Free-electron and thermally mediated pathways of photodamage in nonlinear microscopy

Photodamage in nonlinear microscopy of transparent tissues starts at irradiances 1.5 times above the autofluorescence imaging level. Although the free-electron density is low, their energy suffices to break bonds in water, DNA and the backbone and side chains of proteins. We explored photodamage kinetics using physical indicators (hyperfluorescence, plasma luminescence, bubble formation). By plotting threshold values in (irradiance/radiant exposure) space, we identified a “safe” region for microscopy. Thermomechanical effects become relevant in melanin-containing tissue. Two-photon excitation of retinal fluorophores allows monitoring metabolic transformations. We analyze the thermomechanical damage pathways in retinal imaging, and discuss strategies for mitigating such damage.