We demonstrate an accurate quantitative characterization of absolute two‐ and three‐photon absorption (2PA and 3PA) action cross sections of a genetically encodable fluorescent marker Sypher3s. Both 2PA and 3PA action cross sections of this marker are found to be remarkably high, enabling high‐brightness, cell‐specific two‐ and three‐photon fluorescence brain imaging. Brain imaging experiments on sliced samples of rat's cortical areas are presented to demonstrate these imaging modalities. The 2PA action cross section of Sypher3s is shown to be highly sensitive to the level of pH, enabling pH measurements via a ratiometric readout of the two‐photon fluorescence with two laser excitation wavelengths, thus paving the way toward fast optical pH sensing in deep‐tissue experiments.

We present all-optical high-resolution temperature measurements in the brain of freely behaving mice using an implantable thermometer that integrates a reconnectable two-section fiber-optic probe and a microcrystal of diamond with germanium-vacancy (GeV) centers. The local brain temperature is read out from the photoluminescence spectrum of GeV centers coupled to the guided modes of the optical fiber implanted in a mouse brain. This fiber thermometer is shown to enable brain temperature measurements with an accuracy within 0.15 °C at a level of laser power low enough to keep laser-induced heating of the brain well below 0.1 °C.

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Cognitive tests on representative groups of freely behaving transgenic mice are shown to enable a quantitative characterization of reconnectable implantable fiber-optic neurointerfaces for optogenetic neurostimulation. A systematic analysis of such tests provides a robust quantitative measure for the cognitive effects induced by fiber-optic neurostimulation, validating the performance of fiber-optic neurointerfaces for long-term optogenetic brain stimulations and showing no statistically significant artifacts in the behavior of transgenic mice due to interface implantation.