Practical Aspects of Quantitative Confocal Microscopy

Confocal microscopes are in principle well suited for quantitative imaging. The 3D fluorophore distribution in a specimen is transformed by the microscope optics and detector into the 2D intensity distribution of a digital image by a linear operation, a convolution. If multiple 2D images of the specimen at different focal planes are obtained, then the original 3D distribution in the specimen can be reconstructed. This reconstruction is a low-pass spatially filtered representation of the original, but quantitatively preserves relative fluorophore concentrations, with of course some limitations on accuracy and precision due to aberrations and noise. Given appropriate calibration, absolute fluorophore concentrations are accessible. A few simple guidelines are given for setting up confocal microscopes and checking their performance. With a little care, the images collected should be suitable for most types of quantitative analysis.