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Retina  |   August 2013
Wide-Field Laser Ophthalmoscopy for Mice: A Novel Evaluation System for Retinal/Choroidal Angiogenesis in Mice
Author Affiliations & Notes
  • Shintaro Nakao
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Ryoichi Arita
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Takahito Nakama
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Hiroshi Yoshikawa
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Shigeo Yoshida
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Hiroshi Enaida
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Ali Hafezi-Moghadam
    Center for Excellence in Functional and Molecular Imaging, Brigham and Women's Hospital, and Department of Radiology, Harvard Medical School, Boston, Massachusetts
  • Takaaki Matsui
    Ohshima Hospital of Ophthalmology, Fukuoka, Japan
  • Tatsuro Ishibashi
    Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan
  • Correspondence: Shintaro Nakao, Department of Ophthalmology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan; snakao@med.kyushu-u.ac.jp
Investigative Ophthalmology & Visual Science August 2013, Vol.54, 5288-5293. doi:10.1167/iovs.13-11946
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      Shintaro Nakao, Ryoichi Arita, Takahito Nakama, Hiroshi Yoshikawa, Shigeo Yoshida, Hiroshi Enaida, Ali Hafezi-Moghadam, Takaaki Matsui, Tatsuro Ishibashi; Wide-Field Laser Ophthalmoscopy for Mice: A Novel Evaluation System for Retinal/Choroidal Angiogenesis in Mice. Invest. Ophthalmol. Vis. Sci. 2013;54(8):5288-5293. doi: 10.1167/iovs.13-11946.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose.: The purpose of this study was to investigate the application of wide-field laser ophthalmoscopy (Optos) for the evaluation of established models of angiogenesis and the healthy retina in mice.

Methods.: To investigate whether angiogenesis and leakage in the retina and choroid can be evaluated with Optos, we used two models of angiogenesis: oxygen-induced retinopathy (OIR) and laser-induced choroidal neovascularization (CNV). Fundus imaging and fluorescein angiography (FAG) were performed with the Optos system without a contact lens. Furthermore, to evaluate in vivo leukocyte infiltration in these models, we injected acridine orange (AO) and performed imaging using Optos.

Results.: In vivo fundus imaging with Optos did not require any additional optical device. Additionally, Optos enabled us to repeatedly obtain high-resolution color images and FAG images in the OIR model as well as in the CNV model in mice. Through a combination of Optos imaging and AO fluorography, the number and location of the infiltrating leukocytes could be identified in these models.

Conclusions.: Optos is a wide-viewing imaging tool for the noninvasive in vivo evaluation of common angiogenesis models, oxygen-induced retinopathy and laser-induced choroidal neovascularization, as well as the healthy retina in mice.

