Various mechanisms produce chemical mediators that can recruit granulocytes to inflammatory sites. Intravital imaging with 2-photon microscopy can lead to the formation of singlet oxygen and reactive oxygen species (ROS) when tissue endogenous fluorophores, such as NAD(P)H and FAD, act as photosensitizers in photo-oxidative processes.30,31 ROS are oxygen-containing molecules with unpaired electrons in their outer orbit, that is, metabolites of oxygen that possess strong oxidizing capabilities. In a healthy organism, they are mainly produced by the mitochondrial respiratory chain. At low concentrations, the ROS function as signaling molecules that regulate cell growth, the adhesion of cells toward other cells, differentiation, and apoptosis.32 An enhanced production of ROS can pose a severe threat to cells and, if not controlled by sophisticated antioxidant machinery, ultimately leads to cell death. Necrotic cell death is followed by the liberation of a plethora of DAMPs which act as chemical mediators and lead to chemotaxis of eosinophil and neutrophil granulocytes to the site of lesion.33,34 For imaging at the same fluorescence brightness, production of ROS seems to be more pronounced with the ultra-broadband pulses. Many more PMNLs were recruited into the epithelium compared to imaging with 220 fs pulses. Granulocytes are among the first blood-borne cells that travel into damaged organs in which they phagocytose particulate material, such as cell debris. During phagocytosis, however, they also generate ROS. This functional response, termed an oxidative burst, contributes to the host’s defense, but it can also result in collateral damage of host tissue. Thus, these cells are central to both repair and destruction of tissues following sterile injury.35,36 Degranulation of granulocytes, as seen in A2P microscopy with the ultra-broadband laser pulses, resulted in the generation of highly fluorescent material within the tissue, quiescent PMNLs, and destruction of these immune cells (Figs. 11, 12, Video 6). These highly fluorescent areas are not identical with the described hyperfluorescence, as these areas did not spread during further irradiation and hyperfluorescence was only detected at higher excitation flux.