Digital imaging fluorescence microscopy: analytical purpose and low light level intensity

It is shown that the problems encountered in the use of numerical analysis on digitized images obtained from low-light-level fluorescence imaging using intensified TV cameras to quantify fluorescence intensities can be overcome by an appropriate treatment of the digitized image. A protocol that corrects for the dark signal and pixel-to-pixel response variations, which are the sources of the problem, is presented. The efficiency of the protocol is exemplified by the evaluation of nuclear fluorescence intensities of 300 3T3 fibroblasts of the same population sample.<>