Introduction
Animal models are essential for understanding the pathogenesis of retinal and choroidal diseases. 1 Mice are the most widely used experimental animals, among other reasons because of their small size. 
Oxygen-induced retinopathy (OIR) is generally used in in vivo mouse models of retinal neovascular disease such as retinopathy of prematurity (ROP). 2 The retinal nonperfusion area, neovascular lesions, or leukocyte infiltration in the angiogenesis model is then commonly evaluated in flat mounts. 3  
Choroidal neovascularization (CNV) in animals was first discovered at ruptured sites of Bruch's membrane that were induced through laser photocoagulation. 4 Laser-induced CNV is commonly used because it resembles the lesions in age-related macular degeneration (AMD). This technique was then adapted to mice and used as in knockout and transgenic mice for mechanistic studies of wet AMD. 5 CNV lesions are also generally evaluated in flat mounts. Since flat mounts allow quantification of nonperfusion and neovascular areas only after enucleation, dynamic retinal or choroidal changes over time cannot be studied using this technique. 
The murine fundus has been studied using a confocal scanning laser ophthalmoscope (SLO). 6,7 The field of view could extend up to the ora serrata, but the image has to be compensated. 6  
Recently, an ultra-wide-field SLO called Optos 200TX (Optos PLC, Dunfermline, Scotland, UK) with a green (532 nm) and a red (633 nm) laser was developed. The green laser scans from the sensory retina to the pigment epithelial layers, and the red laser scans from the RPE to the choroid. The other features of the system consist of a wide angle (up to 200°), the ability to operate in nonmydriatic conditions, and the ability to perform fluorescein angiography (FAG) at 488 nm. 8 Optos provides ultra-wide-field retinal imaging that may first present itself in the periphery, which would remain undetected with other traditional techniques. The efficacy of Optos in diabetic retinopathy and rhegmatogenous retinal detachment has been reported. 911 In this study, we evaluated the usefulness of this novel technique in mice. 
Materials and Methods
Mice
C57BL/6J mice (aged 7–10 weeks and 4 days) were purchased from Kyudo Co. (Saga, Japan). All experimental procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Fundus Image Acquisition
Pupils were dilated with topical phenylephrine 2.5% and tropicamide 0.5%, which were applied 60 minutes and 30 minutes, respectively, prior to eye examination. Whiskers were shaved, and one drop of oxybuprocaine 0.4% was applied to each eye immediately prior to examination. Mice were anesthetized by an intraperitoneal injection of 15 mg/kg ketamine and 7 mg/kg xylazine just before examination. The cornea was kept moist using saline. Mice were manually held in front of the Optos. Fundus images were taken with the Optos Panoramic 200TX system (Optos PLC) without a contact lens and at the indicated wave lengths. Angiography was performed after intraperitoneal injection of 12 μL/g 2.5% fluorescein sodium (Alcon, Freiburg, Germany). Images were taken during the early (15–30 seconds), middle (1 minute), and late phase (2–3 minutes) of fluorescein perfusion relative to the time of injection and used for quantitative evaluations. 
Mouse Model of OIR
OIR was induced in C57BL/6J mice as described previously. 2 Briefly, litters of postnatal day 7 (P7) C57BL/6J pups along with their mothers were placed in a 75 ± 2% oxygen atmosphere (hyperoxia) for 5 days and then returned to room air at age P12. After the pups were exposed to room air and the avascular areas of retina became hypoxic, intraretinal physiological revascularization of the avascular areas and preretinal pathological neovascularization (NV) developed simultaneously. Pathological NV reached its maximum at P17. 
Whole-Mount Immunofluorescence
Mice were euthanized by cervical dislocation, and the eyes were enucleated for flat mount. The mouse eyes were fixed with 4% paraformaldehyde for 30 minutes at 4°C. For whole-mount preparation, the retinas were microsurgically exposed by removing other portions of the eye. Tissues were washed with PBS three times for 5 minutes and then placed in methanol for 20 minutes. Tissues were incubated overnight at 4°C with fluorescein-labeled isolectin B4 (1:150 dilution; Vector Laboratories, Burlingame, CA) in PBS containing 10% goat serum and 1% Triton X-100. Tissues were washed four times for 20 minutes in PBS. Radial cuts were then made in the retina. Retinal flat mounts were prepared on glass slides using a mounting medium (TA-030-FM, Mountant Permafluor; Lab Vision Corporation, Fremont, CA). The flat mounts were examined by fluorescence microscopy with a ×4 objective lens. The field of view in the setting is 3623 × 2728 μm, and the pixel count is 1360 × 1024 (2.66 μm/pixel). The digital images were recorded using a fluorescent microscope (BZ-9000; Keyence Corp., Osaka, Japan) with standardized illumination and contrast. 
Analysis of Magnification Error
The magnification percentage was evaluated with Optos and flat-mount images in adult healthy mice. To analyze the magnification error, two retinal vessels were selected. For instance, when the magnification at the superior area was measured, vessels at 45° and 315° were appropriate. The distance from the optic nerve to a branch of two vessels was shown as papilla diameter with the Optos image. The distance between two vessels in the Optos image was compared to that in a flat-mount image. 
In Vivo Leukocyte Transmigration Assay
The animals were anesthetized and intraperitoneally injected with 10 μL/g acridine orange (AO) (1 mg/mL; A6014; Sigma-Aldrich, St. Louis, MO). The AO concentration in the intravascular leukocytes and the endothelial cells significantly diminished 30 minutes after dye injection due to a washout effect. In contrast, transmigrated leukocytes retained their staining, which allowed their visualization. At 2 hours after AO injection, AO-positive transmigrated leukocytes (outside the vessels) were observed with a blue excitation light (488 nm). 
Mouse Model of Laser-Induced CNV
CNV was induced in C57BL/6J mice as described previously. 5 Briefly, the pupils were dilated with topical phenylephrine 2.5% and tropicamide 0.5%, and photocoagulation injuries to the retina were achieved using a krypton laser (50-μm spot size; 0.05 seconds duration; 350–400 mW), a slit-lamp delivery system, and a cover glass as a contact lens. One week after laser photocoagulation, CNV was examined using FAG. 
Results
High-Resolution Images of the Mouse Fundus
First, we examined whether retinal images in normal adult mice could be taken by Optos using the same approach as in human patients. Mice were manually held in front of the Optos (Fig. 1A). The eye was located with the position of the green sign, and the picture was taken (Fig. 1B). We were able to visualize the optic disc, retinal vessel, and peripheral retina with high resolution (1.29 μm/pixel: width 3900 pixels, height 3072 pixels) at a quality comparable to that with human imaging, and without any additional optical device such as a contact lens (Fig. 1C). Color images could be taken conveniently within seconds (Fig. 1C). The color image was saved at higher quality. Based on the optical properties we know from humans, the green laser displays the retina (Fig. 1D), while the red laser displays the deeper tissue (Fig. 1E). 
Figure 1
 
