Erik Mason

In Vivo Imaging and Morphometry of the Human Pre-Descemet's Layer and Endothelium With Ultrahigh-Resolution Optical Coherence Tomography.

PURPOSE To visualize in vivo and quantify the thickness of the posterior corneal layers: the acellular pre-Descemets layer (PDL), Descemets membrane (DM), and endothelium (END) in healthy subjects, using ultrahigh-resolution optical coherence tomography (UHR-OCT). METHODS A research-grade, 800-nm UHR-OCT system with 0.95-μm axial resolution in corneal tissue was used to image in vivo the posterior cornea in healthy subjects. The system offers approximately 98 dB sensitivity for 680 μW optical power incident on the cornea and 34,000 A-scans/s image acquisition rate. This study comprised 20 healthy subjects, aged 20 to 60 years. The thickness of the PDL, DM, and END layers was measured both with a custom, automatic segmentation algorithm and manually. RESULTS The boundaries and structure of the posterior corneal layers were clearly visible in the UHR-OCT images. The average thickness was measured to be 6.6 ± 1.4 μm (PDL), 10.4 ± 2.9 μm (DM), and 4.8 ± 0.4 μm (END), which agrees well with published data from ex vivo studies. Both the END and DM thickness showed minor spatial variations, whereas the PDL showed up to 2× thickness change for different locations on the same cross-sectional corneal image or over the entire imaged region of the cornea. CONCLUSIONS Our data indicate that all three layers of the posterior cornea can be clearly visualized in vivo and their thicknesses measured precisely with UHR-OCT. Although the PDL thickness showed large spatial variations, the thickness of the DM and END layers was consistent over the entire imaged region of the cornea.

Correlation of Visually Evoked Functional and Blood Flow Changes in the Rat Retina Measured With a Combined OCT+ERG System

Purpose To correlate visually evoked functional and blood flow changes in the rat retina measured simultaneously with a combined optical coherence tomography and electroretinography system (OCT+ERG). Methods Male Brown Norway (n = 6) rats were dark adapted and anesthetized with ketamine/xylazine. Visually evoked changes in the retinal blood flow (RBF) and functional response were measured simultaneously with an OCT+ERG system with 3-μm axial resolution in retinal tissue and 47-kHz image acquisition rate. Both single flash (10 and 200 ms) and flicker (10 Hz, 20% duty cycle, 1- and 2-second duration) stimuli were projected onto the retina with a custom visual stimulator, integrated into the OCT imaging probe. Total axial RBF was calculated from circular Doppler OCT scans by integrating over the arterial and venal flow. Results Temporary increase in the RBF was observed with the 10- and 200-ms continuous stimuli (∼1% and ∼4% maximum RBF change, respectively) and the 10-Hz flicker stimuli (∼8% for 1-second duration and ∼10% for 2-second duration). Doubling the flicker stimulus duration resulted in ∼25% increase in the RBF peak magnitude with no significant change in the peak latency. Single flash (200 ms) and flicker (10 Hz, 1 second) stimuli of the same illumination intensity and photon flux resulted in ∼2× larger peak RBF magnitude and ∼25% larger RBF peak latency for the flicker stimulus. Conclusions Short, single flash and flicker stimuli evoked measureable RBF changes with larger RBF magnitude and peak latency observed for the flicker stimuli.

Structural, functional and blood perfusion changes in the rat retina associated with elevated intraocular pressure, measured simultaneously with a combined OCT+ERG system

