Line-scanning SD-OCT for in-vivo, non-contact, volumetric, cellular resolution imaging of the human cornea and limbus.

In-vivo, non-contact, volumetric imaging of the cellular and sub-cellular structure of the human cornea and limbus with optical coherence tomography (OCT) is challenging due to involuntary eye motion that introduces both motion artifacts and blur in the OCT images. Here we present the design of a line-scanning (LS) spectral-domain (SD) optical coherence tomography system that combines 2 × 3 × 1.7 µm (x, y, z) resolution in biological tissue with an image acquisition rate of ∼2,500 fps, and demonstrate its ability to image in-vivo and without contact with the tissue surface, the cellular structure of the human anterior segment tissues. Volumetric LS-SD-OCT images acquired over a field-of-view (FOV) of 0.7 mm × 1.4 mm reveal fine morphological details in the healthy human cornea, such as epithelial and endothelial cells, sub-basal nerves, as well as the cellular structure of the limbal crypts, the palisades of Vogt (POVs) and the blood microvasculature of the human limbus. LS-SD-OCT is a promising technology that can assist ophthalmologists with the early diagnostics and optimal treatment planning of ocular diseases affecting the human anterior eye.

In-vivo functional and structural retinal imaging using multiwavelength photoacoustic remote sensing microscopy.

Many important eye diseases as well as systemic disorders manifest themselves in the retina. Retinal imaging technologies are rapidly growing and can provide ever-increasing amounts of information about the structure, function, and molecular composition of retinal tissue in-vivo. Photoacoustic remote sensing (PARS) is a novel imaging modality based on all-optical detection of photoacoustic signals, which makes it suitable for a wide range of medical applications. In this study, PARS is applied for in-vivo imaging of the retina and estimating oxygen saturation in the retinal vasculature. To our knowledge, this is the first time that a non-contact photoacoustic imaging technique is applied for in-vivo imaging of the retina. Here, optical coherence tomography is also used as a well-established retinal imaging technique to navigate the PARS imaging beams and demonstrate the capabilities of the optical imaging setup. The system is applied for in-vivo imaging of both microanatomy and the microvasculature of the retina. The developed system has the potential to advance the understanding of the ocular environment and to help in monitoring of ophthalmic diseases.

Functional photoacoustic remote sensing microscopy using a stabilized temperature-regulated stimulated Raman scattering light source.

Stimulated Raman scattering (SRS) has been widely used in functional photoacoustic microscopy to generate multiwavelength light and target multiple chromophores inside tissues. Despite offering a simple, cost-effective technique with a high pulse repetition rate; it suffers from pulse-to-pulse intensity fluctuations and power drift that can affect image quality. Here, we propose a new technique to improve the temporal stability of the pulsed SRS multiwavelength source. We achieve this by lowering the temperature of the SRS medium. The results suggest that a decrease in temperature causes an improvement of temporal stability of the output, considerable rise in the intensity of the SRS peaks, and significant increase of SRS cross section. The application of the method is shown for in vivo functional imaging of capillary networks in a chicken embryo chorioallantois membrane using photoacoustic remote sensing microscopy.

Three-dimensional virtual histology in unprocessed resected tissues with photoacoustic remote sensing (PARS) microscopy and optical coherence tomography (OCT).

Histological images are critical in the diagnosis and treatment of cancers. Unfortunately, current methods for capturing these microscopy images require resource intensive tissue preparation that may delay diagnosis for days or weeks. To streamline this process, clinicians are limited to assessing small macroscopically representative subsets of tissues. Here, a combined photoacoustic remote sensing (PARS) microscope and swept source optical coherence tomography system designed to circumvent these diagnostic limitations is presented. The proposed multimodal microscope provides label-free three-dimensional depth resolved virtual histology visualizations, capturing nuclear and extranuclear tissue morphology directly on thick unprocessed specimens. The capabilities of the proposed method are demonstrated directly in unprocessed formalin fixed resected tissues. The first images of nuclear contrast in resected human tissues, and the first three-dimensional visualization of subsurface nuclear morphology in resected Rattus tissues, captured with a non-contact photoacoustic system are presented here. Moreover, the proposed system captures the first co-registered OCT and PARS images enabling direct histological assessment of unprocessed tissues. This work represents a vital step towards the development of a rapid histological imaging modality to circumvent the limitations of current histopathology techniques.

Functional and structural ophthalmic imaging using noncontact multimodal photoacoustic remote sensing microscopy and optical coherence tomography.

Early diagnosis of ocular diseases improves the understanding of pathophysiology and aids in accurate monitoring and effective treatment. Advanced, multimodal ocular imaging platforms play a crucial role in visualization of ocular components and provide clinicians with a valuable tool for evaluating various eye diseases. Here, for the first time we present a non-contact, multiwavelength photoacoustic remote sensing (PARS) microscopy and swept-source optical coherence tomography (SS-OCT) for in-vivo functional and structural imaging of the eye. The system provides complementary imaging contrasts of optical absorption and optical scattering, and is used for simultaneous, non-contact, in-vivo imaging of murine eye. Results of vasculature and structural imaging as well as melanin content in the retinal pigment epithelium layer are presented. Multiwavelength PARS microscopy using Stimulated Raman scattering is applied to enable in-vivo, non-contact oxygen saturation estimation in the ocular tissue. The reported work may be a major step towards clinical translation of ophthalmic technologies and has the potential to advance the diagnosis and treatment of ocular diseases.

Label-free, non-contact, in vivo ophthalmic imaging using photoacoustic remote sensing microscopy.

