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.

MULTISCALE CORRELATION OF MICROVASCULAR CHANGES ON OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY WITH RETINAL SENSITIVITY IN DIABETIC RETINOPATHY.

PURPOSE: To assess global, zonal, and local correlations between vessel density changes measured by optical coherence tomography angiography and retinal sensitivity measured by microperimetry across diabetic retinopathy severity. METHODS: Diabetic patients and nondiabetic controls underwent optical coherence tomography angiography imaging and microperimetry testing. Pearson's correlation was used to assess associations between average sensitivity and skeletonized vessel density (SVD) or foveal avascular zone area centrally. Linear mixed effects modeling was used to assess relationships between local SVD measurements and their spatially corresponding retinal sensitivity measurements. RESULTS: Thirty-nine eyes from 39 participants were imaged. In all slabs, there was a statistically significant positive correlation between retinal sensitivities and SVDs on both global and zonal scales. No statistically significant correlation was found between central retinal sensitivities and the foveal avascular zone areas. Assessment of 1,136 spatially paired retinal sensitivity and SVD measurements revealed a statistically significant local relationship; this seemed to be driven by eyes with proliferative diabetic retinopathy that had reduced retinal sensitivities. CONCLUSION: This study supports positive correlations between SVD and retinal sensitivity at global and zonal spatial scales in diabetic eyes. However, our analysis did not find evidence of statistically significant correlations between retinal sensitivity and SVD on a local scale until advanced diabetic retinopathy.

FULL-THICKNESS MACULAR HOLE SIZE BY HYPERTRANSMISSION SIGNAL ON SPECTRAL-DOMAIN OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To assess the full-thickness macular hole (FTMH) size using the choroidal hypertransmission signal on spectral-domain optical coherence tomography and to compare this method to the standard aperture measurement of the minimum aperture size at the level of the neurosensory retina. DESIGN: Cross-sectional study of retrospective data. METHODS: Eyes with FTMH imaged on spectral-domain optical coherence tomography were included. Two independent masked graders used the device's built-in caliper tool to measure the FTMH minimum aperture size at the level of the neurosensory retina and the size of the corresponding hypertransmission signal below the level of the retinal pigment epithelium/Bruch membrane complex. To assess the reproducibility of the hypertransmission measurement in tilted scans, two measurements were obtained and compared; the first was traced parallel to the retinal pigment epithelium (parallel hypertransmission), and the second was horizontal to the image frame (horizontal hypertransmission), both using Image J software. RESULTS: A total of 31 eyes were enrolled. The mean FTMH minimum aperture size was smaller compared with both the choroidal parallel hypertransmission and horizontal hypertransmission measurements (mean ± SD: 335.7 ± 139.5 µm, 376.7 ± 150.6 µm, 375.1 ± 150.0 µm, respectively. P < 0.001 for both comparisons). CONCLUSION: The proposed hypertransmission measurement is a feasible and reproducible alternative to assess FTMH size and could provide the basis for an automated FTMH measurement on cross-sectional spectral-domain optical coherence tomography scans, as presented in this study, or on the spectral-domain optical coherence tomography volumetric data set by using an en face projection.

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.

QUANTIFICATION OF RETINAL CAPILLARY NONPERFUSION IN DIABETICS USING WIDE-FIELD OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To combine advances in high-speed, wide-field optical coherence tomography angiography (OCTA) with image processing methods for semiautomatic quantitative analysis of capillary nonperfusion in patients with diabetic retinopathy (DR). METHODS: Sixty-eight diabetic patients (73 eyes), either without retinopathy or with different degrees of retinopathy, were prospectively recruited for volumetric swept-source OCTA imaging using 12 mm × 12 mm fields centered at the fovea. A custom, semiautomatic software algorithm was used to quantify areas of capillary nonperfusion. RESULTS: The mean percentage of nonperfused area was 0.1% (95% confidence interval: 0.0-0.4) in the eyes without DR; 2.1% (95% confidence interval: 1.2-3.7) in the nonproliferative DR eyes (mild, moderate, and severe), and 8.5% (95% confidence interval: 5.0-14.3) in the proliferative DR eyes. The percentage of nonperfused area increased in a statistically significant manner from eyes without DR, to eyes with nonproliferative DR, to eyes with proliferative DR. CONCLUSION: Capillary nonperfusion area in the posterior retina increases with increasing DR severity as measured by swept-source OCTA. Quantitative analysis of retinal nonperfusion on wide-field OCTA may be useful for early detection and monitoring of disease in patients with diabetes and DR.