Acute elevation of intraocular pressure (IOP) to ischemic and non-ischemic levels can cause temporary or permanent changes in the retinal morphology, function and blood flow/blood perfusion. Previously, such changes in the retina were assessed separately with different methods in clinical studies and animal models. In this study, we used a combined OCT+ ERG system in combination with Doppler OCT and OCT angiography (OCTA) imaging protocols, in order to evaluate simultaneously and correlate changes in the retinal morphology, the retinal functional response to visual stimulation, and the retinal blood flow/blood perfusion, associated with IOP elevation to ischemic and non-ischemic levels in rats. Results from this study suggest that the inner retina responds faster to IOP elevation to levels greater than 30 mmHg with significant reduction of the total retinal blood flow (TRBF), decrease of the capillaries’ perfusion and reduction of the ON bipolar cells contribution to the ERG traces. Furthermore, this study showed that ischemic levels of IOP elevation cause an additional significant decrease in the ERG photoreceptor response in the posterior retina. Thirty minutes after IOP normalization, retinal morphology, blood flow and blood perfusion recovered to baseline values, while retinal function did not recover completely.

The Effect of Acutely Elevated Intraocular Pressure on the Functional and Blood Flow Responses of the Rat Retina to Flicker Stimulation

Purpose To evaluate the effect of acutely elevated intraocular pressure (IOP) on the functional and blood flow responses of the rat retina to flicker stimulation. Methods Brown Norway (n = 15) rats were dark-adapted before ketamine/xylazine anesthesia. IOP was raised acutely in one eye to ∼45 mm Hg with a vascular loop. In 11 rats, white light flicker stimulus (10 Hz, 2 seconds duration, 0.80 log scotopic cd·s/m2) was applied before and during IOP elevation, and 10 minutes after loop removal. Changes in the total retinal blood flow (TRBF) and retinal function induced by the visual stimulus were measured simultaneously with a combined optical coherence tomography (OCT) + electroretinography (ERG) system. Systemic blood pressure was measured in the remaining four rats frequently from 10 to 90 minutes post anesthesia injection. Results The systemic blood pressure remained at 99 ± 4 mm Hg throughout the measurements (n = 4). Under normal IOP, the TRBF was 5.6 ± 1.9 μL/min, and the average retinal blood vessel size (BVS) in the vicinity of the optic nerve head (ONH) was 44.1 ± 4.5 μm. During IOP elevation, the TRBF was significantly lower (3.8 ± 1.2 μL/min, P < 0.01) and the BVS was significantly smaller (35.1 ± 2.6 μm, P < 0.01). Both TRBF and BVS returned to baseline within ∼10 minutes from removal of the vascular loop. The flicker-induced TRBF change measured under normal IOP (6.0 ± 3.3%) was reduced significantly to 0.1 ± 0.3% (P < 0.01) during IOP elevation, and recovered to 5.9 ± 1.7% within 10 minutes after loop removal. During IOP elevation, the magnitude of the ERG second harmonic component (SHC) decreased to 55% of its baseline value (P < 0.01) and remained significantly smaller than baseline (P < 0.01). Conclusions Acute IOP elevation to 45 mm Hg caused suppression of the retinal functional and TRBF response to flicker stimulation.

In-vivo imaging of the palisades of Vogt and the limbal crypts with sub-micrometer axial resolution optical coherence tomography

A research-grade OCT system was used to image in-vivo and without contact with the tissue, the cellular structure and microvasculature of the healthy human corneo-scleral limbus. The OCT system provided 0.95 µm axial and 4 µm (2 µm) lateral resolution in biological tissue depending on the magnification of the imaging objective. Cross-sectional OCT images acquired tangentially from the inferior limbus showed reflective, loop-like features that correspond to the fibrous folds of the palisades of Vogt (POV). The high OCT resolution allowed for visualization of individual cells inside the limbal crypts, capillaries extending from the inside of the POVs fibrous folds and connecting to a lateral grid of micro-vessels located in the connective tissue directly below the POV, as well as reflections from individual red blood cells inside the capillaries. Difference in the reflective properties of the POV was observed among subjects of various pigmentation levels of the POV. Morphological features observed in the high resolution OCT images correlated well with histology. The ability to visualize the limbal morphology and microvasculature in-vivo at cellular level can aid the diagnostics and treatment of limbal stem cell dysfunction and dystrophies.

