Rapid examination of lung tissues by nonlinear microscopy.

OBJECTIVES: Traditional histopathology is a time-intensive and labor-intensive process involving tissue formalin fixation, paraffin embedding, and microtoming into thin sections for H&E staining. Frozen section analysis is a modality used during surgery to quickly evaluate tissue, but it has limitations, such as the size and number of the specimens that can be analyzed as well as difficulties with fatty and bony tissues. Our objective was to investigate the performance of nonlinear microscopy, a fluorescence microscopy technique, for the rapid examination of resected lung tumors. METHODS: In this proof-of-principle study, nonlinear microscopy imaging of resected lung tissue was performed on a total of 73 tissue specimens collected from 13 patients who underwent lobectomy, segmentectomy, or wedge resection for pulmonary nodules. RESULTS: Two pathologists reviewed the digital nonlinear microscopy images in comparison to the corresponding histopathologic H&E slides from a variety of pulmonary pathologies. CONCLUSIONS: This study demonstrated that nonlinear microscopy readily replicates traditional H&E staining for both lung tumors and nonneoplastic pulmonary structures. Nonlinear microscopy provides many advantages over frozen section analysis and is an optical imaging platform that has the potential to augment rapid pathologic evaluation of resected tissues in the age of digital pathology.

Data retrieval from archival renal biopsies using nonlinear microscopy.

Thorough examination of renal biopsies may improve understanding of renal disease. Imaging of renal biopsies with fluorescence nonlinear microscopy (NLM) and optical clearing enables three-dimensional (3D) visualization of pathology without microtome sectioning. Archival renal paraffin blocks from 12 patients were deparaffinized and stained with Hoechst and Eosin for fluorescent nuclear and cytoplasmic/stromal contrast, then optically cleared using benzyl alcohol benzyl benzoate (BABB). NLM images of entire biopsy fragments (thickness range 88-660 µm) were acquired using NLM with fluorescent signals mapped to an H&E color scale. Cysts, glomeruli, exudative lesions, and Kimmelstiel-Wilson nodules were segmented in 3D and their volumes, diameters, and percent composition could be obtained. The glomerular count on 3D NLM volumes was high indicating that archival blocks could be a vast tissue resource to enable larger-scale retrospective studies. Rapid optical clearing and NLM imaging enables more thorough biopsy examination and is a promising technique for analysis of archival paraffin blocks.

Rapid Examination of Nonprocessed Renal Cell Carcinoma Using Nonlinear Microscopy.

CONTEXT.­: Histology, the traditional method of examining surgical tissue under a microscope, is a time-consuming process involving the fixation of tissue in formalin, dehydration, embedding in paraffin, and cutting into thin sections for hematoxylin-eosin (H&E) staining. Frozen section analysis is a faster alternative used in surgery to quickly evaluate tissue, but it has limitations, such as the size of the specimens that can be analyzed and difficulties with fatty and bony tissues. OBJECTIVE.­: To rapidly examine nonprocessed kidney tumors using nonlinear microscopy (NLM), a fluorescence microscopy technique that can rapidly visualize fresh or fixed, rapidly stained, nonprocessed tissue resembling H&E histology. This technology eliminates the need for fixation, embedding, microtome sectioning, or slide preparation. DESIGN.­: In this study, a total of 190 tissue specimens were collected from 46 patients who underwent partial or radical nephrectomy. RESULTS.­: Two genitourinary pathologists confirmed that diagnostically important features present in the H&E images could also be identified in the NLM images. CONCLUSIONS.­: The results of this study demonstrated that NLM had a high degree of correspondence with H&E staining for the classical variants of renal cell carcinoma. NLM offers several clinical benefits, such as facilitating rapid renal cell carcinoma diagnosis, assessment of targeted kidney biopsies for both tumor and medical kidney diseases, and collection of fresh renal cell carcinoma tissue for molecular studies.

Optical Coherence Tomography Angiography Analysis Toolbox: A Repeatable and Reproducible Software Tool for Quantitative Optical Coherence Tomography Angiography Analysis.