Color retinal imaging using Optos 200TX in mice. (A) Mice were manually held in front of the Optos device. (B) Optos display image. Mouse eyes were located by the position of the green sign. (C) Fundus color image using the Optos Panoramic 200TX system without any additional optical device. Images were of sufficient quality to evaluate retinal fundus up to the peripheral area. (D) Retinal images with green laser scans. (E) Images with red laser scans.
Figure 1
 
Color retinal imaging using Optos 200TX in mice. (A) Mice were manually held in front of the Optos device. (B) Optos display image. Mouse eyes were located by the position of the green sign. (C) Fundus color image using the Optos Panoramic 200TX system without any additional optical device. Images were of sufficient quality to evaluate retinal fundus up to the peripheral area. (D) Retinal images with green laser scans. (E) Images with red laser scans.
FAG Optos Images in the Mouse
FAG is a useful technique for detection of nonperfused areas, new vessels, and vascular leakage. Optos allowed FAG of the retinal vasculatures in mice (Fig. 2A). FAG using Optos showed wide-field retinal vessels with high resolution and quality (width 3900 pixels, height 3072 pixels) compared to the Heidelberg Retina Angiograph (HRA; Heidelberg Engineering, GmbH, Dossenheim, Germany) (width 768 pixels, height 868 pixels), for example, showing details of retinal capillaries (Fig. 2B). The period for the fluorescein dye to reach the retina after a peritoneal injection (peritoneal cavity-to-retina time) was between 10 and 15 seconds. Thus, the early phase was characterized as the time period when the retinal vessels are completely filled (between 15 and 30 seconds). Four minutes after the peritoneal injection, the fluorescein washout images with residual hyperfluorescent staining were identified. After 48 hours, flat mounts were created and compared with the FAG images of the same eyes. Strikingly, Optos images showed a resolution comparable to that of the lectin-labeled flat-mount images (Fig. 2C). The visible area and the magnification percentage were also evaluated. The detectable area of a photographic image using Optos was 67.2 ± 8.43% of the whole retina as seen in flat mounts. The Optos image became drawn out in a circumferential direction with increasing distance from the optic disc by a factor of 1.2 at six papilla diameters, and 1.4 at more than eight papilla diameters (Fig. 3). 
Figure 2
 
Optos fluorescein angiograph and flat mount of retinal vessels. (A) Fluorescein angiography (FAG) images of the retinal vasculatures in mice using the Optos Panoramic 200TX. Images could depict wide-field retinal vessels with high resolution and quality. (B) Magnified view of panoramic FAG image displayed details of retinal capillary vessels. (C) Flat-mount image with fluorescein-labeled lectin after 48 hours with angiography. (D) Magnified view of flat-mount image. Optos images had resolution comparable to that of lectin-labeled flat-mount images. The retinal artery could be visualized and evaluated by Optos but not with flat-mount images.
Figure 2
 
Optos fluorescein angiograph and flat mount of retinal vessels. (A) Fluorescein angiography (FAG) images of the retinal vasculatures in mice using the Optos Panoramic 200TX. Images could depict wide-field retinal vessels with high resolution and quality. (B) Magnified view of panoramic FAG image displayed details of retinal capillary vessels. (C) Flat-mount image with fluorescein-labeled lectin after 48 hours with angiography. (D) Magnified view of flat-mount image. Optos images had resolution comparable to that of lectin-labeled flat-mount images. The retinal artery could be visualized and evaluated by Optos but not with flat-mount images.
Figure 3
 
