Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

Summary

A method is described for the preparation of single living photoreceptor cells from different vertebrate species for fluorescence imaging. The method can be used to image the fluorescence of endogenous fluorophores, such as NADH or vitamin A, or that of exogenously added fluorescent dyes sensitive to Ca²⁺ or other factors.

Abstract

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells^1-4. Rod photoreceptors are responsible for vision in dim light, cones in bright light. Phototransduction takes place in the outer segment of the photoreceptor cell, a specialized compartment that contains a high concentration of visual pigment, the primary light detector. The visual pigment is composed of a chromophore, 11-cis retinal, attached to a protein, opsin. A photon absorbed by the visual pigment isomerizes the chromophore from 11-cis to all-trans. This photoisomerization brings about a conformational change in the visual pigment that initiates a cascade of reactions culminating in a change in membrane potential, and bringing about the transduction of the light stimulus to an electrical signal. The recovery of the cell from light stimulation involves the deactivation of the intermediates activated by light, and the reestablishment of the membrane potential. Ca²⁺ modulates the activity of several of the enzymes involved in phototransduction, and its concentration is reduced upon light stimulation. In this way, Ca²⁺ plays an important role in the recovery of the cell from light stimulation and its adaptation to background light.

Another essential part of the recovery process is the regeneration of the visual pigment that has been destroyed during light-detection by the photoisomerization of its 11-cis chromophore to all-trans^5-7. This regeneration begins with the release of all-trans retinal by the photoactivated pigment, leaving behind the apo-protein opsin. The released all-trans retinal is rapidly reduced in a reaction utilizing NADPH to all- trans retinol, and opsin combines with fresh 11-cis retinal brought into the outer segment to reform the visual pigment. All-trans retinol is then transferred out of the outer segment and into neighboring cells by the specialized carrier Interphotoreceptor Retinoid Binding Protein (IRBP).

Fluorescence imaging of single photoreceptor cells can be used to study their physiology and cell biology. Ca²⁺-sensitive fluorescent dyes can be used to examine in detail the interplay between outer segment Ca²⁺ changes and response to light^8-12 as well as the role of inner segment Ca²⁺ stores in Ca²⁺ homeostasis^13,14. Fluorescent dyes can also be used for measuring Mg²⁺ concentration¹⁵, pH, and as tracers of aqueous and membrane compartments¹⁶. Finally, the intrinsic fluorescence of all-trans retinol (vitamin A) can be used to monitor the kinetics of its formation and removal in single photoreceptor cells^17-19.

Protocol

1. Preparation of Sylgard-covered dishes, experimental chambers, and razor blades

1. 35 mm Falcon Petri dishes coated with Sylgard elastomer are needed for the proper chopping of an isolated retina to obtain single photoreceptor cells. The elastomer is prepared according to the supplier's instructions and a small amount is poured into each dish to cover its bottom with a layer. Replace the dish covers after coating and store them. In a few days' time the elastomer hardens and the dishes are ready.
2. Isolated photoreceptors need to stick to the bottom of the experimental chamber so that they are immobilized during the course of an imaging experiment. ....

Discussion

If healthy isolated cells are not obtained, the problem lies either with the isolation or health of the retina or with its chopping. Typically, after removing the front of the eye and the vitreous, the retina readily lifts off the pigment epithelium. If it does not, try to peel it off starting from the periphery of the eyecup. If it is still difficult to separate, a likely possibility is that the animal has not been dark-adapted for an adequate period of time, or the red light is too bright. Ensure proper dark-adapta.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dark room (100-150 ft2)
Red lights19			Online stores
Infrared light sources andinfrared image viewers	FJW Optical Systems, Inc.
Dissecting microscope19			Outfitted with infrared viewers
Epifluorescence microscope enclosed in a light-tight cage19
Dissecting tools(scissors, forceps, blade holder)	Roboz Surgical Instruments Co.
Sylgard elastomer	Essex (Charlotte, NC)	Sylgard 184 elastomer kit
Poly-L-ornithine (0.01%)	Sigma-Aldrich	P4957
Poly-L-lysine (0.1%)	Sigma-Aldrich	P8920	Dilute to 0.01%
Experimental chambers	Warner Instruments	D3512P
Petri dishes, plastic pipettes	Fisher Scientific