BACKGROUND AND OBJECTIVE: The purpose of this article is to demonstrate the optical coherence tomography angiography (OCTA) Analysis Toolkit (OAT), a custom-designed software package, as a repeatable and reproducible tool for computing OCTA metrics across different devices. MATERIALS AND METHODS: Fourteen participants were imaged using three devices. Foveal avascular zone, vessel index, vessel length index, and vessel diameter index were calculated using the OAT. Repeatability and reproducibility were assessed using the coefficient of variation and interclass correlation coefficient (ICC). RESULTS: Analysis of identical images demonstrated perfect levels of repeatability for all metrics (coefficient of variation 0%), which was a consequence of the software being deterministic (ie, producing the same outputs for the same inputs). Foveal avascular zone ICC values were in the excellent-to-good range (ICC > 0.6) for all devices. All values for vessel index (VI), vessel length index, and vessel diameter index fell in the good-to-fair (ICC > 0.4) or excellent-to-good range, except for vessel index analysis in the Cirrus device (ICC = 0.34). CONCLUSIONS: The OAT appears to be a reliable tool that may enable comparison between OCTA data sets acquired on different imaging instruments, thereby facilitating a more consistent approach to OCTA analysis. [Ophthalmic Surg Lasers Imaging Retina 2023;54:114-122.].

Rapid histological imaging of bone without microtome sectioning using nonlinear microscopy.

Tissue preparation for histologic evaluation of bone is particularly lengthy, limiting its use in intraoperative or intraprocedural histological evaluation. Nonlinear microscopy (NLM) is an optical sectioning microscopy method that can visualize pathology in freshly excised tissue without requiring physical microtome sectioning. This study describes a rapid protocol for NLM imaging of bone and associated cartilage. NLM imaging was performed on 71 specimens of normal bone as well as arthritic, malignant and inflammatory bone tissue from 40 patients who underwent joint replacement, amputation, bone marrow biopsy or autopsy. Specimens ranged in size from core needle biopsies to transections of entire femoral heads. Specimens were stained with acridine orange and sulforhodamine 101, nuclear and cytoplasmic/stromal fluorescent dyes, for 5 min, then rinsed for 30 s. NLM fluorescent images were displayed using colors analogous to hematoxylin and eosin (H&E) to facilitate interpretation. Pathologists examined NLM images of the specimens in real time by rapidly translating the specimen to areas of interest, similar to a standard transmission light microscope. By adjusting the NLM focus depth, images from a few-µm-thick layer could be obtained down to ~100 µm below the tissue surface, analogous to serial sectioning. Following real-time NLM imaging, the tissue was processed for conventional paraffin histology, and H&E slides were compared to recorded NLM images. Similarities and differences between NLM and paraffin H&E were assessed. NLM enabled visualization of normal bone architecture, including the lamellar matrix and osteocytes of trabecular bone, articular cartilage, as well as pathological bone features such osteoarthritis, osteomyelitis, and malignancy with an appearance resembling the paraffin H&E. Differences such as changes in cell border sharpness, cellular and nucleolar size, and color patterns were noted, suggesting that training is required for accurate evaluation of bone pathology with NLM. Irregular surface contours and debris generated by gross tissue preparation of bone can make some regions difficult to evaluate with NLM, but the ability to perform rapid three-dimensional translation and sub-surface imaging reduced these problems. NLM is a promising technique for rapid evaluation of bone pathology. Further studies assessing diagnostic performance are warranted.

Real-time diagnosis and Gleason grading of prostate core needle biopsies using nonlinear microscopy.

