Cellular Resolution Panretinal Imaging of Optogenetic Probes Using a Simple Funduscope

Abstract

The new system could prove to be a basic tool for non-invasive in vivo small animal retinal imaging in a wide array of translational vision applications, including the tracking of fluorescently tagged cells and the expression of gene-therapy and optogenetic vectors.

Holographic optogenetic stimulation of patterned neuronal activity for vision restoration

“…To address this possibility, we have recently begun exploring cellular-resolved in vivo retinal imaging 36 and its integration with holographic projection systems for cellular-resolution optogenetic targeting (Fig. 7).…”

“…We constructed a custom system based on the design of the in-vitro holographic system described above, combined with an endoscope-based small animal fundus imager 36,45 . To acquire fundus fluorescence images, we used a white light illumination source (Intensilight C-HGFI, Nikon, Japan), a blue excitation filter (XF1068, Omega Optical), a green emission filter (MF535-22, Thorlabs), and an SLR camera (D5000 with Nikkor 80-200 F/2.8 AF-D lens, Nikon, Japan).…”

“…To acquire fundus fluorescence images, we used a white light illumination source (Intensilight C-HGFI, Nikon, Japan), a blue excitation filter (XF1068, Omega Optical), a green emission filter (MF535-22, Thorlabs), and an SLR camera (D5000 with Nikkor 80-200 F/2.8 AF-D lens, Nikon, Japan). The imaging resolution (B20 mm) was derived as described in Schejter et al 36 The holographic pattern was generated using a ferroelectric liquid-crystal micro-display (SXGA-R3, ForthDD, Scotland) and a blue DPSS laser light source (473 nm; Lambdapro Technologies, China), and projected through the back aperture of the endoscope using a 3:1 demagnifying Kepler telescope. The dimensions of spots projected on the optic disc were measured (a relatively large area of uniform expression-spot size was compensated for the imaging PSF).…”

In vivo characterization of genetic expression of virus-transduced calcium indicators in retinal ganglion cells using a low-cost funduscope

“…Compared with these relatively complex and expensive imaging techniques, fundus imaging in fluorescence mode, is cost-effective for screening purposes or characterization of the gene expression in transduced neurons [7][8]. In addition, the user-friendly examination procedure facilitates the training and execution time needed to perform the examination.…”

“…We developed a custom endoscope-based fundus system for monitoring and characterizing in vivo bright-field and fluorescence retinal images, by adapting a low-cost and simple fundus system based on the concepts proposed by Paques et al [7] and Schejter et al [8]. To monitor the genetic expression of GECI GCaMP6f [9] transduced in mouse RGCs by adeno-associated viral vector (AAV), we performed long-term tracking following intravitreal injection.…”