A Study in Green

I have recently been working towards an in vitro GCaMP imaging system. This came about when I inherited a patch/imaging rig from a colleague who moved on to pastures new. The rig was set up for calcium imaging using calcium-sensitive dyes, such as Fura, and included a nice Zeiss inverted microscope, perfusion stage and Hamamatsu Orca 2 camera.

However, rather than chemical indicators of calcium activity, I wanted to use our genetically encoded calcium indicators. Luckily, the excitation/emission spectra for GCaMP align very closely with GFP, which meant that I could use the GFP filters for imaging. I was starting tests with a technician in the lab, and then one day the camera just wouldn’t switch on (seems the control box died – to be fair it’s quite old, but still costs several thousand pounds to replace).

Well, that was about a year and a half ago, and for obvious reasons put a complete halt to further imaging studies. Which is a shame, because the GCaMP imaging provides a number of features that can make it desirable over electrophysiology (Table 1).

Luckily, my supervisor had a sizeable pot of grant cash that had been earmarked for electrophysiology equipment, but could instead be rerouted towards a new imaging camera. So after some research, I found a couple of very good (but also very expensive, >£15k) cameras to test out, the BSI Express from Teledyne Photometrics and the Fusion BT from Hamamatsu. These are both equivalent high-end CMOS cameras, which means they have outstanding sensitivity, imaging speed and resolution.

My plan was to only buy what was absolutely needed, and use what was in place until we want to/can afford to upgrade the system (Figure 1A). I arranged loans of the two cameras I was interested in; the BSI express came first so that’s the one I’ll show today.

The light source we have is a Prior white light with excitation filter changer. I purchased 2 excitation filters for this experiment at 470 nm and 410 nm (Figure 1B), which represent Ca2+-dependent and Ca2+-independent excitation wavelengths of GCaMP, respectively.

Having set up the system, it was time to test it out. Reaching for the low-hanging fruit, I found that one of our mouse lines that we had been gifted by a collaborator included a GCaMP3 reporter (we were actively trying to breed the reporter out, but in the mean time we had a bunch of cre and GCaMP3 double-positive mice that would otherwise not be used). The mouse line in question is a GLP1R-cre mouse, which means that all the GCaMP-expressing cells should have GLP1R. Therefore, the obvious experiment to validate the system was to apply the GLP1R agonist Exendin-4.

Anyway, I took a video, drew regions of interest round identifiable neurones and plotted the change in fluorescence in response to 1 µM Exendin-4 (Figure 2).

All in all, the in vitro GCaMP imaging system worked well, despite a number of problems along the way that I haven’t gone into here. I am very much a convert to this kind of experiment; having spent many years patching individual neurones, it’s lovely how visually obvious these data are. I would highly recommend anyone reading this to look into GCaMP imaging as a quick and easy alternative to patch clamping.