Rapid histologic assessment of fresh prostate biopsies may reduce patient anxiety, aid in biopsy sampling, and enable specimen triaging for molecular/genomic analyses and research that could benefit from fresh tissue analysis. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can produce high-resolution images of freshly excised tissue resembling formalin-fixed paraffin-embedded (FFPE) H&E. NLM enables evaluation of tissue up to ~100 µm below the surface, analogous to serial sectioning, but without requiring microtome sectioning. One hundred and seventy biopsies were collected from 63 patients who underwent in-bore MRI or MRI/ultrasound fusion biopsy procedures. Biopsies were stained in acridine orange and sulforhodamine 101, a nuclear and cytoplasmic/stromal fluorescent dye, for 45 s. Genitourinary pathologists evaluated the biopsies using NLM by translating the biopsies in real time to areas of interest and NLM images were recorded. After NLM evaluation, the biopsies were processed for standard FFPE H&E and similarities and differences between NLM and FFPE H&E were investigated. Accuracies of NLM diagnoses and Gleason scores were calculated using FFPE histology as the gold standard. Pathologists achieved a 92.4% sensitivity (85.0-96.9%, 95% confidence intervals) and 100.0% specificity (94.3-100.0%) for detecting carcinoma compared to FFPE histology. The agreement between the Grade Group determined by NLM versus FFPE histology had an unweighted Cohen's Kappa of 0.588. The average NLM evaluation time was 2.10 min per biopsy (3.08 min for the first 20 patients, decreasing to 1.54 min in subsequent patients). Further studies with larger patient populations, larger number of pathologists, and multiple institutions are warranted. NLM is a promising method for future rapid evaluation of prostate needle core biopsies.

Tethered capsule en face optical coherence tomography for imaging Barrett's oesophagus in unsedated patients.

OBJECTIVE: Barrett's oesophagus (BE) screening outside the endoscopy suite can identify patients for surveillance and reduce mortality. Tethered capsule optical coherence tomography (OCT) can volumetrically image oesophageal mucosa in unsedated patients and detect features of BE. We investigated ultrahigh-speed tethered capsule swept-source OCT (SS-OCT), improved device design, developed procedural techniques and measured capsule contact, longitudinal pullback non-uniformity and patient toleration. DESIGN: OCT was performed in 16 patients prior to endoscopic surveillance/treatment. Unsedated patients swallowed the capsule with sips of water and the tether was pulled back to image the oesophagus. SS-OCT at 1 000 000 A-scans/s enabled imaging 10 cm oesophageal lengths in 10 s with 30 µm transverse and 8 µm axial resolution. Capsule contact, longitudinal image coverage and patient toleration were assessed. RESULTS: Nine patients had non-dysplastic BE, three had ablative treatment-naïve neoplasia and four had prior ablation for dysplasia. Dry swallows facilitated capsule transit through the lower oesophageal sphincter (LES), and waiting 10 s before pullback reduced swallow induced LES relaxation. Slow nasal inhalation facilitated capsule retrieval and minimised gag reflex. The procedure was well tolerated. Ultrahigh-speed SS-OCT generated cross-sectional and subsurface en face images showing BE features, while subsurface en face images were required to assess the gastro-oesophageal junction. Candidate features of dysplasia were also identified which could inform follow-up endoscopy/biopsy. BE features were seen in all patients with histologically confirmed BE. Mean capsule contact over BE was 75%±27% for all patients and better in short segment BE. Mean longitudinal image coverage over BE was 59%±34% and better for long segment BE. CONCLUSIONS: Ultrahigh-speed tethered capsule SS-OCT can image en face and cross-sectional mucosal features over wide areas. Device and procedure optimisation improved performance. BE features could be identified in all patients, but limited capsule contact and longitudinal coverage could cause sampling errors for focal pathologies.

Macular and Peripapillary Optical Coherence Tomography Angiography Metrics Predict Progression in Diabetic Retinopathy: A Sub-analysis of TIME-2b Study Data.

PURPOSE: To identify optical coherence tomography angiography (OCTA)-derived vessel metrics of the macula and optic nerve head (ONH) that predict diabetic retinopathy (DR) disease progression. DESIGN: Secondary analysis of clinical trial data. METHODS: This was a sub-analysis of prospectively collected data from 73 subjects that participated in the TIME-2b study (Aerpio Pharmaceuticals), a multicenter clinical trial for patients with moderate-to-severe DR treated with AKB-9778 and followed over a 12-month period. Eligible subjects were tested every 3 months with color fundus photography, spectral-domain OCT, and slit-lamp biomicroscopy. OCTA of the macula and ONH was obtained for a subset of patients enrolled at participating sites. En face, full-depth retinal projections centered at the macula were analyzed for multiple metrics including foveal avascular zone (FAZ) area and perimeter, nonperfusion area, vessel density (VD), and presence of intraretinal microvascular abnormalities (IRMA). VD of the radial peripapillary capillaries was evaluated in 4 quadrants surrounding the optic disc for ONH images. Progression was defined as a ≥2-step increase in DR severity scale score or development of diabetic macular edema. RESULTS: Over a follow-up period of 12 months, 15 of 73 (20.5%) subjects progressed. At pretreatment baseline, larger FAZ area, presence of IRMA, and reduced peripapillary VD in the superior temporal and inferior temporal regions were significantly associated with increased odds of progression. CONCLUSIONS: FAZ area and temporal peripapillary VD are predictors of DR progression. OCTA metrics may improve progression risk assessment in DR when compared to established risk factors alone.

