Kowalczuk, Laura, Laforest, Timothe, Kunzi, Mathieu, Dos Santos, Florentino Caetano, Moulin, Alexandre P., Zografos, Leonidas, Lassiaz, Patricia, Behar-Cohen, Francine F., and Moser, Christophe
Purpose : Based on oblique partially coherent illumination of transparent samples, we developed a simple custom Optical Phase Imaging (OPI) microscope providing a label-free, semi-quantitative phase contrast imaging. The aim of this study was to explore this ex-vivo modality for retinal imaging and correlate it with standard clinical images and fluorescence microscopy. Methods : Multimodal macular imaging was performed on the flat-mounted retina of an eye presenting an epiretinal membrane with cystoid macular edema, enucleated for a peripheral melanoma. After glial fibrillary acidic protein (GFAP) - aquaporin (AQP)-4 – collagen (Col)-IV co-immuno-labeling and nuclei staining, the retina was cleared by index matching in a medium of refractive index (RI) 1.46 to decrease scattering for high-resolution deep-tissue ex vivo imaging. We performed a comparison of the clinical examinations obtained by Optical Coherence Tomography-Angiography and fluorescein angiography before enucleation, with the images obtained with confocal microscopy and OPI microscopy. Ex-vivo imaging of the retina mounted in a medium with a lower RI (1.40), close to the mean RI of Muller glial cell (MGC), was then repeated to better view the latter cells. Results : The retinal vessels were used as landmarks for correlating all imaging modalities. OPI microscopy allowed for different contrast imaging depending on the RI of the mounting medium. With the high RI medium (1.46), deep contrast imaging of nuclei and intraretinal cysts was obtained. The solution with a RI of 1.4 provided an improvement in the contrast of the retinal structures, from the inner layer (AQP4-positive MGC, epi-retinal membrane, nerve fibers surrounded by GFAP-positive astrocytes) to the photoreceptor segments. No AQP4 labeling was observed inside the cyst. AQP4-positive, GFAP-negative cells were visualized on the ColIV-labeled epi-retinal membrane, demonstrating that the membrane is made of retinal Muller glial cells. Conclusions : This morphological correlative imaging study demonstrated OPI on numerous cellular structures of a human retina by tuning the tissue RI. This label-free in-depth imaging modality offers a new research tool to study the cellular origin of retinal diseases.