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Here’s another microscopy blog we enjoy:
All fluorophores bleach, some just bleach faster or slower than others. This can cause phototoxicity in your cells, and/or alter fluorescent intensity independent of your experimental variable(s). Neither of these are good things, particularly if you want to quantify your fluorescence and acquire meaningful data. Thus if you do any kind of fluorescence imaging, it is really important that you use a shutter on your excitation light source(s), to control light exposure to your samples. For quantitative fluorescence work, ideally you want to minimize and precisely equalize your samples’ light exposures, so that all experimental conditions (that will be compared in a single run) have seen the “same” amount of light exposure (and thus photobleaching).
The best ways to do this are: 1. Use only as much excitation light power as you need to get a decent image (and don’t saturate pixels!), 2. Minimize and equalize samples’ light exposure before taking data (for example, when scanning samples to focus or to find a field of view for imaging), AND 3. Use a shutter synchronized with your camera’s exposure time so that (especially) for data capture, the samples are ONLY exposed to the intense excitation light when the camera is actually acquiring an image.
Veroptics has many simple shutter solutions for fluorescence microscopy, so please CONTACT US and we will be happy to walk you through the process of synchronizing shutters with your image acquisition. It’s easier than you think, and we can help!
