The overall goal of this procedure is to prepare single living photoreceptor cells for fluorescence imaging. This is accomplished by first isolating a dark adapted retina. The second step of the procedure is to isolate single living photoreceptor cells.
The third step of the procedure is the transfer of the cells in an experimental chamber. The fourth step of the procedure is the placement of the chamber on the microscope stage. The final step of the procedure is the selection of a healthy cell For fluorescence imaging, ultimately results can be obtained that show realtime changes in cell metabolism, signaling, intracellular trafficking or oxidation through imaging of the cell fluorescence.
This method can help answer key questions in the vertebrate photoreceptor field, such as clearance of toxic byproducts, so the detection of light. The implications of this technique extend toward the therapy of the diseases of the retina because it is can be used to study the photoreceptor cells from mouse models of the diseases. Before beginning the protocol, you need to prepare various materials in advance.
First, 35 millimeter Falcon Petri dishes with just enough cigar elastomer to coat the dish need to be prepared a few days in advance so the elastomer can harden. The experimental chambers are modified 35 millimeter Petri dishes with a 12 millimeter diameter glass bottom. The experimental chambers need to be sticky, so cells are immobilized at 200 microliters of 0.01%poly L lysine or poly L ornithine to the bottom of the chamber.
Then cover it with a paper towel and allow it to dry for a few hours. When the chambers are dry, wash them with distilled water and store in a closed box. Clean the chambers after experiments by washing them with 100%ethanol and carefully scrubbing away debris with a cotton tipped applicator.
Afterwards, wash the chambers with distilled water. Let them dry and recoat. Next, using a metal cutter, prepare double-edged razor blades by cutting each into eight smaller blades.
Lastly, prepare a physiological solution suitable for the species from which you will be obtaining photoreceptor cells without added glucose, the physiological solution can be kept in a tightly sealed bottle for a few months. On the day of the experiment, have the mice dark adapted before they're sacrificed. It is best to dark.
Adapt the animals overnight in a dark room and sacrifice them at the end of their night cycle. However, placing the animal in the dark for at least two hours will suffice while waiting. Make a few preparations.
First, make a physiological solution with five millimoles per liter glucose. The solution is easily contaminated, so it must always be made fresh. Next, fill 2 35 millimeter Petri dishes halfway with the physiological solution.
After the animal is dark adapted, sacrifice it and remove the eyes using a pair of forceps and scissors. Next, place the eyes under an infrared sensitive camera equipped with a zoom lens illuminate with dim or infrared light and use a monitor to view what is under the camera. Begin dissecting by removing any leftover tissue from the outside surface of the eye using iris, scissors and dmat forceps.
Then with the front of the eye facing upwards, cut below the level of the aura serrata and remove the anterior third of the eye, including the cornea, iris, and the lens. Transfer the isolated eye cup back into the Petri dish filled with physiological solution. Be sure that only dim Redd or infrared light is illuminating the bench.
Use a pair of forceps to pinch and carefully remove the vitreous while leaving the retina behind. Then using fine forceps, carefully separate the retina from the rest of the eye cup by pinching off or cutting any attachments, gently lift the retina with the forceps, separating it fully from the eye cup. Transfer the retina to the second petri dish with a plastic transfer.
Pipette now store the retina in the dark before proceeding with isolation of the photoreceptor cells, open the light tight box containing the retinas. Dark adapted retinas are pink under room lights and bleach within a few minutes. If you want to work with dark adapted cells, all subsequent procedures have to be carried out under dim Redd or infrared light, begin isolating cells by cutting out a small section of retina using a pair of iris scissors.
Using a plastic pipette, transfer the section to a sill guard covered dish. The volume of the solution containing the retina piece should be about 250 microliters. Now, attach a small razor blade to a holder at an approximately 45 degree angle.
Use the side of the blade to flatten the piece of retina on the cigar layer and then gently mince the retina into smaller pieces while keeping it stuck to the cigar layer. Gradually a lot of the tissue, including isolated cells, get suspended into the solution. Transfer 200 microliters of the suspended tissue into an experimental chamber, leaving behind any larger pieces of retina.
Keep the chamber with the isolated cells in a light tight box. After 10 minutes, cells will settle. Then add two to three milliliters of physiological solution.