Optos 200TX magnification rate in the mouse retina. The magnification percentage was evaluated with Optos images and flat-mount images in adult healthy mice. The Optos image became drawn out in a circumferential direction with increasing distance from optic disc (by a factor of 1.2 times with six papilla diameters and 1.4 times with more than eight papilla diameters, n = 4 each).
Figure 3
 
Optos 200TX magnification rate in the mouse retina. The magnification percentage was evaluated with Optos images and flat-mount images in adult healthy mice. The Optos image became drawn out in a circumferential direction with increasing distance from optic disc (by a factor of 1.2 times with six papilla diameters and 1.4 times with more than eight papilla diameters, n = 4 each).
Leakage in the Mouse OIR Model With Optos
OIR is a commonly used in vivo mouse model of retinal neovascular diseases. 2 Retinal nonperfused area, neovascular lesions, and leukocyte infiltration in this model have been commonly evaluated by flat mount. 3 Optos also made it possible to obtain both color images (Fig. 4A) and FAG images (Figs. 4B–D) in OIR mice. The images demonstrated multiple areas of capillary nonperfusion in the early phase (Fig. 4B) and neovascular fronds in the middle phase (Fig. 4C). Retinal vascular permeability changes were also detected in the late phase (Fig. 4D). 
Figure 4
 
Optos images of the oxygen-induced retinopathy mouse model. (A) Fundus color image using the Optos Panoramic 200TX system in the OIR mouse. (BD) Fluorescein angiography (FAG) images using Optos in the OIR mouse. (B) FAG image in the early phase (capillary nonperfusion, white arrow). (C) Middle phase (neovascular fronds, white arrow). (D) Late phase (fluorescein leakage due to retinal vascular permeability change, white arrow). Neovascular activity can be assessed and monitored over time in the OIR model.
Figure 4
 
Optos images of the oxygen-induced retinopathy mouse model. (A) Fundus color image using the Optos Panoramic 200TX system in the OIR mouse. (BD) Fluorescein angiography (FAG) images using Optos in the OIR mouse. (B) FAG image in the early phase (capillary nonperfusion, white arrow). (C) Middle phase (neovascular fronds, white arrow). (D) Late phase (fluorescein leakage due to retinal vascular permeability change, white arrow). Neovascular activity can be assessed and monitored over time in the OIR model.
AO-Positive Leukocytes in Optos
Leukocyte infiltration is an important component of inflammatory angiogenesis. 12,13 In vivo AO staining with subsequent flat-mount preparations has been used to examine extravasated leukocytes in the retina and in angiogenic or peripheral areas. 3 Using Optos, we were able to detect infiltrated AO-positive leukocytes in vivo (Figs. 5A–D). Optos could continuously zoom images and display the location and number of infiltrated AO-positive leukocytes (Figs. 5B, 5D). The number of infiltrating AO-positive leukocytes in the OIR retinas was significantly higher than in normal mice retinas (Fig. 5E). In addition to the images from infiltrating AO-positive leukocytes, Optos also made it possible to obtain FAG images. Before fluorescein injection, the number and location of the infiltrated AO-positive leukocytes were identified (Supplementary Fig. S1A). FAG images then showed neovascular fronds (Supplementary Figs. S1B, S1C) and fluorescein leakage (Supplementary Fig. S1D), as well as the position of the infiltrated AO-positive leukocytes. 
Figure 5
 
Leukocyte transmigration assay using the Optos 200TX, infiltrated acridine orange (AO)-positive leukocytes in Optos images. AO-positive leukocytes were assessed 2 hours after intraperitoneal injection. (A) AO-positive leukocytes visualized in an adult healthy mouse. (B) Magnified view in adult healthy mouse, displaying the location of infiltrated AO-positive leukocytes. (C) AO-positive leukocytes visualized in an OIR mouse. (D) Magnified view of OIR mouse. (E) The number of infiltrated AO-positive leukocytes in adult healthy mice and OIR mice. AO-positive leukocytes were remarkably increased in OIR (*P < 0.05 compared with adult healthy mice; n = 8 each).
Figure 5
 
