This report describes an integrated multimodality imaging system that combines PAM, optical coherence tomography OCT , and fluorescence microscopy FM to evaluate angiogenesis in larger animal eyes. High-resolution in vivo imaging was performed in live rabbit eyes with vascular endothelial growth factor VEGF -induced retinal neovascularization RNV.
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The results demonstrate that our multimodality imaging system can non-invasively visualize RNV in both albino and pigmented rabbits to determine retinal pathology using PAM and OCT and verify the leakage of neovascularization using FM and fluorescein dye. This work presents high-resolution visualization of angiogenesis in rabbits using a multimodality PAM, OCT, and FM system and may represent a major step toward the clinical translation of the technology. Retinal neovascularization RNV represents a major cause of vision loss and blindness and is a common complication of numerous retinal diseases, including proliferative diabetic retinopathy, retinopathy of prematurity, sickle cell retinopathy, and retinal vein occlusions 1 , 2 , 3 , 4 , 5.
FA permits visualization of retinal circulation in detail, but its role in studying choroidal circulation is limited due to free permeation of fluorescein in choroidal vessels. However, OCTA does not exhibit leakage, provides limited visualization of microaneurysms, and has a restricted field of view, often with artifacts. This suggests that a multimodality optical imaging approach that can combine the advantages of OCT with additional functional and molecular information would be very beneficial in the field of ophthalmology As a hybrid biomedical imaging method, photoacoustic microscopy PAM has the unique capability to non-invasively explore the optical absorption properties in biological tissues with high spatial and temporal resolution PAM has been widely used in preclinical research, including studies of tumor 12 , brain 13 , 14 , bone 15 , liver 16 , and joint tissues A nanosecond-pulse-duration laser is used to achieve localized thermoelastic expansion of the target tissue.
This causes acoustic waves to be emitted from the target area, which can be detected with ultrasound transducers and reconstructed to obtain photoacoustic imaging with high contrast and resolution Due to the optical transparency, the eye and retina are considered very suitable for the application of PAM 19 , 20 , 21 , A multimodality imaging system with integrated OCT and PAM used to evaluate normal rabbit eyes has recently been described 23 , Whereas PAM has been employed in corneal neovascularization 25 and tumor microvasculature 26 , high-resolution PAM retinal imaging, particularly in large animal eyes, faces numerous technical challenges, including ultrasound signal attenuation with distance, particularly for high-frequency components, corneal dehydration, and optical aberrations, and thus most studies have been limited to mice and rats.
In Fig. Both the main vessels and vascular branches in the rabbit retina can be easily distinguished using PAM. FM images were acquired after PAM. Fluorescein sodium was used as a dye to obtain the FM image with nm incident light. After the laser finished scanning the area, the fluorescence signals obtained at each point were combined to reconstruct an FM image, which is shown in Fig.
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Although, we can distinguish the retinal vessels in the FM image, the strong background signal from dense choroidal vessels deceased the contrast of the image. The cross-section of the main vessels and the vascular branches of the retinal vasculature are indicated by white arrows. After removing the signal from the choroidal layer, the photoacoustic signal acquired from retinal vessels was reconstructed into 3D imaging see Video. The 3D structure of the normal retinal vessels is shown in Fig.
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The same images were acquired with pigmented rabbits in vivo to determine whether melanin would impede the acquisition of high-quality images. Melanin has a broad absorption peak, and its proximity to the vasculature previously caused difficulty in performing PAM imaging.
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In addition, melanin from the retinal pigment epithelium RPE overlies the choroid and choriocapillaris and thus could possibly block the diffuse choroidal hyperfluorescence since fluorescein is freely permeable in choroidal vessels. As shown in Fig. The corresponding FA image for the same area, shown in Fig. The PAM image shows that the retinal vasculature is visible more clearly than for the albino rabbit Fig. The FM image also has improved contrast compared to the albino rabbit due to less choroidal hyperfluorescence, which allows improved visualization of the microvasculature Fig.
