Multiphoton Microscopy
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Multiphoton microscopy is a powerful in vivo imaging technique because of its ability to image in three dimensions at high resolutions. It is made possible by a nonlinear dependence on excitation intensity, which localizes the resulting signal to the focal volume of the excitation light. Unlike conventional fluorescence microscopy, which requires a single photon to excite a fluorophore, multiphoton microscopy requires two (or three) photons with longer wavelengths to generate fluorescence. These longer excitation wavelengths allow multiphoton microscopy to achieve imaging depths significantly greater than confocal microscopy.
![**Two-photon microscopy of cortical vessels and neurons:** Image stacks ($400\times400\times750$ µm$^3$) of microvasculature labeled with Texas Red and neurons expressing yellow fluorescent protein. Cross-sectional maximum intensity projections at various depths ([Perillo, 2016](/publication/perillo-2016/)).](/project/multiphoton-microscopy/fiber_stack_hufaf9ccd166904af98289254865fcc4d3_1448636_a34f05b6b89fefc10512afd1dd6a5805.webp)
We design and build custom two- and three-photon fluorescence microscopes to study the complex vascular and neuronal structures of the cerebral cortex in mice. We also use two-photon phosphorescence lifetime microscopy to precisely measure oxygen concentrations in the neurovasculature. These systems allow us to visualize the cortex at depths and resolutions pushing the limits the optical imaging.
![**Large field-of-view imaging with resonant scanner:** Top and side maximum intensity projections of large stack ($1160\times1160$ µm) created by stitching together multiple two-photon acquisitions. Speckle contrast image of surrounding surface vasculature ([Engelmann, 2022](/publication/engelmann-2022)).](/project/multiphoton-microscopy/resonant_hu758e1bf39f6ee0257c9d6041ba6e8ad2_411686_8d7fb053d6797951bd38e27121b6aef6.webp)