Leukocyte transmigration assay using the Optos 200TX, infiltrated acridine orange (AO)-positive leukocytes in Optos images. AO-positive leukocytes were assessed 2 hours after intraperitoneal injection. (A) AO-positive leukocytes visualized in an adult healthy mouse. (B) Magnified view in adult healthy mouse, displaying the location of infiltrated AO-positive leukocytes. (C) AO-positive leukocytes visualized in an OIR mouse. (D) Magnified view of OIR mouse. (E) The number of infiltrated AO-positive leukocytes in adult healthy mice and OIR mice. AO-positive leukocytes were remarkably increased in OIR (*P < 0.05 compared with adult healthy mice; n = 8 each).
Optos in the Mouse CNV Model
Laser-induced CNV is commonly used as a model of wet AMD. 5,14,15 The lesions are generally evaluated in flat-mount staining. We could show high-quality images of CNV using Optos in the early phase (Fig. 6A) and leakage in the middle to late phase (Figs. 6B, 6C). At the same time, our images showed infiltrating AO-positive leukocytes that accumulated around the choroidal neovascular lesions (Fig. 6D). 
Figure 6
 
Laser-induced choroidal neovascularization model using Optos. Choroidal neovascularization (CNV) was induced with krypton laser photocoagulation (50-μm spot size; 0.05 seconds duration; 350–400 mW). (AC) Fluorescein angiography (FAG) images using the Optos Panoramic 200TX system in the CNV mouse model. (A) FAG image in early phase, (B) middle phase, and (C) late phase. Fluorescein leakage was demonstrated from the middle to the late phase. (D) The infiltrated acridine orange (AO)-positive leukocytes were also visualized in the CNV model with high quality and resolution.
Figure 6
 
Laser-induced choroidal neovascularization model using Optos. Choroidal neovascularization (CNV) was induced with krypton laser photocoagulation (50-μm spot size; 0.05 seconds duration; 350–400 mW). (AC) Fluorescein angiography (FAG) images using the Optos Panoramic 200TX system in the CNV mouse model. (A) FAG image in early phase, (B) middle phase, and (C) late phase. Fluorescein leakage was demonstrated from the middle to the late phase. (D) The infiltrated acridine orange (AO)-positive leukocytes were also visualized in the CNV model with high quality and resolution.
Discussion
Angiogenesis is a key component of retinal/choroidal diseases such as diabetic retinopathy, ROP, and AMD. 16 The pathogenesis of retinal NV is partially revealed in the OIR mouse model. 3,17 Laser-induced CNV is also generally used as an animal model of wet AMD. 14,15,18 Understanding the molecular changes underlying retinal/choroidal neovascularization is a priority. 
Recently, several in vivo imaging methods of the mouse fundus have been developed and introduced. 6,7,19,20 It has been reported that confocal scanning laser ophthalmoscopy (cSLO) via HRA could be useful for detailed fundus imaging in mice. 6 Furthermore, HRA2 (Heidelberg Engineering, GmbH) is also available to perform imaging of the mouse retina. 7,20 An advantage of Optos is the autofocus mechanism, whereas the focus in HRA is manual. 
A previous paper 20 showed that the resolution of HRA2 images was 0.61 μm/pixel, whereas our study revealed that the resolution of Optos images was 1.29 μm/pixel. This study revealed that Optos resolution was adequate for investigating retinal or choroidal angiogenesis, as well as AO-stained leukocyte infiltration in mice. However, the resolution would be inferior to that with the HRA2 device. 
The field angle of imaging is important for understanding the fundus in mouse models. The image frame of HRA2 represents approximately 40% of the total retina with a 55° wide-field lens. 7 The largest field of view is reported to be 1520 × 1520 μm. 6 In contrast, Optos was able to depict 67.2 ± 8.43% of the whole retina in the mouse. This wide-angle feature could be useful in various mouse models. 
In humans, the Optos green laser scan showed the retina, while the red laser scan can display the choroid. However, the Optos red laser could not depict choroidal structure appropriately. This might be due to the pigment of the C57BL/6J mouse strain as reported with HRA. 6 A recent paper showed that a novel optical angiography system could visualize choroidal vascular network. 19 Optos, on the other hand, may not be useful for choroidal imaging. 
Optos allows FAG in the mouse retina, revealing a wider field of the retinal vessels with high resolution. Details of the retinal capillaries are revealed, comparable with the resolution of ex vivo flat mounts commonly used to evaluate retinal nonperfusion and neovascular lesions in OIR. Moreover, dynamic events, such as vascular leakage, cannot be evaluated in flat mount. Our Optos FAG images in the retinal vasculature showed resolution comparable to that of the flat-mount images. However, Optos covers a smaller area of the retina than the whole retinal flat mount. Therefore, flat mount may still be suitable for some experimental conditions. Optos images also reveal the curvature and tortuosity of the retinal vessels, as well as the growth of new vessels. Optos makes it possible to obtain FAG images of OIR, such as capillary nonperfusion, neovascular fronds, and retinal vascular permeability changes in color. CNV lesions and leakage also could be evaluated in Optos, and the quality was high. A feature of Optos FAG is that the neovascular activity is assessed through measurement of retinal vascular leakage, which may become a new tool in the evaluation of OIR and CNV. Another advantage of the technique is that dynamic vascular changes are studied over time, which could be useful for evaluation of new drugs. 
The infiltration of AO-positive leukocytes was visualized in Optos, which displayed their number and location. Leukocyte infiltration is a key component of angiogenesis in inflammatory diseases. 3,5,12,13,18,21 A major advantage of using Optos is that FAG and AO fluorography are obtained simultaneously with the wide viewing field. This technical advancement allowed us to show the proximity of the infiltrated AO-positive leukocytes around neovascular vessels in OIR and CNV. The ability to study infiltrated leukocytes and angiogenic vessels at the same time may provide new insights into the pathogenesis of neovascular diseases. 
The present study introduces a new mouse fundus evaluation system using the ultra-wide-field laser ophthalmoscope, Optos 200TX. We demonstrate the power and advantageous features of the new technology in experimental animal models such as OIR and CNV. This method may become a standard evaluation system for fundus imaging in experimental animals. 
Supplementary Materials
Acknowledgments
We thank Chuo Sangio Co. for the technical support. We also thank Jane Lin and Gary Guzik for editorial support. 
Supported by grants from JSPS KAKENHI, Grant-in-Aid for Young Scientists (A) No. 25713057 (SN) and Okinaka Memorial Institute for Medical Research, Japan (SN). 
Disclosure: S. Nakao, None; R. Arita, None; T. Nakama, None; H. Yoshikawa, None; S. Yoshida, None; H. Enaida, None; A. Hafezi-Moghadam, None; T. Matsui, None; T. Ishibashi, None 
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Figure 1
 
