The exposure time determines the signal intensity level for the image acquisition. Finding the appropriate exposure time for each filter in an experiment is crucial to acquiring the best image data. Many factors must be considered. Although you want to see the maximum intensity in each wavelength, you must not saturate the camera or photobleach your sample.
Camera saturation occurs when the intensity values reach 4095 counts. The CCD camera is a 12-bit camera with an intensity range of 0 to 4095 counts. For successful deconvolution, a minimum intensity of 50-100 counts above background is recommended.
Photobleaching occurs when the fluorescent dyes lose their emission intensity as they are exposed to illumination. Certain dyes are more susceptible to photobleaching than others. The potential for excessive photobleaching is increased with increased exposure time and decreased by use of a higher value neutral density filter.
There are two ways to decrease illumination, and therefore the likelihood of photobleaching:
Decrease exposure time (intensity level decreases are roughly directly proportional to exposure time decreases)
Increase neutral density value
To use auto focus in an experiment macro