SPATIAL DISTRIBUTION OF CHORIOCAPILLARIS IMPAIRMENT IN EYES WITH CHOROIDAL NEOVASCULARIZATION SECONDARY TO AGE-RELATED MACULAR DEGENERATION: A Quantitative OCT Angiography Study.

PURPOSE: To develop an optical coherence tomography angiography (OCTA)-based framework for quantitatively analyzing the spatial distribution of choriocapillaris (CC) impairment around choroidal neovascularization (CNV) secondary to age-related macular degeneration. METHODS: In a retrospective, cross-sectional study, 400-kHz swept-source OCTA images from 7 eyes of 6 patients with CNV secondary to age-related macular degeneration were quantitatively analyzed using custom software. A lesion-centered zonal OCTA analysis technique-which portioned the field-of-view into zones relative to CNV boundaries-was developed to quantify the spatial dependence of CC flow deficits. RESULTS: Quantitative, lesion-centered zonal analysis of CC OCTA images revealed highest flow-deficit percentages near CNV boundaries, decreasing in zones farther from the boundaries. Optical coherence tomography angiography using shorter (1.5 ms) interscan times revealed more severe flow deficits than OCTA using longer (3.0 ms) interscan times; however, spatial trends were similar for both interscan times. A detailed description of the OCTA processing steps and parameters was provided so as to elucidate their influence on quantitative measurements. CONCLUSION: Impairment of the CC, assessed by flow-deficit percentages, was most prominent closest to CNV boundaries. The lesion-centered zonal analysis technique enabled quantitative CC measurements relative to focal lesions. Understanding how processing steps, imaging/processing parameters, and artifacts can affect quantitative CC measurements is important for longitudinal, OCTA-based studies of disease progression, and treatment response.

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.

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.

Quantitative optical coherence tomography angiography of vascular abnormalities in the living human eye.

Retinal vascular diseases are important causes of vision loss. A detailed evaluation of the vascular abnormalities facilitates diagnosis and treatment in these diseases. Optical coherence tomography (OCT) angiography using the highly efficient split-spectrum amplitude decorrelation angiography algorithm offers an alternative to conventional dye-based retinal angiography. OCT angiography has several advantages, including 3D visualization of retinal and choroidal circulations (including the choriocapillaris) and avoidance of dye injection-related complications. Results from six illustrative cases are reported. In diabetic retinopathy, OCT angiography can detect neovascularization and quantify ischemia. In age-related macular degeneration, choroidal neovascularization can be observed without the obscuration of details caused by dye leakage in conventional angiography. Choriocapillaris dysfunction can be detected in the nonneovascular form of the disease, furthering our understanding of pathogenesis. In choroideremia, OCT's ability to show choroidal and retinal vascular dysfunction separately may be valuable in predicting progression and assessing treatment response. OCT angiography shows promise as a noninvasive alternative to dye-based angiography for highly detailed, in vivo, 3D, quantitative evaluation of retinal vascular abnormalities.

Optical Coherence Tomography-History, Evolution, and Future Prospects: 2023 Lasker-DeBakey Clinical Medical Research Award.

In this Viewpoint, 2023 Lasker-DeBakey Clinical Medical Research Award winners James G. Fujimoto, David Huang, and Eric A. Swanson discuss their invention­optical coherence tomography, which allows rapid detection of diseases of the retina that impair vision.

Optical Coherence Tomography Angiography Characteristics of Iris Melanocytic Tumors.

