The employed imaging camera can only record the total steady-state fluorescence signal; hence the localization of the different fluorescent markers and their individual contributions cannot be determined using this method. It should be noted that, in contrast to confocal scanning with a diffraction limited focal spot size, in widefield illumination the whole cell is exposed to the laser irradiation for the entirety of the measurement. This, in conjunction with the large diameter of the collimated laser beam (several millimeters), leads to no direct comparability of the exposure times and the irradiation levels of the cells in widefield fluorescence image acquisition and the parameters texp/pix and I values, defined for the acquisition of confocal scanning measurements.