Two‐ and three‐photon absorption cross‐section characterization for high‐brightness, cell‐specific multiphoton fluorescence brain imaging

Abstract

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.

A guide to genetically encoded tools for the study of H2O2

“…This approach requires specialized equipment, but has several advantages, the main ones being quantitative readout and much less photobleaching and light scattering effects. Multiphoton microscopy is also increasingly being applied to HyPer and SypHer probes, which allows high-resolution microscopy to be performed even in thick layers of tissues [117][118][119]. HyPer family probes may also be used in popular super resolution imaging techniques, for example, in the live-cell stimulated emission depletion (STED) microscopy [120,121].…”

Single‐beam dual‐color alternate‐pathway two‐photon spectroscopy: Toward an optical toolbox for redox biology

“…In Figures 2d and 4a–f, we present experimental characterization of the 2PEF properties of SypHer3s, HyPer7, and Hypocrates, studied in a well‐controlled setting of a buffer solution with referencing and cross‐calibration as described in Lanin et al [ 58,69 ] As one important check, the 2PEF readout measured as a function of the average power of the laser driver, p , in all the experiments closely followed the p 2 scaling (cf. data points and solid lines in Figure 4c,d).…”

“…[55][56][57] SypHer3s is designed as a highly sensitive genetically encodable fluorescent probe of pH, enabling, as earlier studies show, a quantitative analysis of pH dynamics in mitochondria of living neurons. [55] Adding to the potential of SypHer3s as a genetically encodable fluorescent marker is its large multiphoton absorption cross section, [58] which translates into comfortably detectable two-and three-photon-excited fluorescence (2PEF and 3PEF) signals, enabling optogenetic cell-specific 2PEF and 3PEF imaging of brain cells, subcellular compartments, and important functional brain units, such as gliovascular interfaces. [35,59] HyPer7, on its part, provides a highly sensitive, pH-stable probe for hydrogen peroxide, [56,57] offering means to understand the role of H 2 O 2 as an oxidant in cell migration and mitochondrial function.…”

Single‐beam optogenetic multimodal χ(3)/χ(5) nonlinear microscopy and brain imaging

“…In these studies, HeLa cells were genetically encoded to express SypHer3s [ 32 ] —a high‐fluorescence‐yield, [ 33 ] high‐ σ 3 fluorescent marker. [ 34 ] Due to its enhanced spatial confinement and sectioning ability, [ 11 ] the 3PEF readout enables high‐sharpness, high‐definition detection of HeLa cell boundaries (Figure 2, left). Cell nucleoli and small‐size organelles are also readily visible in 3PEF images.…”