Abstract
Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43®) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.
Highlights
Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass[1]
We apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina
The advantages of objective-type TIRF microscopy are that 1) it provides excellent optical sectioning by restricting excitation light to a narrow region within the focal plane of the objective, thereby minimizing out-of-focus light; 2) since light drops exponentially with distance, movement in a vertical direction can be monitored as a change in fluorescence intensity; 3) efficient light collection through the high numerical aperture objective[1,5]
Summary
Place the filter paper containing the retina in a 35 mm plastic culture dish with hyaluronidase solution and peel off the retina from the filter paper with the help of #7 Dumont tweezers; 11. Cut the retina into [4,5,6] pieces with half of an industrial carbon steel single-edged blade and let it sit in the hyaluronidase solution for 20 minutes; 12. Wash the pieces of retina 3x in low Ca2+ Ringer’s and let them sit in the papain solution for [30-35] minutes; 14. Wash the pieces of retina 3x in low Ca2+ Ringer’s and store them until use at 4 °C in a 35 mm plastic culture dish containing low Ca2+ Ringer’s; 15.
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