Defined power limits prevent cellular damage during longitudinal in vivo 3-photon imaging of mouse cortical gray and white matter

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Presenter(s)

Alexandra Ramirez

Abstract or Description

In vivo 2-photon microscopy (2PM) allows for longitudinal tracking of individual cells over time; however, this method is limited to depths of ~400 µm into the mouse cortex. The development of in vivo 3-photon microscopy (3PM) increases this depth limit to ~1200 µm, which includes the subcortical white matter. Because 3PM requires high pulse energy, the potential for tissue damage is significantly higher than 2PM, and such effects are magnified with longitudinal imaging. In this study, we tracked individual oligodendrocytes over ~3 months with longitudinal 3PM imaging. Following the study, we used correlative post-hoc immunostaining of longitudinally imaged brain regions to assess multiple markers of cellular and molecular stress. We found that, within our empirically determined laser power limits, longitudinal in vivo 3PM imaging did not significantly increase the fluorescence intensities of markers of cellular reactivity in oligodendrocytes, microglia, astrocytes, or neurons when compared to contralateral controls. Then, we analyzed the intensity of molecular markers of oxidative stress and phototoxicity specifically in oligodendrocytes and similarly found no differences between imaged and contralateral cortical hemispheres. In contrast, we found increased markers of cellular reactivity, oxidative stress, and phototoxicity in tissues exposed to elevated laser power limits that resulted in laser-induced tissue damage. Our results broaden the understanding of region-specific differences in oligodendrogenesis and prove that longitudinal in vivo 3PM imaging over multiple months is sustainable without inducing laser damage and cellular stress. These experiments provide important imaging and analysis guidelines for future studies employing longitudinal 3PM imaging in the mouse brain.