One of the most important things to consider when imaging your sample is the exposure time. The exposure time is essentially the length of time the camera spends capturing the image – a longer exposure time will mean a brighter image but also increases the chance of crosstalk and/or of imaging autofluorescence (page 12).

When imaging your sample the first thing to do is to have a look through the eyepieces – does the fluorescence look faint by eye? If so, it may not be real staining, or if it is real it may be difficult to image using a confocal.

If you are confident the fluorescence is the result of real staining choose an exposure time that avoids over saturation. Over saturation can often result in images that cannot be used for any advanced analysis such as comparing protein expression. The exposure time should also be kept to a minimum if you need to avoid photobleaching – this will be the case if you are imaging a live sample over time, or if you are capturing a large Z-stack through your sample. A short exposure time will also minimise crosstalk and autofluorescence.

The settings below are a guide for the exposure times you should observing for your sample using the 20x objective found on the microscopes within QBI. If your exposure times vary greatly from these it is possible you are imaging autofluorescence.

DAPI     20-80msec
Alexa488/GFP  200-800msec
Alexa555/568/594 200-800msec
Alexa647 400-1500msec