Nonlinear microscopy for detection of prostate cancer: analysis of sensitivity and specificity in radical prostatectomies.

Intraoperative evaluation of specimens during radical prostatectomy using frozen sections can be time and labor intensive. Nonlinear microscopy (NLM) is a fluorescence microscopy technique that can rapidly generate images that closely resemble H&E histology in freshly excised tissue, without requiring freezing or microtome sectioning. Specimens are stained with nuclear and cytoplasmic/stromal fluorophores, and NLM evaluation can begin within 3 min of grossing. Fluorescence signals can be displayed using an H&E color scale, facilitating pathologist interpretation. This study evaluates the accuracy of prostate cancer detection in a blinded reading of NLM images compared with the gold standard of formalin-fixed, paraffin-embedded H&E histology. A total of 122 freshly excised prostate specimens were obtained from 40 patients undergoing radical prostatectomy. The prostates were grossed, dissected into specimens of ~10 × 10 mm with 1-4 mm thickness, stained for 2 min for nuclear and cytoplasmic/stromal contrast, and then rinsed with saline for 30 s. NLM images were acquired and multiple images were stitched together to generate large field of view, centimeter-scale digital images suitable for reading. Specimens were then processed for standard paraffin H&E. The study protocol consisted of training, pretesting, and blinded reading phases. After a washout period, pathologists read corresponding paraffin H&E slides. Three pathologists achieved a 95% or greater sensitivity with 100% specificity for detecting cancer on NLM compared with paraffin H&E. Pooled sensitivity and specificity was 97.3% (93.7-99.1%; 95% confidence interval) and 100.0% (97.0-100.0%), respectively. Interobserver agreement for NLM reading had a Fleiss κ = 0.95. The high cancer detection accuracy and rapid specimen preparation suggest that NLM may be useful for intraoperative evaluation in radical prostatectomy.

Retinal Nonperfusion Relationship to Arteries or Veins Observed on Widefield Optical Coherence Tomography Angiography in Diabetic Retinopathy.

Purpose: To evaluate whether retinal capillary nonperfusion is found predominantly adjacent to arteries or veins in eyes with diabetic retinopathy (DR). Methods: Sixty-three eyes from 44 patients with proliferative DR (PDR) or non-PDR (NPDR) were included. Images (12 × 12-mm) foveal-centered optical coherence tomography (OCT) angiography (OCTA) images were taken using the Zeiss Plex Elite 9000. In 37 eyes, widefield montages with five fixation points were also obtained. A semiautomatic algorithm that detects nonperfusion in full-retina OCT slabs was developed, and the percentages of capillary nonperfusion within the total image area were calculated. Retinal arteries and veins were manually traced. Based on the shortest distance, nonperfusion pixels were labeled as either arterial-side or venous-side. Arterial-adjacent and venous-adjacent nonperfusion and the A/V ratio (arterial-adjacent nonperfusion divided by venous-adjacent nonperfusion) were quantified. Results: Twenty-two eyes with moderate NPDR, 16 eyes with severe NPDR, and 25 eyes with PDR were scanned. Total nonperfusion area in PDR (median: 8.93%) was greater than in moderate NPDR (3.49%, P < 0.01). Arterial-adjacent nonperfusion was greater than venous-adjacent nonperfusion for all stages of DR (P < 0.001). The median A/V ratios were 1.93 in moderate NPDR, 1.84 in severe NPDR, and 1.78 in PDR. The A/V ratio was negatively correlated with the total nonperfusion area (r = -0.600, P < 0.0001). The results from the widefield montages showed similar patterns. Conclusions: OCTA images with arteries and veins traced allowed us to estimate the nonperfusion distribution. In DR, smaller nonperfusion tends to be arterial-adjacent, while larger nonperfusion tends toward veins.

