Fyk-Kolodziej, Bozena Hellmer, Chase B. Ichinose, and Tomomi
Department of Anatomy and Cell Biology and Department of Ophthalmology, Wayne State University School of Medicine, Detroit, MI.
Green fluorescent protein (GFP) and its derivatives are broadly used in biomedical experiments for labelling particular cells or molecules. In the mouse retina, the light (~500 nm) used to excite GFP can also lead to photoreceptor bleaching (peak ~500 nm), which diminishes photoreceptor-mediated synaptic transmission in the retinal network. To overcome this problem, we investigated the use of infrared fluorescent protein (iRFP) as a marker since it is excited by light in the near-infrared range that would not damage the photo responsiveness of the retina. Initially, we tested iRFP expression in human embryonic kidney 293 (HEK293) cells to confirm that conventional fluorescence microscopy can detect iRFP fluorescence. We next introduced the iRFP plasmid into adeno-associated virus 2 (AAV-2) and injected the resulting AAV-2 solution into the intraocular space. Retinal neurons were found to successfully express iRFP three weeks post-injection. Light-evoked responses in iRFP-marked cells were assessed using patch clamping, and light sensitivity was found to be similar in iRFP expressing cells and non–iRFP-expressing cells, an indication that iRFP expression and detection do not affect retinal light responsiveness. Taken together, our results suggest iRFP can be a new tool for vision research, allowing for single-cell recordings from an iRFP marked neuron using conventional fluorescence microscopy.
… “Light stimuli were generated using the pE-2 system (CoolLED), which was controlled by the Clampex software.”…
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pE-2: A repeatable, controllable modular system with 20 different LED peaks. Instant on/off and intensity (0-100%) control.
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The international Journal of Life Sciences
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