We present, to the best of our knowledge, the first label-free, non-contact, in vivo imaging of the ocular vasculature using photoacoustic remote sensing (PARS) microscopy. Both anterior and posterior segments of a mouse eye were imaged. Vasculature of the iris, sclera, and retina tissues were clearly resolved. To the best of our knowledge, this is the first study showing non-contact photoacoustic imaging conducted on in vivo ocular tissue. We believe that PARS microscopy has the potential to advance the diagnosis and treatment of ocular diseases.

Towards non-contact photoacoustic imaging [review].

Photoacoustic imaging (PAI) takes advantage of both optical and ultrasound imaging properties to visualize optical absorption with high resolution and contrast. Photoacoustic microscopy (PAM) is usually categorized with all-optical microscopy techniques such as optical coherence tomography or confocal microscopes. Despite offering high sensitivity, novel imaging contrast, and high resolution, PAM is not generally an all-optical imaging method unlike the other microscopy techniques. One of the significant limitations of photoacoustic microscopes arises from their need to be in physical contact with the sample through a coupling media. This physical contact, coupling, or immersion of the sample is undesirable or impractical for many clinical and pre-clinical applications. This also limits the flexibility of photoacoustic techniques to be integrated with other all-optical imaging microscopes for providing complementary imaging contrast. To overcome these limitations, several non-contact photoacoustic signal detection approaches have been proposed. This paper presents a brief overview of current non-contact photoacoustic detection techniques with an emphasis on all-optical detection methods and their associated physical mechanisms.

Comparative Study of Optical Coherence Tomography Angiography and Phase-Resolved Doppler Optical Coherence Tomography for Measurement of Retinal Blood Vessels Caliber.

PURPOSE: To compare the accuracy of Doppler optical coherence tomography (DOCT) and OCT angiography (OCTA) for measuring retinal blood vessel caliber at different flow rates. METHODS: A research-grade 1060-nm OCT system with 3.5-µm axial resolution in retinal tissue and 92,000 A scan/s image acquisition rate was used in this study. DOCT and OCTA measurements were acquired both from a flow phantom and in vivo from retinal blood vessels in six male Brown Norway rats. The total retinal blood flow (TRBF) was modified from baseline to 70% and 20% of baseline by reducing the ocular perfusion pressure (OPP). The retinal blood vessel caliber (RBVC) was measured from OCTA and DOCT images. The caliber measurements were conducted by two separate graders using a custom MATLAB-based image processing algorithm. RESULTS: The RBVC measured with OCTA and DOCT for normal blood flow rates were not significantly different (56.69 ± 12.17 and 57.17 ± 9.46 µm, P = 0.27, respectively). However, significant differences were detected when TRBF was reduced to 70% (55.69 ± 11.56 vs. 50.62 ± 8.85 µm, P < 0.01) and 20% (50.29 ± 9.29 vs. 44.88 ± 7.13 µm, P < 0.01) of baseline. CONCLUSIONS: Reduced TRBF resulted in inaccuracy of the RBVC measurements with DOCT in both the phantom and animal study. This result suggests that OCTA is a more accurate tool for RBVC evaluation when applied to retinal diseases associated with reduced TRBF, such as glaucoma and diabetic retinopathy. TRANSLATIONAL RELEVANCE: Results from this study are directly applicable to clinical studies of retinal blood flow measured with OCTA and DOCT.

250 kHz, 1.5 µm resolution SD-OCT for in-vivo cellular imaging of the human cornea.

We present the first spectral domain optical coherence tomography (SD-OCT) system that combines an isotropic imaging resolution of ~1.5 µm in biological tissue with a 250 kHz image acquisition rate, for in vivo non-contact, volumetric imaging of the cellular structure of the human cornea. OCT images of the healthy human cornea acquired with this system reveal the cellular structure of the corneal epithelium, cellular debris and mucin clusters in the tear film, the shape, size and spatial distribution of the sub-basal corneal nerves and keratocytes in the corneal stroma, as well as reflections from endothelial nuclei. The corneal images presented here demonstrate the potential clinical value of the new high speed, high resolution OCT system for non-invasive diagnostics and monitoring the treatment of corneal diseases.

Dense concentric circle scanning protocol for measuring pulsatile retinal blood flow in rats with Doppler optical coherence tomography.

The variability in the spatial orientation of retinal blood vessels near the optic nerve head (ONH) results in imprecision of the measured Doppler angle and therefore the pulsatile blood flow (BF), when those parameters are evaluated using Doppler OCT imaging protocols based on dual-concentric circular scans. Here, we utilized a dense concentric circle scanning protocol and evaluated its precision for measuring pulsatile retinal BF in rats for different numbers of the circular scans. An spectral domain optical coherence tomography (SD-OCT) system operating in the 1060-nm spectral range with image acquisition rate of 47,000 A-scans/s was used to acquire concentric circular scans centered at the rat's ONH, with diameters ranging from 0.8 to 1.0 mm. A custom, automatic blood vessel segmentation algorithm was used to track the spatial orientation of the retinal blood vessels in three dimensions, evaluate the spatially dependent Doppler angle and calculate more accurately the axial BF for each major retinal blood vessel. Metrics such as retinal BF, pulsatility index, and resistance index were evaluated for each and all of the major retinal blood vessels. The performance of the proposed dense concentric circle scanning protocols was compared with that of the dual-circle scanning protocol. Results showed a 3.8±2.2 deg difference in the Doppler angle calculation between the two approaches, which resulted in ∼7% difference in the calculated retinal BF.

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 POV's 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.