Comparison of nonlinear microscopy and frozen section histology for imaging of Mohs surgical margins.

Mohs surgery uses en face frozen section analysis (FSA) with complete margin examination for the excision of select basal cell carcinomas (BCC), obtaining excellent cosmetic outcomes and extremely low recurrence rates. However, Mohs with FSA is time-consuming because of the need to iteratively perform cryosectioning on sequential excisions. Fluorescent microscopies can image tissue specimens without requiring physical sectioning, potentially reducing the time to perform Mohs surgery. We demonstrate a protocol for nonlinear microscopy (NLM) imaging of surgical specimens that combines dual agent staining, virtual H&E rendering, and video rate imaging. We also introduce a novel protocol that enables micron-level co-registration of NLM images with FSA histology, and demonstrate that NLM can reproduce similar features similar to FSA in BCC specimens with both negative and positive surgical margins. We show that the fluorescent labels can be extracted with conventional vacuum infiltration processing, enabling subsequent immunohistochemistry on fluorescently labeled tissue. This protocol can also be applied to evaluate the performance of NLM compared with FSA in a wide range of pathologies for intraoperative consultation.

Assessment of chronic radiation proctopathy and radiofrequency ablation treatment follow-up with optical coherence tomography angiography: A pilot study.

BACKGROUND: Chronic radiation proctopathy (CRP) occurs as a result of pelvic radiation therapy and is associated with formation of abnormal vasculature that may lead to persistent rectal bleeding. While incidence is declining due to refinement of radiation delivery techniques, CRP remains one of the major complications of pelvic radiation therapy and significantly affects patient quality of life. Radiofrequency ablation (RFA) is an emerging treatment modality for eradicating abnormal vasculature associated with CRP. However, questions remain regarding CRP pathophysiology and optimal disease management. AIM: To study feasibility of optical coherence tomography angiography (OCTA) for investigating subsurface vascular alterations in CRP and response to RFA treatment. METHODS: Two patients with normal rectum and 8 patients referred for, or undergoing endoscopic RFA treatment for CRP were imaged with a prototype ultrahigh-speed optical coherence tomography (OCT) system over 15 OCT/colonoscopy visits (2 normal patients, 5 RFA-naïve patients, 8 RFA-follow-up visits). OCT and OCTA was performed by placing the OCT catheter onto the dentate line and rectum without endoscopic guidance. OCTA enabled depth-resolved microvasculature imaging using motion contrast from flowing blood, without requiring injected dyes. OCTA features of normal and abnormal microvasculature were assessed in the mucosa and submucosa. Blinded reading of OCTA images was performed to assess the association of abnormal rectal microvasculature with CRP and RFA treatment, and rectal telangiectasia density endoscopic scoring. RESULTS: OCTA/OCT images are intrinsically co-registered and enabled depth-resolved visualization of microvasculature in the mucosa and submucosa. OCTA visualized normal vascular patterns with regular honeycomb patterns vs abnormal vasculature with distorted honeycomb patterns and ectatic/tortuous microvasculature in the rectal mucosa. Normal arterioles and venules < 200 µm in diameter versus abnormal heterogenous enlarged arterioles and venules > 200 µm in diameter were visualized in the rectal submucosa. Abnormal mucosal vasculature occurred in 0 of 2 normal patients and 3 of 5 RFA-naïve patients, while abnormal submucosal vasculature occurred more often, in 1 of 2 normal patients and 5 of 5 RFA-naïve patients. After RFA treatment, vascular abnormalities decreased, with abnormal mucosal vasculature observed in 0 of 8 RFA-follow-up visits and abnormal submucosal vasculature observed in only and 2 of 8 RFA-follow-up visits. CONCLUSION: OCTA visualizes depth-resolved microvascular abnormalities in CRP, allowing assessment of superficial features which are endoscopically visible as well as deeper vasculature which cannot be seen endoscopically. OCTA/OCT of the rectum can be performed in conjunction with, or independently from endoscopy. Further studies are warranted to investigate if OCTA/OCT can elucidate pathophysiology of CRP or improve management.