Visually evoked changes in the rat retinal blood flow measured with Doppler optical coherence tomography (Conference Presentation)

Visually evoked changes of retinal blood flow can serve as an important research tool to investigate eye disease such as glaucoma and diabetic retinopathy. In this study we used a combined, research-grade, high-resolution Doppler OCT+ERG system to study changes in the retinal blood flow (RBF) and retinal neuronal activity in response to visual stimuli of different intensities, durations and type (flicker vs single flash). Specifically, we used white light stimuli of 10 ms and 200 ms single flash, 1s and 2s for flickers stimuli of 20% duty cycle. The study was conducted in-vivo in pigmented rats. Both single flash (SF) and flicker stimuli caused increase in the RBF. The 10 ms SF stimulus did not generate any consistent measurable response, while the 200 ms SF of the same intensity generated ~4% change in the RBF peaking at ~1.5 s after the stimulus onset. Single flash stimuli introduced ~2x smaller change in RBF and ~30% earlier RBF peak response compared to flicker stimuli of the same intensity and duration. Doubling the intensity of SF or flicker stimuli increased the RBF peak magnitude by ~1.5x. Shortening the flicker stimulus duration by 2x increased the RBF recovery rate by 2x, however, had no effect on the rate of RBF change from baseline to peak.

Cellular structure of the healthy and keratoconic human cornea imaged in-vivo with sub-micrometer axial resolution OCT(Conference Presentation)

Keratoconus causes progressive morphological changes in the corneal epithelium (EPI), Bowman’s membrane (BM) and anterior stroma. However, it is still not well understood if KC originates in the corneal epithelium and propagates to the anterior stroma through disruptions of the BM, or vice versa. In this study we used a sub-micrometer axial resolution OCT system to image in-vivo the cellular structure of the EPI layer and the fibrous structure of the BM and the anterior stroma in mild to advanced keratoconics, as well as healthy subjects. The imaging study was approved by the University of Waterloo Human Research Ethics Committee. The OCT system operates in the 800 nm spectral region at 34 kHz image acquisition rate and provides 0.95 um axial and < 2 um lateral resolution in corneal tissue, which is sufficient to visualize the cellular structure of the corneal epithelium and the fibrous structure of the BM. In some subjects, localized thinning and thickening of the EPI layer was observed, while there was no visible damage to the BM or anterior stroma. In other subjects, localized breakage of the stromal collagen fibrils was observed with no significant morphological changes of the corneal EPI.

In-vivo imaging of the morphology and blood perfusion of brain tumours in rats with UHR-OCT (Conference Presentation)

Brain tumors are characterized with morphological changes at cellular level such as enlarged, non-spherical nuclei, microcalcifications, cysts, etc., and are highly vascularized. In this study, two research-grade optical coherence tomography (OCT) systems operating at ~800 nm and ~1060 nm with axial resolution of 0.95 µm and 3.5 µm in biological tissue respectively, were used to image in vivo and ex vivo the structure of brain tumours in rats. Female Fischer 344 rats were used for this study, which has received ethics clearance by the Animal Research Ethics Committees of the University of Waterloo and the University Health Network, Toronto. Brain tumours were induced by injection of rat brain cancer cell line (RG2 glioma) through a small craniotomy. Presence of brain tumours was verified by MRI imaging on day 7 post tumour cells injection. The in vivo OCT imaging session was conducted on day 14 of the study with the 1060 nm OCT system and both morphological OCT, Doppler OCT and OMAG images were acquired from the brain tumour and the surrounding healthy brain tissue. After completion of the imaging procedure, the brains were harvested, fixed in formalin and reimaged after 2 weeks with the 800 nm OCT system. The in vivo and ex vivo OCT morphological images were correlated with H and E histology. Results from this study demonstrate that UHR-OCT can distinguish between healthy and cancerous brain tissue based on differences in structural and vascular pattern.