PURPOSE: To evaluate tumor vasculature with optical coherence tomography angiography (OCTA) in malignant iris melanomas and benign iris lesions. DESIGN: Cross-sectional observational clinical study. PARTICIPANTS: Patients with iris lesions and healthy volunteers. METHODS: Eyes were imaged using OCTA systems operating at 1050- and 840-nm wavelengths. Three-dimensional OCTA scans were acquired. Iris melanoma patients treated with radiation therapy were imaged again after I-125 plaque brachytherapy at 6 and 18 months. MAIN OUTCOME MEASURES: OCT and OCTA images, qualitative evaluation of iris and tumor vasculature, and quantitative vessel density. RESULTS: One eye each of 8 normal volunteers and 9 patients with iris melanomas or benign iris lesions, including freckles, nevi, and an iris pigment epithelial (IPE) cyst, were imaged. The normal iris has radially oriented vessels within the stroma on OCTA. Penetration of flow signal in normal iris depended on iris color, with best penetration seen in light to moderately pigmented irides. Iris melanomas demonstrated tortuous and disorganized intratumoral vasculature. In 2 eyes with nevi there was no increased vascularity; in another, fine vascular loops were noted near an area of ectropion uveae. Iris freckles and the IPE cyst did not have intrinsic vascularity. The vessel density was significantly higher within iris melanomas (34.5%±9.8%, P < 0.05) than in benign iris nevi (8.0%±1.4%) or normal irides (8.0%±1.2%). Tumor regression after radiation therapy for melanomas was associated with decreased vessel density. OCTA at 1050 nm provided better visualization of tumor vasculature and penetration through thicker tumors than at 840 nm. But in very thick tumors and highly pigmented lesions even 1050-nm OCTA could not visualize their full thickness. Interpretable OCTA images were obtained in 82% of participants in whom imaging was attempted. CONCLUSIONS: This is the first demonstration of OCTA in iris tumors. OCTA may provide a dye-free, no-injection, cost-effective method for monitoring a variety of tumors, including iris melanocytic lesions, for growth and vascularity. This could be helpful in evaluating tumors for malignant transformation and response to treatment. Penetration of the OCT beam remains a limitation for highly pigmented tumors, as does the inability to image the entire iris in a single field.

Multicolor lasers using birefringent filters: experimental demonstration with Cr:Nd:GSGG and Cr:LiSAF.

In this study, we numerically and experimentally investigate application of birefringent filters (BRFs) as frequency selective elements in multicolor lasers. A BRF plate made out of crystalline quartz with an arbitrarily oriented optical axis has been explored. Simulation results have shown that compared to regular BRFs where the optical axis lies in the plane of the plates surface, a BRF with an optical axis pointing out of its surface enables design flexibility in filter parameters, providing access to a wider set of free spectral range and bandwidth values. As a result, multicolor operation could be obtained in many wavelength pairs using a single BRF plate. In the experiments a 3-mm thick quartz BRF with an optical axis 45° to the surface plane has been used. With Cr:Nd:GSGG as a laser medium two-color and three-color cw laser operation has been demonstrated in 11 and 3 different transition combinations, respectively. Moreover, two-color laser operation has been demonstrated in 10 different wavelength pairs in Cr:LiSAF. To our knowledge, this study is the first detailed investigation and experimental demonstration of BRFs with tilted optical axis for multicolor operation of solid-state lasers. Compared to other methods, BRFs enable a rich selection of transition pairs and also the ratio of the power in each line could be regulated by fine adjustment of the rotation angle. Implementation of tilted-axis BRFs should boost development of efficient and low-cost multicolor lasers in other gain media as well.

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.

Endoscopic optical coherence tomography angiography microvascular features associated with dysplasia in Barrett's esophagus (with video).

BACKGROUND AND AIMS: Angiogenesis is associated with neoplastic progression of Barrett's esophagus (BE). Volumetric optical coherence tomography angiography (OCTA) visualizes subsurface microvasculature without exogenous contrast agents. We investigated the association of OCTA microvascular features with low-grade dysplasia (LGD) and high-grade dysplasia (HGD). METHODS: Fifty-two patients undergoing BE surveillance or endoscopic eradication therapies for dysplasia were imaged using volumetric OCTA and corresponding histologic diagnoses wre obtained to yield 97 data sets (nondysplastic BE [NDBE], 74; LGD, 10; HGD, 13). After evaluating OCTA image quality, 54 datasets (NDBE, 35; LGD, 8; HGD, 11) from 32 patients were used to develop a training and reading protocol. The association of abnormal vessel branching and heterogeneous vessel size with LGD/HGD and a regular honeycomb vessel pattern with NDBE were investigated. RESULTS: Blinded OCTA reading of 41 OCTA datasets (NDBE, 27; LGD, 7; HGD, 7) was performed by readers with various levels of OCT/OCTA experience including 3 OCT trainees, 1 gastroenterologist, and 2 gastroenterology fellows. Among the 6 readers, OCTA features of abnormal vessel branching and heterogeneous vessel size had an overall 94% sensitivity (95% CI, 89-99) and 69% specificity (95% CI, 62-76) for differentiating LGD/HGD versus NDBE with a mean reading time of 45 seconds per data set and moderate (kappa = .58) interobserver agreement. CONCLUSIONS: Volumetric en face OCTA imaging enables rapid examination of depth resolved microvascular features with near-microscopic resolution. OCTA can visualize microvascular features associated with LGD/HGD with high accuracy, which motivates new technologic advances and future studies investigating the diagnostic performance of OCTA.