In addition, the cross-section of the retinal vessels shown in the OCT image corresponds to the photoacoustic image. This demonstrates not only that high-quality PAM, OCT, and FM of the retinal vasculature is possible with pigmented rabbits but also that the melanin improves the image quality of all 3 modalities.
According to the fundus image in Fig. Whereas the myelinated nerve fibers of the medullary ray previously caused a white streak across the fundus photograph, a network of small, irregular neovascular vessels now covers it, changing the white streak to red.
The FA image in Fig.
The normal, organized retinal vasculature becomes a dense bundle of tortuous blood vessels, indicating that the normal retinal blood vessels are surrounded by RNV. Compared with the normal retinal vasculature without VEGF injection with clearly demarcated vessels, RNV, in contrast, leads to difficulty in distinguishing the blood vessel branches and main vessel.
For the FM image shown in Fig. The leakage of fluorescein sodium from RNV induces a strong diffuse fluorescence signal through the field of view. In addition, fluorescence from the choroidal layer also causes diffuse background fluorescence. As a result of this diffuse leakage from both vascular supplies, the contrast between retinal vessels and the background was too low to be detected by the digitizer.
Although the preretinal fibrovascular tissue covers the retinal vasculature, which will block most of the OCT light, the corresponding irregular structures were shown clearly in the OCT image in Fig. Normal vasculature has thicker, more mature blood vessel walls than RNV, and thus, normal vasculature has more shadowing below it than RNV, as revealed by the OCT image. The vascular structures with less shadowing below them indicate RNV.
Via comparison with the normal retinal vessels in Fig. To eliminate the influence from the choroidal layer, the photoacoustic signal from the retinal layer was exacted from the raw data to perform a 3D reconstruction see Video. To ensure clinical translatability given the significant melanin present in most human eyes, a similar study was performed with pigmented rabbits. Similar to the albino rabbits, pigmented rabbits have a significant increase in vascular tortuosity, which can be distinguished in the PAM image shown in Fig.
Some strong photoacoustic signals around the normal retinal vessels can also be detected; these represent small, irregular neovascularization indicated by green arrows. Although a dense network of tortuous neovascularization grew around the retinal blood vessel layer, PAM still can distinguish each individual vessel.
These OCT images also reveal a network of blood vessels on the inner retinal surface, shown in Fig. FM demonstrates significant progressive hyperfluorescence with blurred margins, consistent with leakage of fluorescein dye, shown in Fig. After we remove the photoacoustic signal from the choroidal layer, 3D reconstruction was performed to obtain a 3D view of the retinal vasculature see Video.
According to the image shown in Fig. For a 7. The real-time PAM B-scan image was displayed with a 3. Due to the selective absorption of hemoglobin, only the cross-section of retinal and choroidal blood vessel can be observed in the PAM B-scan imaging.
The individual retinal blood vessel can be easily distinguished. As the density of choroidal vessels is higher in the choroidal layer than in the retinal layers, the choroidal layer looks more continuous than the retinal layers. In contrast, the retinal blood vessel can still be distinguished clearly in the real-time PAM B-scan image. Points of correspondence between the two sets of images were determined from the PAM images with normal retinal vessels and neovascularization.
Then, the two images were overlaid in different color bands as a composite pseudo color image. Due to the high resolution of PAM and its alternative sensitivity to hemoglobin, PAM has the ability to quantify the RNV by extracting the retinal vessels from different layers and calculating the distribution of neovascularization in the region of interest ROI.
Rather than quantifying the RNV by using the area occupied by the leakage of fluorescein sodium on angiography, as is conventionally performed 27 , PAM can quantify neovascularization by using the number of pixels occupied by RNV at a pixel level, which is more accurate than the conventional fluorescein angiographic method, which is very dependent on the time after fluorescein instillation that images are acquired.
The fill factor of the retinal vessels, which was defined as the percentage of retinal vessels in a special ROI, was applied to quantify the growth of RNV using following equation:. Pseudo color indicates the normal retinal vessels; grayscale shows the retinal vessels after injection with VEGF. The fill factors of normal retinal vessels shown in Fig. The fill factors in pigmented rabbits shown in Fig. ImageJ was used to measure the vessel sizes of the main vessels, vessel branches, and neovascularization in the same area to further quantify the differences between normal retinal vessels and VEGF-induced RNV.
