomarkers. Here, we demonstrate that the topical application and endoscopic imaging of a multiplexed cocktail of receptor-targeted SERS NPs enables the rapid detection of tumors in an orthotopic rat model of esophageal cancer. Antibody-conjugated SERS NPs were topically applied on the lumenal surface of the rat esophagus to target EGFR and HER2, and a miniature spectral endoscope featuring rotational scanning and axial pull-back was employed to comprehensively image the NPs bound on the lumen of the esophagus. Ratiometric analyses of specific vs. nonspecific binding enabled the visualization of tumor locations and the quantification of biomarker expression in agreement with immunohistochemistry and flow cytometry validation data.

    2015

    • Comparing High-Resolution Microscopy Techniques for Potential Intraoperative Use in Guiding Low-Grade Glioma Resections

      Meza, D., Wang, D., Wang, Y., Borwege, S., Sanai, N., Liu, J. T. C.

      LASERS IN SURGERY AND MEDICINE

      ABSTRACT
      The early detection and biological investigation of esophageal cancer would benefit from the development of advanced imaging techniques to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and the investigation of cancer progression through the sensitive and multiplexed phenotyping of cell-surface biomarkers. Here, a miniature endoscope featuring rotational scanning and axial pull back has been developed for 2D spectral imaging of SERS NPs topically applied on the lumenal surface of the rat esophagus. Raman signals from low-pM concentrations of SERS NP mixtures are demultiplexed in real time to accurately calculate the concentration and ratio of the NPs. Ex vivo and in vivo experiments demonstrate the feasibility of topical application and imaging of multiplexed SERS NPs along the entire length of the rat esophagus.
    • Characterizing the beam steering and distortion of Gaussian and Bessel beams focused in tissues with microscopic heterogeneities

      Chen, Y., Liu, J. T. C.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      The complete removal of cancerous tissue is a central aim of surgical oncology, but is difficult to achieve in certain cases, especially when the removal of surrounding normal tissues must be minimized. Therefore, when post-operative pathology identifies residual tumor at the surgical margins, re-excision surgeries are often necessary. An intraoperative approach for tumor-margin assessment, insensitive to nonspecific sources of molecular probe accumulation and contrast, is presented employing kinetic-modeling analysis of dual-probe staining using surface-enhanced Raman scattering nanoparticles (SERS NPs). Human glioma (U251) and epidermoid (A431) tumors were implanted subcutaneously in six athymic mice. Fresh resected tissues were stained with an equimolar mixture of epidermal growth factor receptor (EGFR)-targeted and untargeted SERS NPs. The binding potential (BP; proportional to receptor concentration) of EGFR - a cell-surface receptor associated with cancer - was estimated from kinetic modeling of targeted and untargeted NP concentrations in response to serial rinsing. EGFR BPs in healthy, U251, and A431 tissues were 0.06 ± 0.14, 1.13 ± 0.40, and 2.23 ± 0.86, respectively, which agree with flow-cytometry measurements and published reports. The ability of this approach to quantify the BP of cell-surface biomarkers in fresh tissues opens up an accurate new approach to analyze tumor margins intraoperatively.
    • A Real-Time Clinical Endoscopic System for Intraluminal, Multiplexed Imaging of Surface-Enhanced Raman Scattering Nanoparticles

      Garai, E., Sensarn, S., Zavaleta, C. L., Loewke, N. O., Rogalla, S., Mandella, M. J., Felt, S. A., Friedland, S., Liu, J. T., Gambhir, S. S., Contag, C. H.