ULTRAHIGH SPEED SWEPT SOURCE OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY OF RETINAL AND CHORIOCAPILLARIS ALTERATIONS IN DIABETIC PATIENTS WITH AND WITHOUT RETINOPATHY.

PURPOSE: To investigate the utility of ultrahigh speed, swept source optical coherence tomography angiography in visualizing retinal microvascular and choriocapillaris (CC) changes in diabetic patients. METHODS: The study was prospective and cross-sectional. A 1,050 nm wavelength, 400 kHz A-scan rate swept source optical coherence tomography prototype was used to perform volumetric optical coherence tomography angiography of the retinal and CC vasculatures in diabetic patients and normal subjects. Sixty-three eyes from 32 normal subjects, 9 eyes from 7 patients with proliferative diabetic retinopathy, 29 eyes from 16 patients with nonproliferative diabetic retinopathy, and 51 eyes from 28 diabetic patients without retinopathy were imaged. RESULTS: Retinal and CC microvascular abnormalities were observed in all stages of diabetic retinopathy. In nonproliferative diabetic retinopathy and proliferative diabetic retinopathy, optical coherence tomography angiography visualized a variety of vascular abnormalities, including clustered capillaries, dilated capillary segments, tortuous capillaries, regions of capillary dropout, reduced capillary density, abnormal capillary loops, and foveal avascular zone enlargement. In proliferative diabetic retinopathy, retinal neovascularization above the inner limiting membrane was visualized. Regions of CC flow impairment in patients with proliferative diabetic retinopathy and nonproliferative diabetic retinopathy were also observed. In 18 of the 51 of eyes from diabetic patients without retinopathy, retinal mircrovascular abnormalities were observed and CC flow impairment was found in 24 of the 51 diabetic eyes without retinopathy. CONCLUSION: The ability of optical coherence tomography angiography to visualize retinal and CC microvascular abnormalities suggests it may be a useful tool for understanding pathogenesis, evaluating treatment response, and earlier detection of vascular abnormalities in patients with diabetes.

100-nm tunable femtosecond Cr:LiSAF laser mode locked with a broadband saturable Bragg reflector.

We report broad tunability of a femtosecond (fs) diode-pumped Cr:LiSAF laser mode-locked with a broadband saturable Bragg reflector (SBR). The SBR had seven pairs of AlxOy(n∼1.5) and Al0.17Ga0.83As(n∼3.5) layers in its Bragg stack, enabling a 250 nm reflectivity bandwidth around 850 nm. A 6-nm-thick strained In0.15Ga0.85As quantum well placed between Al0.17Ga0.83As cladding layers was used as a broadband saturable absorber in the 800-920 nm wavelength range. The laser was pumped by 6 single mode diodes-four at 640 nn and two at 660 nm. Mode locking was self-starting and fs pulses could be continuously tuned from 800 nm to 905 nm by an intracavity birefringent filter with an out-of-plane optic axis. The pulse widths varied from 70 fs to 255 fs as the laser was tuned. The laser had an 85.5 MHz repetition rate and the output power varied from 80 mW to 180 mW with tuning.

Assessment of breast pathologies using nonlinear microscopy.

