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* Context.--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. Objective.--To present an image atlas of OTLS images of prostate core needle biopsies. Design.--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. Results.--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. Conclusions.--Three-dimensional OTLS microscopy images have a similar appearance to traditional hematoxylineosin 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. (Arch Pathol Lab Med. 2019;143:1069-1075; doi: 10.5858/arpa.2018-0466-OA), Light-sheet microscopy is one of a family of emerging imaging techniques for slide-free, nondestructive ex vivo microscopy. Ex vivo microscopy gives pathologists the ability to examine fresh or fixed tissue [...]

* Context.--In vivo microscopy (IVM) allows direct, real-time visualization of tissue histology in living patients without the need for tissue removal, processing, or staining. The IVM technologies in clinical use include confocal microscopy and optical coherence tomography. These technologies also show promise for use with pathology specimens (ex vivo microscopy [EVM]). However, few systems designed for EVM are commercially available, at least in part because of the lack of defined minimal functional requirements (FRs). Objective.--To develop minimal FRs for likely high-volume pathology applications of EVM. Design.--The IVM Committee of the College of American Pathologists identified potential EVM pathology applications based on the published literature. A subcommittee of IVM and EVM early adopters and experts then defined FRs for the most likely EVM applications. Results.--Potential EVM applications include assessment of margins, adequacy of needle biopsies and aspirates for diagnosis, and transplant tissues; selection of tissue for molecular studies or biorepository; and guidance in block selection from gross specimens. The first 3 applications were selected for development of FRs. The FRs were identified based on existing laboratory practices and guidelines and input from experts in the field and included device footprint and portability, specimen preparation, imaging time, field of view or resolution, morphologic diagnostic capability, yield, accuracy, ease of use, safety, and cost. Conclusions.--Consensus was achieved on FRs that would accommodate the selected EVM applications. Publication and dissemination of those FRs will provide guidance to engineers, researchers, and vendors on how to optimally adapt IVM technologies for EVM for widespread adoption by pathologists. (Arch Pathol Lab Med. 2019;143:1052-1057; doi: 10.5858/arpa.2018-0482-OA), In vivo microscopy (IVM) is the use of novel optical imaging modalities, many of which are US Food and Drug Administration-approved for clinical use, to produce cellular (or near-cellular) histologic [...]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Functional implication of stromal heterogeneity in the prostate remains incompletely understood. Using lineage tracing and light-sheet imaging, we show that some fibroblast cells at the mouse proximal prostatic ducts and prostatic urethra highly express Lgr5. Genetic ablation of these anatomically restricted stromal cells, but not nonselective ablation of prostatic stromal cells, rapidly induces prostate epithelial turnover and dedifferentiation that are reversed following spontaneous restoration of the Lgr5+ stromal cells. RNA sequencing (RNA-seq) analysis indicates that ablating the Lgr5+ stromal cells activates a mechanosensory response. Ablating the Lgr5+ stromal cells impairs the control of prostatic ductal outlet, increases prostate tissue stiffness, and activates the mitogen-activated protein kinase (MAPK). Suppressing MAPK overrides the elevated epithelial proliferation. In summary, the Lgr5+ stromal cells regulate prostate tissue homeostasis and maintain its functional integrity in a long-distance manner. Our study implies that the cells at organ junctions most likely control organ homeostasis by sustaining a balanced mechanoforce. [Display omitted] • Stromal cells at the junction of mouse prostate and urethra highly express Lgr5 • Ablating Lgr5+ stromal cells impairs the control of prostatic ductal outlet • Ablating Lgr5+ stromal cells activates a mechanosensory response • Ablating Lgr5+ stromal cells induces epithelial turnover and dedifferentiation Wei et al. show that the stromal cells at the junction of mouse prostate and urethra highly express Lgr5. Genetically ablating these cells impairs the control of prostatic ductal outlet, increases prostate tissue stiffness, activates the mitogen-activated protein kinase (MAPK)-mediated mechanosensory response, and rapidly induces prostate epithelial turnover and dedifferentiation. [ABSTRACT FROM AUTHOR]

