Is 3 really better than 2? In vivo functional calcium imaging of cortical circuits by two- and three-photon microscopy.

Functional calcium imaging with genetically encoded calcium indicators (GECIs) has become a cornerstone of preclinical in vivo circuit analysis. Yet, due to light scattering, two-photon (2P) functional calcium imaging has been largely confined to superficial cortical layers in rodent models. Three-photon (3P) excitation offers deeper penetration due to reduced scattering of longer excitation wavelengths, but lower repetition rates of 3P laser systems impose a significant limitation on temporal resolution. Therefore, 3P calcium imaging of cortical microcircuits is still in its infancy. In this study, we performed a back-to-back comparison of 2P and 3P functional calcium imaging in the visual cortex of awake, head-fixed mice. We assessed the efficacy of 3P imaging to capture the local functional microcircuit dynamics in the visual cortex. 3P imaging not only revealed neuronal somata and neuropil in layers V/VI with robust morphological SNR but also generated label-free contrast of the blood vessels via third harmonic generation. Functionally, in superficial layers, a comparable functional architecture was assessed with both methods; while 3P detected lower firing frequencies, the underlying circuit dynamics, such as functional connectivity, remained consistent with 2P recordings. Notably in layers V/VI, while 2P failed to resolve the functional circuit state, with 3P a functional readout of neuronal circuits became achievable. These results demonstrate that, despite its current temporal limitations, 3P calcium imaging can reliably capture the functional architecture across all cortical layers, providing unmatched depth penetration and vascular contrast.NEW & NOTEWORTHY This study explores the capability of three-photon functional imaging to resolve functional cortical architecture in mice. By a quantitative, back-to-back comparison in awake head-fixed mice, we demonstrate that three-photon imaging is equally capable compared to two-photon imaging at superficial layers (II/III) and allows functional assessment of deep cortical layers (V/VI), beyond the reach of conventional two-photon microscopy.