Comparing histologic evaluation of prostate tissue using nonlinear microscopy and paraffin H&E: a pilot study.

Rapid histological assessment of large areas of prostate tissue is required for many intraoperative consultation scenarios such as margin evaluation. Nonlinear microscopy (NLM) enables imaging of large (whole mount) specimens without freezing or cryotoming. This study demonstrates rapid histological imaging of unsectioned prostate cancer surgical specimens using nonlinear microscopy and compares features of prostate pathology to standard paraffin embedded H&E histology. Fresh or formalin fixed specimens were stained in 2.5 min with fluorescent nuclear and stromal dyes. Nonlinear microscopy images of unsectioned tissues were generated by nonlinear (two-photon) excitation of the fluorophores, where fluorescence is only emitted from tissue at the microscope focus, avoiding the need for physical sectioning. The images were displayed in real time using a color scale similar to H&E, then tissues were processed for standard paraffin embedded H&E histology. Seventy nonlinear microscopy and corresponding paraffin H&E images of fresh and fixed prostate specimens (15 cancer, 55 benign) from 24 patients were read by genitourinary pathologists to assess if nonlinear microscopy could achieve an equivalent evaluation to paraffin embedded H&E histology. Differences between nonlinear microscopy images and paraffin H&E slides, including cytoplasmic color and stromal density, were observed, however nonlinear microscopy images could be interpreted with minimal training. Nonlinear microscopy enabled visualization of benign, atrophic and hyperplastic glands and stroma, ejaculatory ducts, vasculature and inflammatory changes. Nonlinear microscopy enabled identification of typical and variants of adenocarcinoma, as well as Gleason patterns. Perineural invasion and extraprostatic extension could also be assessed. Nonlinear microscopy images closely resemble paraffin H&E slides and enable rapid assessment of normal prostate architecture, benign conditions, and carcinoma in freshly excised and fixed specimens. Nonlinear microscopy can image large regions of tissue, equivalent to multiple frozen section tissue blocks, within minutes because cryotoming/microtoming are not required, making it a promising technique for intraoperative consultation.

Assessment of Barrett's esophagus and dysplasia with ultrahigh-speed volumetric en face and cross-sectional optical coherence tomography.

BACKGROUND: This study aimed to evaluate the use of ultrahigh-speed volumetric en face and cross-sectional optical coherence tomography (OCT) with micromotor catheters for the in vivo assessment of Barrett's esophagus and dysplasia. METHODS: 74 OCT datasets with correlated biopsy/endoscopic mucosal resection histology (49 nondysplastic Barrett's esophagus [NDBE], 25 neoplasia) were obtained from 14 patients with Barrett's esophagus and a history of dysplasia and 30 with NDBE. The associations between irregular mucosal patterns on en face OCT, absence of mucosal layering, surface signal > subsurface, and > 5 atypical glands on cross-sectional OCT vs. histology and treatment history were assessed by three blinded readers. RESULTS: Atypical glands under irregular mucosal patterns occurred in 75 % of neoplasia (96 % of treatment-naïve neoplasia) vs. 30 % of NDBE datasets (43 % of short- and 18 % of long-segment NDBE). Mucosal layering was absent in 35 % of neoplasia and 50 % of NDBE datasets, and surface signal > subsurface occurred in 29 % of neoplasia and 30 % of NDBE datasets. CONCLUSIONS: Atypical glands under irregular mucosal patterns are strongly associated with neoplasia, suggesting potential markers for dysplasia and a role in pathogenesis.

Multiscale nonlinear microscopy and widefield white light imaging enables rapid histological imaging of surgical specimen margins.

