Photoacoustic microscopy (PAM) can be an effective imaging modality to visualize retinal vein occlusion during laserinduced photocoagulation on major retinal veins. Developments in the vessels could be observed before and after laser irradiation due to the change of the optical absorption spectrum of the target vessels. However, the suitable wavelength to achieve high contrast PAM images of occluded vessels is unclear. This study evaluates the effect of wavelength on PAM imaging to evaluate the photocoagulation lesions on the rabbit to optimize the wavelength for imaging. Retinal vein occlusion (RVO) was created using a 532 nm millisecond pulse duration green light with concurrent intravenous administration of Rose Bengal (5 mg/kg) in New Zealand rabbits. Imaging was acquired by the PAM system at various wavelengths ranging from 520 nm to 590 nm. In addition, the thermal lesion was also confirmed using optical coherence tomography (OCT). A group of 20 retinal veins was irradiated for 0.5 s at a laser fluence of 850 W/cm2 (power = 150 mW, beam diameter = 75 μm). Twenty shots of the laser were applied to each major vein. PAM results showed that the thermal lesion was obviously visualized and exhibited lower contrast in comparison with untreated vessels posttreatment. Photoacoustic spectroscopy exhibited that the highest PA contrast of vessels treated with Rose Bengal laserinduced RVO occurred at a wavelength of 563, 570, and 578 nm, which was higher than the PA amplitude at lower and longer wavelengths respectively. The use of multi-wavelength PAM can provide a better method for visualization and evaluation of retinal vein occlusions.
Spectral domain optical coherence tomography (SD-OCT) and photoacoustic microscopy (PAM) were integrated to improve the visualization of retinal vein occlusion (RVO) and retinal neovascularization (RNV). RVO and RNV were observed in living, New Zealand rabbits by Rose Bengal laser-induced retinal vein thrombosis. Multimodal imaging techniques including PAM, OCT, fluorescein angiography (FA), and color fundus photographs were utilized to observe and analyze changes in the retinal vasculature. Spectral domain OCT identified RNV cross-sectional structures, and progressive changes in retinal anatomy due to angiogenesis from the photothermal treatment were monitored at 4, 28, 35, 49, and 90 days post-laser. Compared to alternative methods, PAM in vivo high-contrast imaging possesses the capabilities to accurately visualize the treatment margins of occluded vasculature and areas of RNV. Results were obtained using a laser energy of 80 nJ, which is half a dose below the American National Standards Institute safety limit. The PAM system also demonstrated an increased depth of penetration, which provided high resolution images of the choroid and retinal vasculature when the optical absorption of hemoglobin was used to improve the visualize blood flow. Current modalities of imaging possess the ability to visualize RNV and RVO in two-dimensional and three-dimensional angiography. However, the system integrating both PAM and OCT can better visualize the depth and position of microvessels as well as surrounding structures. This specific multimodal ocular imaging technique may therefore be an improved technology to document minute changes in rabbit eye retinal vasculature while proving to be both safe and efficient.
The current study investigates the beneficial combination of optical coherence tomography (OCT) and photoacoustic microscopy (PAM) as a safe method for observing retinal and choroidal vasculature. A recent addition to the field has been the integration of gold nanoparticles (AuNPs) to provide enhanced contrast in OCT and PAM images. The improved analysis of capillaries is the result of the strong optical scattering and optical absorption of gold nanoparticles due to surface plasmon resonance. Femtosecond laser ablation created the ultra-pure colloidal gold nanoparticles, which were then capped with polyethylene glycol (PEG). The AuNPs were administered to thirteen New Zealand rabbits to determine the advantages of this technology, while also investigating the safety and biocompatibility. The study determines that the synthesized PEG-AuNPs (20.0 ± 1.5 nm) were beneficial in enhancing contrast in PAM and OCT images without demonstrating cytotoxic effects to bovine retinal endothelial cells. In living rabbits, the administered PEG-AuNPs resulted in an 82% increased signal for PAM and a 45% increased signal for OCT in the retinal and choroidal vessels. A histology and biodistribution report determined that the AuNPs had mostly accumulated in the liver and spleen. TUNEL staining and histology established that no cell injury or death in the lung, liver, kidney, spleen, heart, or eyes had occurred up to 1 week after receiving a dose of AuNP. The nanoparticle technology, therefore, provides an effective and safe method to enhance contrast in ocular imaging, resulting in improved visualization of retinal microvasculature.
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