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

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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.

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Modified from (Ricard and Debarbieux 2014)

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CH is supported by an F30 Fellowship (NS093719 from NIH NINDS), as well as the UCLA Medical Scientist Training Program (NIH NIGMS training grant GM08042). CP-C is supported by W81XWH-14-1-0433 (USAMRMC, DOD), a Developmental Disabilities Translational Research Program Grant (#20160969 from John Merck), SFARI Awards 295438 and 513155CP (Simons Foundation) and 5R01HD054453 (NICHD/NIH). GL is supported by the Agence Nationale de la Recherche (ANR-15-CE37-0004 “SmellBrain” and ANR-15-NEUC-0004 “Circuit_OPL”).

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Correspondence to Daniel Fiole.

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GL is supported by the life insurance company AG2R-La-Mondiale (“Vivons vélo pour l’Institut Pasteur”).

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All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted. This article does not contain any studies with human participants performed by any of the authors.

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Biophysical properties of two-photon–suitable probes in alphabetical order: peak wavelength of two-photon action cross section (λ2PA); peak two-photon action cross section (σ2φ) ; peak wavelength of molecular brightness (λε_max) ; peak molecular brightness (εmax) ; fluorescence wavelength (λfluo). (1) : calculated from the σ2 and φ values (2) : data from commercial provider (XLSX 45 KB)

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Ricard, C., Arroyo, E.D., He, C.X. et al. Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells. Brain Struct Funct 223, 3011–3043 (2018).

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