Rapid intraoperative assessment of breast excision specimens is clinically important because up to 40% of patients undergoing breast-conserving cancer surgery require reexcision for positive or close margins. We demonstrate nonlinear microscopy (NLM) for the assessment of benign and malignant breast pathologies in fresh surgical specimens. A total of 179 specimens from 50 patients was imaged with NLM using rapid extrinsic nuclear staining with acridine orange and intrinsic second harmonic contrast generation from collagen. Imaging was performed on fresh, intact specimens without the need for fixation, embedding, and sectioning required for conventional histopathology. A visualization method to aid pathological interpretation is presented that maps NLM contrast from two-photon fluorescence and second harmonic signals to features closely resembling histopathology using hematoxylin and eosin staining. Mosaicking is used to overcome trade-offs between resolution and field of view, enabling imaging of subcellular features over square-centimeter specimens. After NLM examination, specimens were processed for standard paraffin-embedded histology using a protocol that coregistered histological sections to NLM images for paired assessment. Blinded NLM reading by three pathologists achieved 95.4% sensitivity and 93.3% specificity, compared with paraffin-embedded histology, for identifying invasive cancer and ductal carcinoma in situ versus benign breast tissue. Interobserver agreement was κ = 0.88 for NLM and κ = 0.89 for histology. These results show that NLM achieves high diagnostic accuracy, can be rapidly performed on unfixed specimens, and is a promising method for intraoperative margin assessment.

SWEPT-SOURCE OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY REVEALS CHORIOCAPILLARIS ALTERATIONS IN EYES WITH NASCENT GEOGRAPHIC ATROPHY AND DRUSEN-ASSOCIATED GEOGRAPHIC ATROPHY.

PURPOSE: To investigate choriocapillaris (CC) alteration in patients with nascent geographic atrophy (nGA) and/or drusen-associated geographic atrophy (DAGA) using swept-source optical coherence tomography angiography (OCTA). METHODS: A 1,050-nm wavelength, 400 kHz A-scan rate swept-source optical coherence tomography prototype was used to perform volumetric swept-source optical coherence tomography angiography over 6 mm × 6 mm fields of view in patients with nGA and/or DAGA. The resulting optical coherence tomography (OCT) and OCTA data were analyzed using a combination of en face and cross-sectional techniques. Variable interscan time analysis (VISTA) was used to differentiate CC flow impairment from complete CC atrophy. RESULTS: A total of 7 eyes from 6 patients (mean age: 73.8 ± 5.7 years) were scanned. Seven areas of nGA and three areas of DAGA were identified. Analysis of cross-sectional OCT and OCTA images identified focal alterations of the CC underlying all seven areas of nGA and all three areas of DAGA. En face OCTA analysis of the CC revealed diffuse CC alterations in all eyes. Variable interscan time analysis processing suggested that the observed CC flow alterations predominantly corresponded to flow impairment rather than complete CC atrophy. CONCLUSION: The OCTA imaging of the CC revealed focal CC flow impairment associated with areas of nGA and DAGA, as well as diffuse CC flow impairment throughout the imaged field. En face OCT analysis should prove useful for understanding the pathogenesis of nGA and DAGA and for identifying the formation of nGA and DAGA as endpoints in therapeutic trials.

AN AUTOMATIC, INTERCAPILLARY AREA-BASED ALGORITHM FOR QUANTIFYING DIABETES-RELATED CAPILLARY DROPOUT USING OPTICAL COHERENCE TOMOGRAPHY ANGIOGRAPHY.

PURPOSE: To develop a robust, sensitive, and fully automatic algorithm to quantify diabetes-related capillary dropout using optical coherence tomography (OCT) angiography (OCTA). METHODS: A 1,050-nm wavelength, 400 kHz A-scan rate swept-source optical coherence tomography prototype was used to perform volumetric optical coherence tomography angiography imaging over 3 mm × 3 mm fields in normal controls (n = 5), patients with diabetes without diabetic retinopathy (DR) (n = 7), patients with nonproliferative diabetic retinopathy (NPDR) (n = 9), and patients with proliferative diabetic retinopathy (PDR) (n = 5); for each patient, one eye was imaged. A fully automatic algorithm to quantify intercapillary areas was developed. RESULTS: Of the 26 evaluated eyes, the segmentation was successful in 22 eyes (85%). The mean values of the 10 and 20 largest intercapillary areas, either including or excluding the foveal avascular zone, showed a consistent trend of increasing size from normal control eyes, to eyes with diabetic retinopathy but without diabetic retinopathy, to nonproliferative diabetic retinopathy eyes, and finally to PDR eyes. CONCLUSION: Optical coherence tomography angiography-based screening and monitoring of patients with diabetic retinopathy is critically dependent on automated vessel analysis. The algorithm presented was able to automatically extract an intercapillary area-based metric in patients having various stages of diabetic retinopathy. Intercapillary area-based approaches are likely more sensitive to early stage capillary dropout than vascular density-based methods.