Functional imaging of visual cortical layers and subplate in awake mice with optimized three-photon microscopy

Abstract

Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. Here, we developed a custom three-photon microscope optimized to image a vertical column of the cerebral cortex > 1 mm in depth in awake mice with low (<20 mW) average laser power. Our measurements of physiological responses and tissue-damage thresholds define pulse parameters and safety limits for damage-free three-photon imaging. We image functional visual responses of neurons expressing GCaMP6s across all layers of the primary visual cortex (V1) and in the subplate. These recordings reveal diverse visual selectivity in deep layers: layer 5 neurons are more broadly tuned to visual stimuli, whereas mean orientation selectivity of layer 6 neurons is slightly sharper, compared to neurons in other layers. Subplate neurons, located in the white matter below cortical layer 6 and characterized here for the first time, show low visual responsivity and broad orientation selectivity.

Label-free characterization of visual cortical areas in awake mice via three-photon microscopy reveals correlations between functional maps and structural substrates

“…1B-D). Taking the blood vessel architecture and retinotopic map as a reference, we utilized a custom-made three-photon microscope 19 (Fig. 1E-F) to perform depth-resolved THG imaging at 1300 nm in each cortical region, including the cortex and white matter spanning >1 mm thickness ( Fig.…”

“…where Esurf is the laser energy at the surface, z is the ablation depth, EAL is the effective attenuation length of cortex, w0 is the one-photon 1/e 2 radius at the focal plane which can be calculated from the three-photon point-spread function 19 , and F is the fluence at the focal plane. To calculate the threshold fluence and EAL, Eq.…”

“…Multiphoton microscopy has revolutionized neuroscience by providing subcellular resolution as well as capabilities for deep-brain imaging [17][18][19] in mice. However, there are limitations to both two-and three-photon microscopy with respect to maximum imaging depth, depending on signalto-noise ratio, as well as optical properties such as scattering and absorption lengths of specific brain regions 19,20 . Several of these features are related to structural components of the cortex, such as cytoarchitecture and myeloarchitecture 21,22 .…”

“…We recently showed that the EAL can be estimated either with third-harmonic generation (THG) imaging of blood vessels and myelin fibers at 1300 nm excitation wavelength in the cortex and in the white matter, respectively, or focally ablating tissue at multiple depths in the primary visual cortex (V1) of awake mice 19 . THG imaging is a label-free three-photon microscopy technique that is sensitive to index of refraction and third order susceptibility changes 27,28 .…”

“…THG imaging is a label-free three-photon microscopy technique that is sensitive to index of refraction and third order susceptibility changes 27,28 . Previous studies have shown that THG could be used to image blood vessels 19,29 , neurons and their processes 30,31 , and myelin 22 in the vertebrate central nervous system. Thus, THG imaging of blood vessels and myelin fibers in awake mouse brain can provide in vivo structural information.…”