Color retinal imaging using Optos 200TX in mice. (A) Mice were manually held in front of the Optos device. (B) Optos display image. Mouse eyes were located by the position of the green sign. (C) Fundus color image using the Optos Panoramic 200TX system without any additional optical device. Images were of sufficient quality to evaluate retinal fundus up to the peripheral area. (D) Retinal images with green laser scans. (E) Images with red laser scans.
Figure 1
 
Color retinal imaging using Optos 200TX in mice. (A) Mice were manually held in front of the Optos device. (B) Optos display image. Mouse eyes were located by the position of the green sign. (C) Fundus color image using the Optos Panoramic 200TX system without any additional optical device. Images were of sufficient quality to evaluate retinal fundus up to the peripheral area. (D) Retinal images with green laser scans. (E) Images with red laser scans.
Figure 2
 
Optos fluorescein angiograph and flat mount of retinal vessels. (A) Fluorescein angiography (FAG) images of the retinal vasculatures in mice using the Optos Panoramic 200TX. Images could depict wide-field retinal vessels with high resolution and quality. (B) Magnified view of panoramic FAG image displayed details of retinal capillary vessels. (C) Flat-mount image with fluorescein-labeled lectin after 48 hours with angiography. (D) Magnified view of flat-mount image. Optos images had resolution comparable to that of lectin-labeled flat-mount images. The retinal artery could be visualized and evaluated by Optos but not with flat-mount images.
Figure 2
 
Optos fluorescein angiograph and flat mount of retinal vessels. (A) Fluorescein angiography (FAG) images of the retinal vasculatures in mice using the Optos Panoramic 200TX. Images could depict wide-field retinal vessels with high resolution and quality. (B) Magnified view of panoramic FAG image displayed details of retinal capillary vessels. (C) Flat-mount image with fluorescein-labeled lectin after 48 hours with angiography. (D) Magnified view of flat-mount image. Optos images had resolution comparable to that of lectin-labeled flat-mount images. The retinal artery could be visualized and evaluated by Optos but not with flat-mount images.
Figure 3
 
