Rats were anesthetized and the pupils were dilated as previously described.19 (link),20 (link) Color fundus photography and fluorescein angiography were performed using a fundus imaging system (Micron III system; Phoenix Research Labs, Pleasanton, CA, USA). For fluorescein angiography, rats received intraperitoneal injection of 10% fluorescein sodium at a dose of 0.8 mL/kg (Akorn). The fluorescein images were taken immediately after administration and followed for 5 minutes.
Micron 3 system
Manufactured by Phoenix Pharmaceuticals
Sourced in United States
The Micron III system is a state-of-the-art laboratory equipment designed for precise and efficient sample analysis. It utilizes advanced technology to perform high-resolution microscopy, enabling accurate measurements and detailed observations of microscopic samples.
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6 protocols using micron 3 system
1
Fundus Imaging and Fluorescein Angiography in Rats
2
Retinal Autofluorescence Imaging in Mice
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Ophthalmic examination was performed from 1 to 5 months of age on six mice per genotype using the Micron III system (Phoenix Research Laboratories, Pleasanton, CA, USA). The pupils were dilated with tropicamide (Mydriaticum, Théa à l'international Produits, Clermont-Ferrand, France) and phenylephrine (Néosynéphrine; Europhta, Monaco) prior to placing the animals in front of the Micron III device under Isoflurane anesthesia (Axience, Paris, France). During the procedure, eyes were kept moist with 0.9% NaCl. We made use of an excitation filter at 482 nm and an emission filter at 536 nm to detect autofluorescent structures in the retina.
3
Multimodal Imaging of Retinal Structure and Function
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FP, SD-OCT, and FFA images were acquired sequentially using the Phoenix Micron III system (Tempe, AZ, USA), according to the manufacturer’s instructions. The mice were anesthetized with a mixture of ketamine and xylazine, followed by a topical administration of 0.125% atropine for pupil dilation. For improved visualization of the fundus, a topical drop of 2% Methocel® (OmniVision, Switzerland) was administered before ophthalmoscopic observation. After SD-OCT scanning, an intraperitoneal injection of sodium fluorescein (10 mg/kg) was administered prior to FFA observation. To quantify structural changes, retinal thickness (or retinal sublayers) was automatically computed using the Insight software, as described previously [22 (link)]. The FFA images for the quantification analysis were saved in the 16-pixel format. After the removal of the fluorescence signal in the retinal vessels, the remaining areas (in terms of the number of affected pixels) are considered the leakage regions. The total leakage area was calculated using ImageJ (U.S. National Institutes of Health, Bethesda, MD, USA) [23 (link)].
4
Multimodal Imaging of Retinal Hypoxia
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Fundus images were taken with a Micron III system (Phoenix Research Labs, Pleasanton, CA, USA). Angiography was performed with rhodamine B (100 mg/ml, 200 mg/kg mouse, i.p.) that nicely fits to the TRITC filter of the Micron system, or with fluorescein and a SLO (HRA1, Heidelberg Engineering, Heidelberg, Germany).
Hypoxic retinal areas were visualized by the Hypoxyprobe Red549 Kit (Hypoxyprobe, Burlington, MA, USA). In brief, pimonidazol was dissolved at a concentration of 20 mg/ml and used intraperitoneally at 60 mg/kg. After 3 h, mice were perfused with 2% paraformaldehyde. Retinal flatmounts were prepared and stained with an antibody raised against hypoxyprobe followed by an antibody raised against Col4 (1:250, polyclonal, ab6586, Abcam, Cambridge, UK) or by staining with lectin (10 μg/ml FITC-lectin (BSI) from Griffonia simplicifolia, L9381, Sigma, Taufkirchen, Germany) in order to stain vessels.
Paraffin sections were prepared for histological examination by standard methods after formalin fixation, paraffin embedding and staining with hematoxylin and eosin (HE).
Hypoxic retinal areas were visualized by the Hypoxyprobe Red549 Kit (Hypoxyprobe, Burlington, MA, USA). In brief, pimonidazol was dissolved at a concentration of 20 mg/ml and used intraperitoneally at 60 mg/kg. After 3 h, mice were perfused with 2% paraformaldehyde. Retinal flatmounts were prepared and stained with an antibody raised against hypoxyprobe followed by an antibody raised against Col4 (1:250, polyclonal, ab6586, Abcam, Cambridge, UK) or by staining with lectin (10 μg/ml FITC-lectin (BSI) from Griffonia simplicifolia, L9381, Sigma, Taufkirchen, Germany) in order to stain vessels.
Paraffin sections were prepared for histological examination by standard methods after formalin fixation, paraffin embedding and staining with hematoxylin and eosin (HE).
5
Retinal Vascular Leakage Imaging Protocol
6
Retinal Vascular Damage Assessment
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To assess vascular damage in the retina, fundus imaging and fluorescein angiography were performed using the Micron III system (Phoenix Research Laboratories, Pleasanton, CA, United States) as described (Koirala et al., 2013 (link); Chakraborty et al., 2020 (link)). Mice were anesthetized with ketamine/xylazine (84, 7 mg kg–1)) and eyes were dilated with 1% cyclopentolate eye drops (Family Medicine Pharmacy, University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States). One drop of 2.5% Gonak (McKesson Medical Surgical, Richmond, VA, United States) was applied to each eye. Bright field images were collected, and then animals were injected intraperitoneally with 100 μL of 1% (w/v) fluorescein sodium (Sigma-Aldrich, St. Louis, MO, United States) for angiography. Angiogram images were captured using a GFP filter. All fundus images were captured using StreamPix software (Phoenix Research Labs, Bend, OR, United States). We observed repeated patterns of constriction in some retinal vessels, to semiquantitatively assess this phenotype, the number of affected vessels in each image was counted by an observer blinded to age and genotype.
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