At this stage, the isolated cells can be loaded with a fluorescent dye like URA two If desired, the cells can now be taken to the microscope stage for experiments. If you are using an oil immersion lens, add a drop of oil and then position the chamber on the stage of the epi fluorescence microscope. If you are working with dark adapted cells, illumination should only be from a dim red or infrared light source.
Now adjust the environmental conditions of the chamber. Turn on the vacuum suction for removing the solution from the chamber. Then turn on the physiological solution for perfusing the chamber.
Finally, adjust the temperature controllers for the chamber temperature, the lens temperature, and the incoming solution temperature to obtain a chamber temperature of 37 degrees Celsius. Now put the condenser lens in place. Turn the infrared light on, close the curtains and begin the experiment with the infrared light turned on inside the microscope cage.
Focus on the bottom of the experimental chamber using the camera in live mode. Adjust the settings for viewing images in live mode. Use the IR channel and set the bin factor and exposure time to be able to move the stage in smooth fashion while seeing clear images of the cells from the infrared image.
You can judge the health of the photoreceptor cells prior to experimentation and identify healthy rod photoreceptors in this case. From a mouse retina, you will find many broken off mouse rod outer segments, which lack cell bodies. A healthy isolated mouse rod photoreceptor cell has an outer segment and ellipsoid and a nucleus.
After you select a cell for experimentation, switch the camera to image acquisition mode, select the size of the field. You want to capture the channel for image, capture the spinning and the exposure time for an infrared image of the cell, select the transmitted infrared light channel and set spinning and exposure time. For a fluorescence image of the cell, select the appropriate arrangement of excitation.
Filter dichroic mirror and emission filter. For measurements of retinol fluorescence, the open channel corresponds to DPI optics. Set the benning and the exposure time Focus on the cell by previewing the infrared image.
Keep in mind that the focus for imaging with the transmitted infrared light is different from that. For fluorescence, compare the different foci by previewing the fluorescence image. If you need to capture a fluorescence image of a dark adapted cell, you will need to first become familiar with how the infrared image looks.
When the fluorescence is in focus, capture a fluorescence image of the cell in the case of a bleached cell, the rod outer segment chose strong retinol fluorescence. Under dappy optics, quantify the outer segment fluorescence by first defining regions of interest for the outer and the background. The mean outer segment.
Fluorescence intensity is obtained by subtracting the mean fluorescence intensity of the background, ROI. From that of the outer segment, RO, I become familiar with the infrared image of the cell. When the fluorescence image in focus, you can capture an infrared image of the cell and focus separately.
Familiarize yourself with what a healthy cell looks like under infrared. By cross-checking the fluorescence images under DPI and fitzy optics ensure that such cells are able to generate retinol after exposure to light. These are rod and cone photoreceptors, isolated from a salamander retina.
These cells are very large, typically between 30 and 50 micrometers long and can survive for several hours. After isolation, the cells have not been damaged and they're healthy because they possess intact lipids. The region where the mitochondria are concentrated salamander retinas contain two different types of rods and several types of cones, but the overwhelming majority of the cells obtained by the procedure are the red rods and red sensitive cones.
The other photoreceptor cell types can be identified by their distinctive morphology, but it is best to confirm their identity by checking their sensitivity to light of different wavelengths. When the cells are viewed under DPI optics, this concentration of mitochondria gives a strong fluorescent signal due to the presence of NADH. If missing or damaged, then the cell is generally unfit for experimentation.
This damaged cell has a swollen cell body and a condensed nucleus. When that same damaged cell is viewed under FSE optics, it shows a strong fad signal in the ellipsoid region, originating from oxidized flavin, nucleotides, and flavoprotein. Another criterion for the health of isolated photoreceptors is their ability to generate all trans retinol or vitamin A in their outer segments upon stimulation by light, the generation of vitamin A requires substantial amounts of N-A-D-P-H, which depends on intact metabolic machinery.
Once mastered, this technique can be done in 30 minutes if it is performed properly Following this procedure. Other methods such as real-time imaging of cell fluorescence can be used in order to answer questions like dependence of retinol formation on different enzymes and metabolic factors.