The ability to histologically assess surgical specimens in real-time is a long-standing challenge in cancer surgery, including applications such as breast conserving therapy (BCT). Up to 40% of women treated with BCT for breast cancer require a repeat surgery due to postoperative histological findings of close or positive surgical margins using conventional formalin fixed paraffin embedded histology. Imaging technologies such as nonlinear microscopy (NLM), combined with exogenous fluorophores can rapidly provide virtual H&E imaging of surgical specimens without requiring microtome sectioning, facilitating intraoperative assessment of margin status. However, the large volume of typical surgical excisions combined with the need for rapid assessment, make comprehensive cellular resolution margin assessment during surgery challenging. To address this limitation, we developed a multiscale, real-time microscope with variable magnification NLM and real-time, co-registered position display using a widefield white light imaging system. Margin assessment can be performed rapidly under operator guidance to image specific regions of interest located using widefield imaging. Using simulated surgical margins dissected from human breast excisions, we demonstrate that multi-centimeter margins can be comprehensively imaged at cellular resolution, enabling intraoperative margin assessment. These methods are consistent with pathology assessment performed using frozen section analysis (FSA), however NLM enables faster and more comprehensive assessment of surgical specimens because imaging can be performed without freezing and cryo-sectioning. Therefore, NLM methods have the potential to be applied to a wide range of intra-operative applications.

Rapid histopathological imaging of skin and breast cancer surgical specimens using immersion microscopy with ultraviolet surface excitation.

Rapid histopathological evaluation of fresh, unfixed human tissue using optical sectioning microscopy would have applications to intraoperative surgical margin assessment. Microscopy with ultraviolet surface excitation (MUSE) is a low-cost optical sectioning technique using ultraviolet illumination which limits fluorescence excitation to the specimen surface. In this paper, we characterize MUSE using high incident angle, water immersion illumination to improve sectioning. Propidium iodide is used as a nuclear stain and eosin yellow as a counterstain. Histologic features of specimens using MUSE, nonlinear microscopy (NLM) and conventional hematoxylin and eosin (H&E) histology were evaluated by pathologists to assess potential application in Mohs surgery for skin cancer and lumpectomy for breast cancer. MUSE images of basal cell carcinoma showed high correspondence with frozen section H&E histology, suggesting that MUSE may be applicable to Mohs surgery. However, correspondence in breast tissue between MUSE and paraffin embedded H&E histology was limited due to the thicker optical sectioning in MUSE, suggesting that further development is needed for breast surgical applications. We further demonstrate that the transverse image resolution of MUSE is limited by the optical sectioning thickness and use co-registered NLM to quantify the improvement in MUSE optical sectioning from high incident angle water immersion illumination.

Microscope-Integrated Intraoperative Ultrahigh-Speed Swept-Source Optical Coherence Tomography for Widefield Retinal and Anterior Segment Imaging.

BACKGROUND AND OBJECTIVE: To demonstrate the feasibility of retinal and anterior segment intraoperative widefield imaging using an ultrahigh-speed, swept-source optical coherence tomography (SS-OCT) surgical microscope attachment. PATIENTS AND METHODS: A prototype post-objective SS-OCT using a 1,050-nm wavelength, 400 kHz A-scan rate, vertical cavity surface-emitting laser (VCSEL) light source was integrated to a commercial ophthalmic surgical microscope after the objective. Each widefield OCT data set was acquired in 3 seconds (1,000 × 1,000 A-scans, 12 × 12 mm2 for retina and 10 × 10 mm2 for anterior segment). RESULTS: Intraoperative SS-OCT was performed in 20 eyes of 20 patients. In six of seven membrane peels and five of seven rhegmatogenous retinal detachment repair surgeries, widefield retinal imaging enabled evaluation pre- and postoperatively. In all seven cataract cases, anterior imaging evaluated the integrity of the posterior lens capsule. CONCLUSIONS: Ultrahigh-speed SS-OCT enables widefield intraoperative viewing in the posterior and anterior eye. Widefield imaging visualizes ocular structures and pathology without requiring OCT realignment. [Ophthalmic Surg Lasers Imaging Retina. 2018;49:94-102.].

Rapid virtual hematoxylin and eosin histology of breast tissue specimens using a compact fluorescence nonlinear microscope.