Three different locations distal, middle, and proximal of each vessel in the yellow dashed boxes were manually measured, and the measurements were averaged to minimize the measurement error.agendapop.cl/wp-content/apps/coce-hackear-instagram-no.php
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All the vessels in the yellow dashed boxes were involved in the statistical calculations. As demonstrated by the presence of vascular endothelial cells and the presence of red blood cells, the irregular small vessels were RNV induced by VEGF injections. Vascular cell nuclei on the inner retinal surface represent areas of RNV and are indicated with green arrows in Fig. The histology results were consistent with the results from the multimodality imaging system.
Green arrows indicate regions of RNV with an abnormal increase in vascular cells present on the inner retinal surface. Compared with previous studies involving small animals, mostly focused on the evaluation of the anterior ocular vasculatures, the proposed multimodal system was uniquely designed for retinas of larger animal eyes, not only for structural imaging but also for functional and molecular imaging.
A telecentric SL LSMBB, Thorlabs, Newton, NJ , which will produce a flat image plane and a spot size that suffers minimal distortion, was utilized to achieve a constant spot size in the focal plane of the telescope configuration. In addition, this system was designed to have high compatibility with multiple wavelengths for functional PAM imaging and molecular imaging utilizing different dyes through the sharing of a tunable optical parametric oscillator OPO laser system for both PAM and FM imaging.
Multimodality imaging provides unique advantages to visualize the anatomy and pathophysiology of diseases. OCT creates an image based on low-coherence interferometry and analyzes the difference in back-scattered light, comparing the tissue to a reference arm. OCT B-scan imaging allows for excellent visualization of the different retinal layers.
Due to the preretinal fibrovascular tissue above the retinal vasculature, OCT has difficulty in distinguishing normal vasculature and neovascularization even with the difference in the blood vessel wall between normal retinal vessels and neovascularization. Melanin and hemoglobin are the two primary endogenous absorbers in the eye.
Working within the optical spectrum sensitive to hemoglobin, PAM can be used to image not only blood vessels, but also bleeding. Although the vessel size and blood flow in neovascularization are smaller than those of normal vessels, PAM is still sufficiently sensitive to detect neovascularization and the network of small, irregular vessels.
Importantly, this study demonstrates that whereas melanin results in a strong PAM signal, the high-resolution nature of this system allows for visualization of neovascularization even in pigmented rabbits. In this multimodality system, FM can also be performed using intravenous injection of fluorescein. High contrast is achieved with FM in pigmented rabbits, where melanin in the RPE blocks the light from the choroid and choriocapillaris vasculature.
The FM images of albino rabbits suffer from diffuse hyperfluorescence, limiting visualization of the RNV, and FM in pigmented rabbits exhibits improved visualization of retinal vasculature and RNV. Multimodality imaging, which can combine the merits and compensate the limitations of each modality to give additional information that cannot be gleaned from a single modality, will be very beneficial in the field of ophthalmology. This study combines PAM, OCT, and FM and presents high-quality visualization of the retinal vasculature and neovascularization in albino and pigmented rabbits in vivo.
This is the first study to perform multimodal imaging, particularly PAM imaging, of RNV in rabbit eyes, and it demonstrates that high-quality, high-resolution images can be achieved below the ANSI safety limit even with the presence of melanin. The current study is also the first that has involved a neovascularization disease model of the retina in large animal eyes with multimodality high-resolution PAM, OCT, and FM imaging and demonstrates in 3D the spatial distribution of the retinal vasculature and neovascularization.
As the PAM signal is attenuated with distance from the detector, the rabbit axial length of Thus, this work presents a significant step in the clinical translation of this technology. The success achieved in this work demonstrates that PAM could play an important role in the diagnosis of RNV-related diseases, such as diabetic retinopathy, sickle cell retinopathy, retinal vein occlusions, and retinopathy of prematurity.
Thus, the blood flow velocity is not a limitation in PAM angiographic imaging.