Cell Level Imaging of Photoreceptors with Retinitis Pigmentosa using Differential Interference Contrast Microscopy

Abstract

 

Purpose

 

Retinitis pigmetosa (RP) is one of the reasons for the degeneration of photoreceptors. The middle band of retina affected by RP becomes debris layer after the death of photoreceptors and layer collapses. Although the fundamental cure has not been discovered, it is very important to diagnose the early stage of RP and to monitor the progress of this disease. Many attempts have been conducted with the various types of apparatus. However, previous methods have limitations including short wavelength range and dyeing requirement. We investigated to observe photoreceptors with RP in a cell level without additional dyeing process.

 

Methods

 

Retina samples of the Royal College of Surgeons (RCS) rats were prepared by flat mount at different lapsed time since the disease occurred. In order to observe the retina with RP at cell level, we set a differential interference contrast (DIC) system, imaging the transparent specimen of photoreceptor with split beams having different polarization. Images formed by the DIC are attributed to the shift of ordinary and extraordinary lights passing through birefringent material. Since DIC uses interference to obtain the image of transparent sample, cell dyeing process is not required.

 

Results

 

As shown in Fig. (A) and (C), the photoreceptors with normal condition are uniformly placed on all surfaces while retina with RP shows sparse circles because retinal degeneration causes the death of rods and mutation of shape in RCS. The degeneration of photoreceptor leads to clearly visible morphological changes assisting that DIC can diagnose RP. The image of retinal section stained with H&E shows good agreement with DIC. The shape mutation and collapse cause the middle band to become a debris layer. The images of DIC have enough resolution for the automated counting of cells by image processing, which shows the decrement in a number of cells within progress.

 

Conclusions

 

This study shows that DIC technique can diagnose and distinguish the progress of the RP. Also, the image of RP-suffered rat obtained using DIC relatively coincides with retinal section and the morphology of photoreceptors at cell level can be easily identified using DIC technique.

 

 

Figure 1. DIC image of normal photoreceptors (A) and photoreceptors with RP (C). Retinal section image stained with H&E of normal retina (B) and retina with RP (D)

 

Keywords: 648 photoreceptors • 551 imaging/image analysis: non-clinical • 702 retinitis