Up to 40% of patients undergoing breast conserving surgery for breast cancer require repeat surgeries due to close to or positive margins. The lengthy processing required for evaluating surgical margins by standard paraffin-embedded histology precludes its use during surgery and therefore, technologies for rapid evaluation of surgical pathology could improve the treatment of breast cancer by reducing the number of surgeries required. We demonstrate real-time histological evaluation of breast cancer surgical specimens by staining specimens with acridine orange (AO) and sulforhodamine 101 (SR101) analogously to hematoxylin and eosin (H&E) and then imaging the specimens with fluorescence nonlinear microscopy (NLM) using a compact femtosecond fiber laser. A video-rate computational light absorption model was used to produce realistic virtual H&E images of tissue in real time and in three dimensions. NLM imaging could be performed to depths of 100 µm below the tissue surface, which is important since many surgical specimens require subsurface evaluation due to contamination artifacts on the tissue surface from electrocautery, surgical ink, or debris from specimen handling. We validate this method by expert review of NLM images compared to formalin-fixed, paraffin-embedded (FFPE) H&E histology. Diagnostically important features such as normal terminal ductal lobular units, fibrous and adipose stromal parenchyma, inflammation, invasive carcinoma, and in situ lobular and ductal carcinoma were present in NLM images associated with pathologies identified on standard FFPE H&E histology. We demonstrate that AO and SR101 were extracted to undetectable levels after FFPE processing and fluorescence in situ hybridization (FISH) HER2 amplification status was unaffected by the NLM imaging protocol. This method potentially enables cost-effective, real-time histological guidance of surgical resections.

Polypoidal Choroidal Vasculopathy on Swept-Source Optical Coherence Tomography Angiography with Variable Interscan Time Analysis.

PURPOSE: To use a novel optical coherence tomography angiography (OCTA) algorithm termed variable interscan time analysis (VISTA) to evaluate relative blood flow speeds in polypoidal choroidal vasculopathy (PCV). METHODS: Prospective cross-sectional study enrolling patients with confirmed diagnosis of PCV. OCTA of the retina and choroid was obtained with a prototype swept-source OCT system. The acquired OCT volumes were centered on the branching vascular network (BVN) and polyps as determined by indocyanine-green angiography (ICGA). The relative blood flow speeds were characterized on VISTA-OCTA. RESULTS: Seven eyes from seven patients were evaluated. Swept-source OCTA enabled detailed enface visualization of the BVN and polyps in six eyes. VISTA-OCTA revealed variable blood flow speeds in different PCV lesion components of the same eye, with faster flow in the periphery of polyps and slower flow in the center of each polyp, as well as relatively slow flow in BVN when compared with retinal vessels. BVNs demonstrated relatively faster blood flow speeds in the larger trunk vessels and relatively slower speeds in the smaller vessels. CONCLUSIONS: Swept-source OCTA identifies polyps in most, but not all, PCV lesions. This limitation that may be related to relatively slow blood flow within the polyp, which may be below the OCTA's sensitivity. VISTA-OCTA showed heterogeneous blood flow speeds within the polyps, which may indicate turbulent flow in the polyps. TRANSLATIONAL RELEVANCE: These results bring relevant insights into disease mechanisms that can account for the variable course of PCV, and can be relevant for diagnosis and management of patients with PCV.

Endoscopic forward-viewing optical coherence tomography and angiography with MHz swept source.

Endoscopic optical coherence tomography (OCT) instruments are mostly side viewing and rely on at least one proximal scan, thus limiting accuracy of volumetric imaging and en face visualization. Previous forward-viewing OCT devices had limited axial scan speeds. We report a forward-viewing fiber scanning 3D-OCT probe with 900 µm field of view and 5 µm transverse resolution, imaging at 1 MHz axial scan rate in the human gastrointestinal tract. The probe is 3.3 mm diameter and 20 mm rigid length, thus enabling passage through the endoscopic channel. The scanner has 1.8 kHz resonant frequency, and each volumetric acquisition takes 0.17 s with 2 volumes/s display. 3D-OCT and angiography imaging of the colon was performed during surveillance colonoscopy.