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.
Highlights
Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers
The excitation wavelength was optimized at 1300 nm to maximize both green fluorescent protein (GFP) and GCaMP6s signals
Since the optics inside the microscope elongated the pulse width on the sample, a two-prism based pre-chirp system was developed to compensate for pulse broadening so that the pulse width on the sample was reduced to 40 fs
Summary
Two-photon microscopy is used to image neuronal activity, but has severe limitations for studying deeper cortical layers. To image all six layers of the cortex with multiphoton microscopy, increasing the excitation wavelength and utilizing threephoton microscopy is reasonable due to increased scattering length at longer wavelengths and reduced out-of-focus background signal[15] Such implementation has been shown to improve the maximum imaging depth in brain tissues for structural imaging[15,16]. We have imaged responses of neurons in the deep layers of V1 and, for the first time, in the subplate, in the white matter below cortical layer 6, and demonstrated their unique response properties These measurements demonstrate the feasibility of optimized three-photon microscopy for live deep-tissue functional imaging at subcellular resolution
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