Effects of photodamage on living organisms during label-free multiphoton imaging.

A: Bright field (BF) and TPEF image of the area in the box in the BF image acquired on living primary human glioblastoma cells in culture, and overly of BF and TPEF images. B: effects of a serial irradiation of 500 scans at laser power of ∼52 mW in the area indicated by the box, leading to increase of TPEF in the cell cytoplasm. C: Label-free multiphoton image and fluorescence image (acquired using a mercury lamp for excitation) of mouse brain cortex; the brain surface is slightly convex, so that the image cuts through different tissue layers: CARS shows the cortical tissue with a blood vessel, while SHG shows the inner meningeal layer on top of the cortex. D: Label-free multiphoton image and fluorescence image acquired in the same region shown in C after 544 scans performed in vivo in the area indicated by the dotted line box; the arrows indicate the localized increase of fluorescence that matches the increase of TPEF.