      PLOS ONE

      ABSTRACT
      Fluorescence image-guided surgery (FIGS), with contrast provided by 5-ALA-induced PpIX, has been shown to enable a higher extent of resection of high-grade gliomas. However, conventional FIGS with low-power microscopy lacks the sensitivity to aid in low-grade glioma (LGG) resection because PpIX signal is weak and sparse in such tissues. Intraoperative high-resolution microscopy of PpIX fluorescence has been proposed as a method to guide LGG resection, where sub-cellular resolution allows for the visualization of sparse and punctate mitochondrial PpIX production in tumor cells. Here, we assess the performance of three potentially portable high-resolution microscopy techniques that may be used for the intraoperative imaging of human LGG tissue samples with PpIX contrast: high-resolution fiber-optic microscopy (HRFM), high-resolution wide-field microscopy (WFM), and dual-axis confocal (DAC) microscopy.Thick unsectioned human LGG tissue samples (n = 7) with 5-ALA-induced PpIX contrast were imaged using three imaging techniques (HRFM, WFM, DAC). The average signal-to-background ratio (SBR) was then calculated for each imaging modality (5 images per tissue, per modality).HRFM provides the ease of use and portability of a flexible fiber bundle, and is simple and inexpensive to build. However, in most cases (6/7), HRFM is not capable of detecting PpIX signal from LGGs due to high autofluorescence, generated by the fiber bundle under laser illumination at 405 nm, which overwhelms the PpIX signal and impedes its visualization. WFM is a camera-based method possessing high lateral resolution but poor axial resolution, resulting in sub-optimal image contrast.Consistent successful detection of PpIX signal throughout our human LGG tissue samples (n = 7), with an acceptable image contrast (SBR >2), was only achieved using DAC microscopy, which offers superior image resolution and contrast that is comparable to histology, but requires a laser-scanning mechanism to achieve optical sectioning.
    • Feasibility of intraoperative fluorescence imaging of squamous cell carcinoma of the oral cavity using an optical PARP1 inhibitor

      Kossatz, S., Irwin, C., Leigh, S., Wang, D., Liu, J., Weber, W., Reiner, T.

      ABSTRACT
      Bessel beams have recently been investigated as a means of improving deep-tissue microscopy in highly scattering and heterogeneous media. It has been suggested that the long depth-of-field and self-reconstructing property of a Bessel beam enables an increased penetration depth of the focused beam in tissues compared to a conventional Gaussian beam. However, a study is needed to better quantify the magnitude of the beam steering as well as the distortion of focused Gaussian and Bessel beams in tissues with microscopic heterogeneities. Here, we have developed an imaging method and quantitative metrics to evaluate the motion and distortion of low-numerical-aperture (NA) Gaussian and Bessel beams focused in water, heterogeneous phantoms, and fresh mouse esophagus tissues. Our results indicate that low-NA Bessel beams exhibit reduced beam-steering artifacts and distortions compared to Gaussian beams, and are therefore potentially useful for microscopy applications in which pointing accuracy and beam quality are critical, such as dual-axis confocal (DAC) microscopy.
    • Quantitative <i>in vivo</i> cell-surface receptor imaging in oncology: kinetic modeling and paired-agent principles from nuclear medicine and optical imaging

      Tichauer, K. M., Wang, Y., Pogue, B. W., Liu, J. T. C.

      PHYSICS IN MEDICINE AND BIOLOGY

      ABSTRACT
      The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.
    • <i>In vivo</i> multiplexed molecular imaging of esophageal cancer via spectral endoscopy of topically applied SERS nanoparticles

      Wang, Y., Kang, S., Khan, A., Bao, P. Q., Liu, J. T. C.

      BIOMEDICAL OPTICS EXPRESS

      ABSTRACT
      The detection of biomarker-targeting surface-enhanced Raman scattering (SERS) nanoparticles (NPs) in the human gastrointestinal tract has the potential to improve early cancer detection; however, a clinically relevant device with rapid Raman-imaging capability has not been described. Here we report the design and in vivo demonstration of a miniature, non-contact, opto-electro-mechanical Raman device as an accessory to clinical endoscopes that can provide multiplexed molecular data via a panel of SERS NPs. This device enables rapid circumferential scanning of topologically complex luminal surfaces of hollow organs (e.g., colon and esophagus) and produces quantitative images of the relative concentrations of SERS NPs that are present. Human and swine studies have demonstrated the speed and simplicity of this technique. This approach also offers unparalleled multiplexing capabilities by simultaneously detecting the unique spectral fingerprints of multiple SERS NPs. Therefore, this new screening strategy has the potential to improve diagnosis and to guide therapy by enabling sensitive quantitative molecular detection of small and otherwise hard-to-detect lesions in the context of white-light endoscopy.
    • Ex-vivo tissue classification of cell surface receptor concentrations using kinetic modeling

      Sinha, L., Wang, Y., Yang, C., Khan, A., Liu, J. T., Tichauer, K. M.

