Eye Drops

All animal procedures were conducted in accordance with the Association for Research in Vision and Ophthalmology (ARVO) statements on the care and used of animals in ophthalmic research. Adult male rd1 mice (>6 weeks of age) were anaesthetised by intraperitoneal injection of ketamine (72 mg/kg) and xylazine (16 mg/kg). The pupils were fully dilated with 1% tropicamide and 2.5% phenylephrine eye drops, and a custom-made ultrafine needle (Hamilton RN needle, 34 gauge) was attached to a 5 μl Hamilton glass syringe and passed at 45 degrees through the pars plana into the vitreous cavity (for intravitreal delivery) or into the subretinal space without retinal perforation (for subretinal delivery). Injections were performed under direct visualisation of the needle tip using an operating microscope (Leica Microsystems), avoiding lenticular contact and blood vessels. Each eye received a total intravitreal dose of 1E+14 genome copies in a 3 μl volume or 2 μl subretinal bleb (6 eyes per vector).

Each subject’s head was stabilized using a chin and forehead rest similar to those found on standard clinical imaging instruments. There was no pupil dilation or control of accommodation using eye drops. A previously described AOSLO was used to image the parafoveal cone mosaic of the right eye.^{28 (link), 29 (link)} The wavelength of the super luminescent diode used for retinal imaging was 775nm, subtending a field of view of 0.96° x 0.96°. The system’s pupil used for imaging was 7.75mm, however the eye’s pupil was undilated and certainly less than this. We thus calculated that the 30μm confocal pinhole of our system was one Airy disk diameter or less. Separate image sequences of 150 frames each were acquired at four parafoveal locations, each approximately 0.65° from the center of fixation (Figure 1). The four parafoveal locations were imaged in a random order, with the subject staying positioned on the chin/forehead rest for each set of four image sequences. Randomization of the imaging order had two potential benefits. First, the image quality may be best at the first location imaged when the tear film might be more evenly distributed across the cornea (though subjects were instructed to blink normally during each imaging set). Second, the randomized order would mitigate any effect in decreased fixational stability over the course of the imaging session, which might result from fatigue. This procedure was repeated 10 times for each subject with a short break after each set of four locations. The image acquisition software had an “active blink removal” algorithm, which discarded frames that had a mean intensity below a specified threshold. This process improved the percentage of frames in the recorded image sequence (always 150 frames) that contained useable retinal image data.
To correct for intraframe distortions within the frames of the raw image sequence due to the sinusoidal motion of the resonant optical scanner, we estimated the distortion from stable images of a Ronchi ruling, and then re-sampled each frame of the raw image sequence over a grid of equally spaced pixels. After desinusoiding, a reference frame was manually selected from within each image sequence, for subsequent registration using custom software. Registration of frames within a given image sequence was performed using a “strip” registration method, in which the frames were registered by dividing the frame of interest into strips, aligning each strip to the location in the reference frame that maximizes the normalized cross correlation between them.³⁰ Once all the frames were registered, the 40 frames with the highest normalized cross correlation to the reference frame were averaged, in order to generate a final registered image with an increased signal to noise ratio for subsequent analysis.