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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.

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.

We derive a physically realistic model for the generation of virtual transillumination, white light microscopy images using epi-fluorescence measurements from thick, unsectioned tissue. We demonstrate this technique by generating virtual transillumination H&E images of unsectioned human breast tissue from epi-fluorescence multiphoton microscopy data. The virtual transillumination algorithm is shown to enable improved contrast and color accuracy compared with previous color mapping methods. Finally, we present an open source implementation of the algorithm in OpenGL, enabling real-time GPU-based generation of virtual transillumination microscopy images using conventional fluorescence microscopy systems.

To characterize qualitative and quantitative features of the choroid in normal eyes using enface swept-source optical coherence tomography (SS-OCT).Fifty-two eyes of 26 consecutive normal subjects were prospectively recruited to obtain multiple three-dimensional 12 x 12 mm volumetric scans using a long-wavelength high-speed SS-OCT prototype. A motion-correction algorithm merged multiple SS-OCT volumes to improve signal. Retinal pigment epithelium (RPE) was segmented as the reference and enface images were extracted at varying depths every 4.13 μm intervals. Systematic analysis of the choroid at different depths was performed to qualitatively assess the morphology of the choroid and quantify the absolute thicknesses as well as the relative thicknesses of the choroidal vascular layers including the choroidal microvasculature (choriocapillaris, terminal arterioles and venules; CC) and choroidal vessels (CV) with respect to the subfoveal total choroidal thickness (TC). Subjects were divided into two age groups: younger (

To describe enhanced vitreous imaging for visualization of anatomic features and microstructures within the posterior vitreous and vitreoretinal interface in healthy eyes using swept-source optical coherence tomography (SS-OCT). The study hypothesis was that long-wavelength, high-speed, volumetric SS-OCT with software registration motion correction and vitreous window display or high-dynamic-range (HDR) display improves detection sensitivity of posterior vitreous and vitreoretinal features compared to standard OCT logarithmic scale display.Observational prospective cross-sectional study.Multiple wide-field three-dimensional SS-OCT scans (500×500A-scans over 12×12 mm2) were obtained using a prototype instrument in 22 eyes of 22 healthy volunteers. A registration motion-correction algorithm was applied to compensate motion and generate a single volumetric dataset. Each volumetric dataset was displayed in three forms: (1) standard logarithmic scale display, enhanced vitreous imaging using (2) vitreous window display and (3) HDR display. Each dataset was reviewed independently by three readers to identify features of the posterior vitreous and vitreoretinal interface. Detection sensitivities for these features were measured for each display method.Features observed included the bursa premacularis (BPM), area of Martegiani, Cloquet's/BPM septum, Bergmeister papilla, posterior cortical vitreous (hyaloid) detachment, papillomacular hyaloid detachment, hyaloid attachment to retinal vessel(s), and granular opacities within vitreous cortex, Cloquet's canal, and BPM. The detection sensitivity for these features was 75.0% (95%CI: 67.8%-81.1%) using standard logarithmic scale display, 80.6% (95%CI: 73.8%-86.0%) using HDR display, and 91.9% (95%CI: 86.6%-95.2%) using vitreous window display.SS-OCT provides non-invasive, volumetric and measurable in vivo visualization of the anatomic microstructural features of the posterior vitreous and vitreoretinal interface. The vitreous window display provides the highest sensitivity for posterior vitreous and vitreoretinal interface analysis when compared to HDR and standard OCT logarithmic scale display. Enhanced vitreous imaging with SS-OCT may help assess the natural history and treatment response in vitreoretinal interface diseases.

