Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells

Abstract

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

Keywords: Calcium imaging; Electroporation; Functional imaging; Intravital; Multicolor microscopy; Two-photon cross section.

Fig. 1

Optical setup for multicolor two-photon microscopy DC: dichroic mirror; NDD: non-descanned detector; OPO: optical parametric oscillator. ¹: Dichroic mirror used to collect fluorescent photons on five non-descanned detectors. Excitatory infrared photons pass through the mirror whereas fluorescence photons are reflected. This mirror must be removed to collect photons in descanned mode. ²: Dichroic mirror used to collect fluorescent photons on a spectral chip in descanned mode. Modified from (Ricard and Debarbieux 2014).

Fig. 2

Wavelength mixing–fluorescence lifetime imaging of endogenous fluorophores When the excitation beams λ₁ = 760 nm and λ ₂ = 1041 nm are synchronized (Δt = 0), a third virtual wavelength for two-photon excitation λ _v = 879 nm is created by wavelength mixing. A time-correlated single photon counting system (TCSPC) is used to perform FLIM. Originally published in Scientific reports (Stringari et al. 2017) under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Fig. 3

Wavelength-mixing optimal combination of two sets of fluorophores Two-photon excitation and emission spectra of two sets of fluorophores that can be efficiently excited by wavelength mixing with λ ₁ = 850 nm and λ ₂ = 1230 nm (full arrows), resulting in λ _v = 1005 nm (dashed arrow). (a) CFP, eGFP, mOrange2, mKate2, eqFP670. (b) Hoechst, eGFP, Kusabira Orange, CMTPX Red, Alexa 647, Atto 680. Originally published in Scientific reports (Rakhymzhan et al. 2017) under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).

Fig. 4

Example of similarity unmixing of more fluorochromes than available channels (a) Images of cells labelled with several fluorophores, showing crosstalk in two detection channels. (b) Extraction of a relative signal distribution in the two channels pixels by pixel. (c) The algorithm calculates similarities between the known fingerprints of each fluorophore and the signature of undefined fluorophores extracted during step (b). (d) Color separation based on the ratios between the fluorophore fingerprints. Originally published in Scientific reports (Rakhymzhan et al. 2017) under a Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/).