Signal Enhancement in Fluorescence Microscopy by Microsecond Pulsed Excitation

Although pulsed illumination is routinely used in multi-photon fluorescence microscopy, it is not quite common for its one-photon counterpart, i.e. (one-photon) confocal fluorescence microscopy. However, prolonged illumination with continuous wave (CW) light source leads to excited (singlet) state absorption (as well as absorption from triplet state) leading to photo-damage. Here, by photo-damage we mean any detrimental (irreversible) effect that leads to reduced fluorescence yield. Stroboscopic or pulsed light illumination has been recently introduced to reduce photo-bleaching as well as photo-toxicity.