To examine the feasibility of automatically segmented choroidal vessels in three-dimensional (3D) 1060-nmOCT by testing repeatability in healthy and AMD eyes and by mapping Haller's and Sattler's layer thickness in healthy eyes.Fifty-five eyes (from 45 healthy subjects and 10 with non-neovascular age-related macular degeneration (AMD) subjects) were imaged by 3D-1060-nmOCT over a 36°x36° field of view. Haller's and Sattler's layer were automatically segmented, mapped and averaged across the Early Treatment Diabetic Retinopathy Study grid. For ten AMD eyes and ten healthy eyes, imaging was repeated within the same session and on another day. Outcomes were the repeatability agreement of Haller's and Sattler's layer thicknesses in healthy and AMD eyes, the validation with ICGA and the statistical analysis of the effect of age and axial eye length (AL) on both healthy choroidal sublayers.The coefficients of repeatability for Sattler's and Haller's layers were 35% and 21% in healthy eyes and 44% and 31% in AMD eyes, respectively. The mean±SD healthy central submacular field thickness for Sattler's and Haller's was 87±56 µm and 141±50 µm, respectively, with a significant relationship for AL (P

To determine the reproducibility of automated segmentation of the three-dimensional (3D) lamina cribrosa (LC) microarchitecture scanned in-vivo using optical coherence tomography (OCT).Thirty-nine eyes (8 healthy, 19 glaucoma suspects and 12 glaucoma) from 49 subjects were scanned twice using swept-source (SS-) OCT in a 3.5×3.5×3.64 mm (400×400×896 pixels) volume centered on the optic nerve head, with the focus readjusted after each scan. The LC was automatically segmented and analyzed for microarchitectural parameters, including pore diameter, pore diameter standard deviation (SD), pore aspect ratio, pore area, beam thickness, beam thickness SD, and beam thickness to pore diameter ratio. Reproducibility of the parameters was assessed by computing the imprecision of the parameters between the scans.The automated segmentation demonstrated excellent reproducibility. All LC microarchitecture parameters had an imprecision of less or equal to 4.2%. There was little variability in imprecision with respect to diagnostic category, although the method tends to show higher imprecision amongst healthy subjects.The proposed automated segmentation of the LC demonstrated high reproducibility for 3D LC parameters. This segmentation analysis tool will be useful for in-vivo studies of the LC.

We used optical coherence tomography (OCT) angiography with a high-speed swept-source OCT system to investigate retinal blood flow changes induced by visual stimulation with a reversing checkerboard pattern. The split-spectrum amplitude-decorrelation angiography (SSADA) algorithm was used to quantify blood flow as measured with parafoveal flow index (PFI), which is proportional to the density of blood vessels and the velocity of blood flow in the parafoveal region of the macula. PFI measurements were taken in 15 second intervals during a 4 minute period consisting of 1 minute of baseline, 2 minutes with an 8 Hz reversing checkerboard pattern stimulation, and 1 minute without stimulation. PFI measurements increased 6.1±4.7% (p = .001) during the first minute of stimulation, with the most significant increase in PFI occurring 30 seconds into stimulation (p

We demonstrate in vivo choriocapillaris and choroidal microvasculature imaging in normal human subjects using optical coherence tomography (OCT). An ultrahigh speed swept source OCT prototype at 1060 nm wavelengths with a 400 kHz A-scan rate is developed for three-dimensional ultrahigh speed imaging of the posterior eye. OCT angiography is used to image three-dimensional vascular structure without the need for exogenous fluorophores by detecting erythrocyte motion contrast between OCT intensity cross-sectional images acquired rapidly and repeatedly from the same location on the retina. En face OCT angiograms of the choriocapillaris and choroidal vasculature are visualized by acquiring cross-sectional OCT angiograms volumetrically via raster scanning and segmenting the three-dimensional angiographic data at multiple depths below the retinal pigment epithelium (RPE). Fine microvasculature of the choriocapillaris, as well as tightly packed networks of feeding arterioles and draining venules, can be visualized at different en face depths. Panoramic ultra-wide field stitched OCT angiograms of the choriocapillaris spanning ∼32 mm on the retina show distinct vascular structures at different fundus locations. Isolated smaller fields at the central fovea and ∼6 mm nasal to the fovea at the depths of the choriocapillaris and Sattler's layer show vasculature structures consistent with established architectural morphology from histological and electron micrograph corrosion casting studies. Choriocapillaris imaging was performed in eight healthy volunteers with OCT angiograms successfully acquired from all subjects. These results demonstrate the feasibility of ultrahigh speed OCT for in vivo dye-free choriocapillaris and choroidal vasculature imaging, in addition to conventional structural imaging.