Abstract: Background: Patients with scleroderma and end-stage lung disease (ESLD) have a very high prevalence of gastroesophageal reflux disease (GERD). Because GERD has been associated with aspiration in those with ESLD, and because those with scleroderma are particularly prone to develop severe GERD, there is some concern that GERD may contribute to shorten survival in patients with scleroderma awaiting lung transplantation. Therefore, we hypothesized that esophageal pH monitoring could predict survival of those with scleroderma and ESLD awaiting lung transplantation and that the severity of reflux can impact survival. Methods: We conducted a retrospective analysis of all scleroderma patients referred for lung transplantation who underwent esophageal manometry and pH monitoring since August 2008. We identified 10 patients in whom we calculated and compared the area under the curve for each receiver operating characteristic curve of the following variables: DeMeester score, forced expiratory volume in 1 s (FEV1), %predicted FEV1, forced vital capacity (FVC), %predicted FVC, diffusion capacity for carbon monoxide (DLco), and %predicted DLco. Results: The DeMeester score nominally outperformed FEV1, FVC, and DLco. Receiver operating characteristic curve analysis was also used to define the optimal DeMeester score (65.2) in differentiating survival status, as determined by maximizing sensitivity and specificity. Based on this value, we calculated the 1-y survival from the time of the esophageal function testing, which was 100% in seven patients with a DeMeester score of <65.2, and 33% in three patients with a score >65.2 (P = 0.01). The latter patients had greater total time pH < 4, greater time pH < 4 in the supine position, greater total episodes of reflux, and higher prevalence of absent peristalsis. The single survivor with a DeMeester score >70 had also proximal reflux, underwent antireflux surgery, and is alive 1201 d after transplant. Conclusions: Our study shows that esophageal pH monitoring can predict survival status in patients with scleroderma awaiting lung transplantation and that the severity of reflux can impact the 1-y survival rate. Therefore, esophageal pH monitoring should be considered early in patients with scleroderma and ESLD, as this test could appropriately identify those in whom laparoscopic antireflux surgery should be performed quicker to prevent GERD and its detrimental effects in patients awaiting lung transplantation. [Copyright &y& Elsevier]

Background: The pathway-focused association approach offers a hypothesis driven alternative to the agnostic genomewide association study. Here we apply the pathway-focused approach to an association study of hypertension, systolic blood pressure (SBP), and diastolic blood pressure (DBP) in 1614 Nigerians with genome-wide data. Methods and Results: Testing of 28 pathways with biological relevance to hypertension, selected a priori, containing a total of 101 unique genes and 4,349 unique single-nucleotide polymorphisms (SNPs) showed an association for the adrenergic alpha 1 (ADRA1) receptor pathway with hypertension (p<0.0009) and diastolic blood pressure (p<0.0007). Within the ADRA1 pathway, the genes PNMT (hypertension Pgene<0.004, DBP Pgene<0.004, and SBP Pgene<0.009, and ADRA1B (hypertension Pgene<0.005, DBP Pgene0.02, and SBP Pgene0.02) displayed the strongest associations. Neither ADRA1B nor PNMT could be the sole mediator of the observed pathway association as the ADRA1 pathway remained significant after removing ADRA1B, and other pathways involving PNMT did not reach pathway significance. Conclusions: We conclude that multiple variants in several genes in the ADRA1 pathway led to associations with hypertension and DBP. SNPs in ADRA1B and PNMT have not previously been linked to hypertension in a genome-wide association study, but both genes have shown associations with hypertension through linkage or model organism studies. The identification of moderately significant (10-2>p>10-5) SNPs offers a novel method for detecting the ''missing heritability'' of hypertension. These findings warrant further studies in similar and other populations to assess the generalizability of our results, and illustrate the potential of the pathway-focused approach to investigate genetic variation in hypertension. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Neuromyelitis optica (NMO) is characterized by selective inflammation of the spinal cord and optic nerves but is distinct from multiple sclerosis (MS). Interferon (IFN)-β mitigates disease activity in MS, but is controversial in NMO, with a few reports of disease worsening after IFN-β therapy in this highly active disease. In systemic lupus erythematosus (SLE), IFNs adversely affect disease activity. This study examines for the first time whether serum IFN-α/β activity and IFN-β-induced responses in peripheral blood mononuclear cells (MNC) are abnormally elevated in NMO, as they are in SLE, but contrast to low levels in MS. Methods: Serum type I IFN-α/β activity was measured by a previously validated bioassay of 3 IFN-stimulated genes (RT-PCR sensitivity, 0.1U/ml) rather than ELISA, which has lower sensitivity and specificity for measuring serum IFNs. IFN responses in PBMNC were assessed by in vitro IFN-β-induced activation of phospho-tyrosine-STAT1 and phospho-serine-STAT1 transcription factors, and MxA proteins using Western blots. Results: Serum IFN-α/β activity was highest in SLE patients, followed by healthy subjects and NMO, but was surprisingly low in therapy-naïve MS. In functional assays in vitro, IFN-β-induced high levels of P-S-STAT1 in NMO and SLE, but not in MS and controls. IFN-β-induced MxA protein levels were elevated in NMO and SLE compared to MS. Conclusions: Serum IFN activity and IFN-β-induced responses in PBMNC are elevated in SLE and NMO patients versus MS. This argues for similarities in pathophysiology between NMO and SLE and provides an explanation for IFN-induced disease worsening in NMO. [Copyright &y& Elsevier]