      ABSTRACT
      Mounting evidence suggests that a more extensive surgical resection is associated with an improved life expectancy for both low-grade and high-grade glioma patients. However, radiographically complete resections are not often achieved in many cases because of the lack of sensitivity and specificity of current neurosurgical guidance techniques at the margins of diffuse infiltrative gliomas. Intraoperative fluorescence imaging offers the potential to improve the extent of resection and to investigate the possible benefits of resecting beyond the radiographic margins. Here, we provide a review of wide-field and high-resolution fluorescence-imaging strategies that are being developed for neurosurgical guidance, with a focus on emerging imaging technologies and clinically viable contrast agents. The strengths and weaknesses of these approaches will be discussed, as well as issues that are being addressed to translate these technologies into the standard of care.
    • Video-rate <i>in vivo</i> fluorescence imaging with a line-scanned dual-axis confocal microscope

      Chen, Y., Wang, D., Khan, A., Wang, Y., Borwege, S., Sanai, N., Liu, J. T. C.

      JOURNAL OF BIOMEDICAL OPTICS

      ABSTRACT
      The development of methods to accurately quantify cell-surface receptors in living tissues would have a seminal impact in oncology. For example, accurate measures of receptor density in vivo could enhance early detection or surgical resection of tumors via protein-based contrast, allowing removal of cancer with high phenotype specificity. Alternatively, accurate receptor expression estimation could be used as a biomarker to guide patient-specific clinical oncology targeting of the same molecular pathway. Unfortunately, conventional molecular contrast-based imaging approaches are not well adapted to accurately estimating the nanomolar-level cell-surface receptor concentrations in tumors, as most images are dominated by nonspecific sources of contrast such as high vascular permeability and lymphatic inhibition. This article reviews approaches for overcoming these limitations based upon tracer kinetic modeling and the use of emerging protocols to estimate binding potential and the related receptor concentration. Methods such as using single time point imaging or a reference-tissue approach tend to have low accuracy in tumors, whereas paired-agent methods or advanced kinetic analyses are more promising to eliminate the dominance of interstitial space in the signals. Nuclear medicine and optical molecular imaging are the primary modalities used, as they have the nanomolar level sensitivity needed to quantify cell-surface receptor concentrations present in tissue, although each likely has a different clinical niche.
    • Quantification of the binding potential of cell-surface receptors in fresh excised specimens via dual-probe modeling of SERS nanoparticles

      Sinha, L., Wang, Y., Yang, C., Khan, A., Brankov, J. G., Liu, J. T. C., Tichauer, K. M.

      SCIENTIFIC REPORTS

      ABSTRACT
      The early detection and biological investigation of esophageal cancer would benefit from the development of advanced imaging techniques to screen for the molecular changes that precede and accompany the onset of cancer. Surface-enhanced Raman scattering (SERS) nanoparticles (NPs) have the potential to improve cancer detection and the investigation of cancer progression through the sensitive and multiplexed phenotyping of cell-surface biomarkers. Here, a miniature endoscope featuring rotational scanning and axial pull back has been developed for 2D spectral imaging of SERS NPs topically applied on the lumenal surface of the rat esophagus. Raman signals from low-pM concentrations of SERS NP mixtures are demultiplexed in real time to accurately calculate the concentration and ratio of the NPs. Ex vivo and in vivo experiments demonstrate the feasibility of topical application and imaging of multiplexed SERS NPs along the entire length of the rat esophagus.

    2014