Validation of an imaging system: steps to evaluate and validate a microscope imaging system for quantitative studies

The processing methods described in this article are general and can be used with any computer with adequate software without reference to a specific experimental apparatus. To achieve appropriately corrected images, the operations must be performed correctly and in a specific order to extract meaningful information from digital images. The operations are subtraction of the dark current, a shading correction after low pass filtering of the shading mask, a geometric correction with a reference grid obtained at the longest wavelength of interest, and finally registration. Since the digital data sets are too large for manual evaluation, statistical criteria must be used to define the analytical capabilities and error limits of the system before the more interesting cellular studies can be undertaken.