High Resolution Imaging of the Adult Zebrafish Retina In vivo

Abstract

 

Purpose

 

To image the zebrafish retina using confocal scanning laser ophthalmoscopy (SLO) and spectral domain optical coherence tomography (OCT).

 

Methods

 

Wild-type and transgenic zebrafish lines expressing GFP in rods, cones, or vascular endothelial cells were imaged. Fish were anesthetized using tricaine (.12-.16 mg/ml), placed in a custom holder, and fitted with a contact lens stabilized on the extraocular surface with semi-viscous fluid. Standard SLO (HRA2, Heidelberg Eng., Inc) and OCT (840HR, Bioptigen, Inc.) systems equipped with wide field objectives (≥50^o FOV) were used to obtain images of retinal morphology at single and/or multiple imaging time points. SLO imaging was performed first followed immediately by OCT. Animals were imaged in room air and recovered in normal tank water.

 

Results

 

Retinal imaging was relatively easy to perform using commercially available systems with minimal (SLO) or no modification (OCT). Anesthesia induction, single eye imaging, and recovery required ~ 6, 5 & 1.5 minutes to perform, respectively. Post-procedure survival rates were high (>95%, n=40) enabling repeated imaging over multiple time points. Contact lens use was essential in obtaining quality images with both imaging systems. SLO images were obtained that revealed both anatomical (vitreoretinal interface, nerve fibers, hyaloid vasculature, photoreceptor mosaic, larval remnant) and GFP labeled structures. OCT imaging performed after SLO provided complementary information for interpretation of SLO observations. Manual z-axis sectioning (i.e. tomography) with SLO could be performed at small increments (~1µm/step) to reveal the depth of the GFP reporter. Baseline imaging of numerous fish (n>300) revealed that some exhibited abnormal retinal morphology unrelated to any treatment and/or manipulations.

 

Conclusions

 

Procedures were developed to perform transpupillary, non-invasive imaging of zebrafish retina. These imaging procedures are useful in establishing normal in vivo retinal morphology, localizing fluorescent protein expression in transgenic models, monitoring the progression of retinal degeneration during experimental studies, and complementing post-mortem microscopic analysis.

 

 

AF-SLO of GFP signal originating from albumin(a), endothelial cell nuclei(b) & cytoplasm (c). IR-SLO of vitreoretinal interface(1d) & cone mosaic showing larvel remnant(1e). AF-SLO of autofluorescence from outer retina(f). OCT of optic nerve & retinal morphology(g).

 

Keywords: 552 imaging methods (CT, FA, ICG, MRI, OCT, RTA, SLO, ultrasound) • 688 retina • 551 imaging/image analysis: non-clinical