Life at 1010 W/cm2: low-damage microscopy in living specimens using multi-photon microscopy

Multi-photon excitation microscopy (Denk, Strickler et al. 1990) provides optical sectioning by excitation confinement alone and therefore allows fluorescence imaging of biological samples with minimal photodamage. Generation of fluorescence requires molecular excitation, which for organic chromophores is always accompanied by the possibility of photochemical side reactions. Such reactions can lead to the destruction of the fluorophore itself (photobleaching) or to damage to surrounding biological molecules (photodynamic damage). Photobleaching and photodynamic damage can often be reduced by the removal of oxygen, which is, however, often incompatible with biological viability. Because only molecules in the focal plane are excited within the multi-photon microscope no spatial discrimination is necessary during the detection process leading to efficient utilization of fluorescence photons. This is different from the confocal microscope, which is very wasteful with fluorescence photons and, in particular for thicker samples, uses only a small fraction of the total fluorescence generated