After our unsuccessful characterization of ChR2 and CatCh using the iVIS Lumina II imaging system, we decided to use a Calcium dye to troubleshoot our channels. At first, we tried imaging our Chr2 and CatCh cells with x-rhod-1 on a Leica TCS SP5 Confocal microscope using the x-rhod-1 calcium dye. However, the imaging did not work, which could possibly be due to the fact that the dye is often localized in mitochondria, while our ChR2 and CatCh are transmembrane proteins. We then decided to switch to Fura-2AM, which is often used in calcium imaging. Fura-2AM is a highly sensitive ratiometric dye that operates at a low wavelength (340 and 380nm) and thus would not interfere with our 473nm laser. When not bound by calcium, Fura-2AM emits at 380nm. When calcium binds to the dye, it emits at 340nm. A camera switched between capturing images at 340 nm and 380 nm filters (Figure 1). It then combined the readouts of these filters to create a 340/380 ratio (Figure 2). The higher the 340/380 ratio, the more calcium there is within the cell.

We transiently transfected our HEK293T cells with either ChR2 or CatCh. On the day of imaging, we washed the cells with PBS then loaded them for approximately 30 minutes with a 5 µM Fura-2AM solution. After loading the dye was complete, we aspirated the Fura-2AM solution and then added a saline imaging solution to the wells.

Our first experiment was a positive control to test that the Fura-2AM dye was working. We loaded cells with the dye, then added ionomycin (an ionophore that stimulates calcium transport across the cell membrane) and imaged the well for 30 minutes. As shown clearly in Figure 3, there was a significant increase in the intracellular calcium, as 340/380 ratio changes from blue and purple to green and yellow (warmer colors indicate a higher ratio). This test shows that our Fura-2AM dye works properly.

We then tested both our ChR2 and CatCh channels with the Fura-2AM setup. We identified several variables that could factor into our imaging results: glass or plastic plates, thin or normal media volume, 2 mM or 90 mM calcium ion concentration, and cell density. After trying every possible combination for both CatCh and ChR2, we determined that using glass plates, thin media, a 90 mM calcium ion concentration, and approximately 70% confluency of cells lead to the best results. We had also originally been transfecting in 100mm dishes, trypsinizing the transfected cells and plating onto 35mm dishes. However, ChR2 and CatCh expression was low – because these are membrane proteins, we hypothesized that the trypsin was cleaving the proteins causing low expression. After transfecting directly into the smaller 35mm dishes, our transfection efficiency was much higher.

As shown in Video 1, pulses of 470nm light caused the 340/380 ratio of cells that were ChR2 positive (shown by yellow fluorescence in the Figure 4 overlay) to increase, meaning calcium was entering the cell. However, the cells also started at a higher 340/380 ratio than the surrounding cells, and after repeated exposure to the blue light, would revert back to lower levels (appearing to burst). This makes us believe the ChR2 is a leaky calcium channel in our given conditions. CatCh performed similarly, but appeared to leak at even lower calcium concentrations. This was an interesting result, as we could not find any literature that would suggest either ChR2 or CatCh would be leaky.

Further literature searches did reveal, however, that we forgot to add all-trans-retinal to our ChR2 and CatCh. All-trans-retinal is a light-sensing co-factor that is required to make the ChR2 light sensitive. We also hypothesize that the leakiness of our channels could be due to stochastic fluctuations in channel conformation combined with the incredibly high calcium ion concentration. We hope to continue to troubleshoot and characterize our receiver cells and channelrhodopsins with the addition of all-trans-retinal, but have been unable to do so due to time constraints.

We also hope to continue our co-culture experiments in which we culture our sender and receiver cells in the same dish, then activate the sender cells, hoping that the luminescence from the Renilla luciferase in close proximity to the ChR2 or CatCh will activate calcium influx and the receiver cell. However, since we have been unable to properly activate the ChR2 or CatCh with a high-powered laser, our focus has been shifted towards characterizing the individual parts first before working on the system as a whole.