Significance: 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. Aim: 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. Approach: 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. Results: 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 %. Conclusions: The 1Hr2Dx method has the potential to improve patient care by providing an accurate preliminary diagnosis within an hour of biopsy. [ABSTRACT FROM AUTHOR]

Introduction. Papillary urothelial carcinomas are currently graded as either low- or high-grade tumors based on World Health Organization (WHO) 2022 guidelines for genitourinary tumors. However, a minority of tumors are mixed-grade tumors, composed predominantly of low-grade cancer with a minor high-grade component. In the 2022 WHO these cancers are recognized as having outcomes comparable to low-grade cancers, although data to date has been limited. Methods. The pathology records of a large academic institution were searched for mixed-grade, non-muscle invasive papillary carcinomas of the bladder and ureter in order to characterize prognosis of these cancers. Results. Of 136 cancers, the majority (n = 104, 76.5%) were solitary, mixed-grade tumors, while 21 (15.4%) had a concurrent low-grade cancer and 11 (8.1%) had multiple mixed-grade tumors at the time of diagnosis. At follow-up (median 48.3 months, range = 1.3 months-18.1 years), 71 cancers recurred (52.2%): 52 (38.2%) as low- or mixed-grade cancers and 18 (13.2%) as high-grade cancers. There were no instances of stage-progression to >pT2. Conclusions. The clinical outcome of mixed-grade carcinomas was similar to what has been reported for low-grade carcinomas. Based on our results, and prior congruent studies of mixed-grade lesions, these lesions may be regarded as a distinct sub-category with a better prognosis than high-grade tumors. [ABSTRACT FROM AUTHOR]

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 The Authors; Published by the American Association for Cancer Research.)

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.

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 cm 2 /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., Competing Interests: A.K.G., N.P.R., and J.T.C.L. are cofounders and shareholders of LightSpeed Microscopy Inc.

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.

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., Competing Interests: Competing Interests: Jonathan T.C. Liu is an inventor for a pending patent: “Raman imaging devices and methods of molecular imaging.” The other authors have declared that no conflict of interest exists.

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 ., (©2017 American Association for Cancer Research.)

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., Competing Interests: Competing interests The authors declare no competing financial interests.

Context: Whole-slide images (WSIs) present a rich source of information for education, training, and quality assurance. However, they are often used in a fashion similar to glass slides rather than in novel ways that leverage the advantages of WSI. We have created a pipeline to transform annotated WSI into pattern recognition training, and quality assurance web application called novel diagnostic electronic resource (NDER)., Aims: Create an efficient workflow for extracting annotated WSI for use by NDER, an attractive web application that provides high-throughput training., Materials and Methods: WSI were annotated by a resident and classified into five categories. Two methods of extracting images and creating image databases were compared. Extraction Method 1: Manual extraction of still images and validation of each image by four breast pathologists. Extraction Method 2: Validation of annotated regions on the WSI by a single experienced breast pathologist and automated extraction of still images tagged by diagnosis. The extracted still images were used by NDER. NDER briefly displays an image, requires users to classify the image after time has expired, then gives users immediate feedback., Results: The NDER workflow is efficient: annotation of a WSI requires 5 min and validation by an expert pathologist requires An additional one to 2 min. The pipeline is highly automated, with only annotation and validation requiring human input. NDER effectively displays hundreds of high-quality, high-resolution images and provides immediate feedback to users during a 30 min session., Conclusions: NDER efficiently uses annotated WSI to rapidly increase pattern recognition and evaluate for diagnostic proficiency.