Micron 3 retinal imaging system

After anesthesia (ketamine 80 mg/kg and xylazine 20 mg/kg), the right eye of the mouse was dilated with 0.125% atropine and treated with a drop of 2% Methocel® (OmniVision, Switzerland). Using an ophthalmoscope, the ocular fundus was focused and positioned circumferentially around the optic disc; FP and FFA images were then acquired using the Phoenix Micron III retinal imaging system (Tempe, AZ). An intraperitoneal injection of sodium fluorescein (10 mg/kg) is essential prior to FFA administration. Following the manufacturer’s instructions, SD-OCT images were scanned vertically or horizontally at the indicated positions. The thickness of the three retinal sublayers was automatically identified and computed using the InSight software. The inner sublayer comprises the nerve fiber layer, ganglion layer, inner plexiform layer (IPL), and inner nuclear layer (INL). The middle sublayer comprises the outer plexiform layer (OPL) and outer nuclear layer (ONL), whereas the outer sublayer comprises the inner and outer segments (IS/OS) of photoreceptors [55 (link)].

To perform fundus examination, mice were anaesthetized by an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (10 mg/kg). The pupils were then dilated with 0.5% tropicamide drops (Mydriacil; Alcon, Geneva, Switzerland), and corneas were kept moist with GenTeal eye gel (Novartis, Basel, Switzerland). Fundus imaging was performed using a Micron III retinal imaging system (Phoenix Research Laboratories, Pleasanton, CA, USA) with StreamPix 5 software. Brightness and contrast adjustments were uniformly applied to images in Adobe Photoshop. Immediately after in vivo imaging, mice were euthanized for tissue collection.

Mice were anesthetized with a steady flow of 1.5 to 3% isofluorane. The eyes were topically anesthetized with one drop of proparacaine, dilated with one drop of a 1:1 mixture of 1% tropicamide and 2.5% phenylephrine. The corneas were kept moist with regular application of 2.5% methylcellulose. Eyes were examined using the Micron III retinal imaging system (Phoenix Research Labs, CA, USA) and raw images were adjusted for levels, enhanced contrast, and sharpened by applying an unsharp mask (100%, 2px, 0) using Photoshop CS6 (Adobe, Ca, USA). For angiography, animals received an intraperitoneal injection of 10 mg/ml fluorescein in PBS at a dose of 10 μl/g body weight. After 1 minute, eyes were imaged with the Micron III using the GFP-A-Basic-000 filter set (Semrock, NY, USA). Spectral domain OCT images were acquired with the Micron Image Guided SD-OCT System (Phoenix Research Labs) by averaging 10 to 20 scans. Levels were adjusted using Photoshop CS6 (Adobe).