© 2002 J.Paul Robinson, Purdue University

Excitation Saturation

•The rate of emission is dependent upon the time the molecule remains within the excitation state (the excited state lifetime f) •Optical saturation occurs when  the rate of excitation exceeds the reciprocal of f •In a scanned image of 512 x 768 pixels (400,000 pixels) if scanned in 1 second requires a dwell time per pixel of 2 x 10-6 sec. •Molecules that remain in the excitation beam for extended periods have higher probability of interstate crossings and thus phosphorescence •Usually, increasing dye concentration can be the most effective means of increasing signal when energy is not the limiting factor (i.e. laser based confocal systems)