Optos 200TX magnification rate in the mouse retina. The magnification percentage was evaluated with Optos images and flat-mount images in adult healthy mice. The Optos image became drawn out in a circumferential direction with increasing distance from optic disc (by a factor of 1.2 times with six papilla diameters and 1.4 times with more than eight papilla diameters, n = 4 each).
Figure 3
 
Optos 200TX magnification rate in the mouse retina. The magnification percentage was evaluated with Optos images and flat-mount images in adult healthy mice. The Optos image became drawn out in a circumferential direction with increasing distance from optic disc (by a factor of 1.2 times with six papilla diameters and 1.4 times with more than eight papilla diameters, n = 4 each).
Figure 4
 
Optos images of the oxygen-induced retinopathy mouse model. (A) Fundus color image using the Optos Panoramic 200TX system in the OIR mouse. (BD) Fluorescein angiography (FAG) images using Optos in the OIR mouse. (B) FAG image in the early phase (capillary nonperfusion, white arrow). (C) Middle phase (neovascular fronds, white arrow). (D) Late phase (fluorescein leakage due to retinal vascular permeability change, white arrow). Neovascular activity can be assessed and monitored over time in the OIR model.
Figure 4
 
Optos images of the oxygen-induced retinopathy mouse model. (A) Fundus color image using the Optos Panoramic 200TX system in the OIR mouse. (BD) Fluorescein angiography (FAG) images using Optos in the OIR mouse. (B) FAG image in the early phase (capillary nonperfusion, white arrow). (C) Middle phase (neovascular fronds, white arrow). (D) Late phase (fluorescein leakage due to retinal vascular permeability change, white arrow). Neovascular activity can be assessed and monitored over time in the OIR model.
Figure 5
 
Leukocyte transmigration assay using the Optos 200TX, infiltrated acridine orange (AO)-positive leukocytes in Optos images. AO-positive leukocytes were assessed 2 hours after intraperitoneal injection. (A) AO-positive leukocytes visualized in an adult healthy mouse. (B) Magnified view in adult healthy mouse, displaying the location of infiltrated AO-positive leukocytes. (C) AO-positive leukocytes visualized in an OIR mouse. (D) Magnified view of OIR mouse. (E) The number of infiltrated AO-positive leukocytes in adult healthy mice and OIR mice. AO-positive leukocytes were remarkably increased in OIR (*P < 0.05 compared with adult healthy mice; n = 8 each).
Figure 5
 
Leukocyte transmigration assay using the Optos 200TX, infiltrated acridine orange (AO)-positive leukocytes in Optos images. AO-positive leukocytes were assessed 2 hours after intraperitoneal injection. (A) AO-positive leukocytes visualized in an adult healthy mouse. (B) Magnified view in adult healthy mouse, displaying the location of infiltrated AO-positive leukocytes. (C) AO-positive leukocytes visualized in an OIR mouse. (D) Magnified view of OIR mouse. (E) The number of infiltrated AO-positive leukocytes in adult healthy mice and OIR mice. AO-positive leukocytes were remarkably increased in OIR (*P < 0.05 compared with adult healthy mice; n = 8 each).
Figure 6
 
Laser-induced choroidal neovascularization model using Optos. Choroidal neovascularization (CNV) was induced with krypton laser photocoagulation (50-μm spot size; 0.05 seconds duration; 350–400 mW). (AC) Fluorescein angiography (FAG) images using the Optos Panoramic 200TX system in the CNV mouse model. (A) FAG image in early phase, (B) middle phase, and (C) late phase. Fluorescein leakage was demonstrated from the middle to the late phase. (D) The infiltrated acridine orange (AO)-positive leukocytes were also visualized in the CNV model with high quality and resolution.
Figure 6
 
Laser-induced choroidal neovascularization model using Optos. Choroidal neovascularization (CNV) was induced with krypton laser photocoagulation (50-μm spot size; 0.05 seconds duration; 350–400 mW). (AC) Fluorescein angiography (FAG) images using the Optos Panoramic 200TX system in the CNV mouse model. (A) FAG image in early phase, (B) middle phase, and (C) late phase. Fluorescein leakage was demonstrated from the middle to the late phase. (D) The infiltrated acridine orange (AO)-positive leukocytes were also visualized in the CNV model with high quality and resolution.
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