Two-photon excitation fluorescent spectral and decay properties of retrograde neuronal tracer Fluoro-Gold.

Matthew Q Miller, Iván Coto Hernández, Jenu V Chacko, Steven Minderler, Nate Jowett

Scientific reports 2021 Sep 10

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Fluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Widefield fluorescence microscopy enables visualization of Fluoro-Gold, but lacks depth discrimination. Though scanning laser confocal microscopy yields volumetric data, imaging depth is limited, and optimal single-photon excitation of Fluoro-Gold requires an unconventional ultraviolet excitation line. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. Deep imaging of Fluoro-Gold-labeled neurons carries potential to advance understanding of the central and peripheral nervous systems, yet its two-photon spectral and temporal properties remain uncharacterized. Herein, we report the two-photon excitation spectrum of Fluoro-Gold between 720 and 990 nm, and its fluorescence decay rate in aqueous solution and murine brainstem tissue. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of Fluoro-Gold labeled facial motor nuclei. Optimal two-photon excitation of Fluoro-Gold within microscope tuning range occurred at 720 nm, while maximum lifetime contrast was observed at 760 nm with mean fluorescence lifetime of 1.4 ns. Whole-mount brainstem explants were readily imaged to depths in excess of 450 µm via immersion in refractive-index matching solution. © 2021. The Author(s).

Citation

Matthew Q Miller, Iván Coto Hernández, Jenu V Chacko, Steven Minderler, Nate Jowett. Two-photon excitation fluorescent spectral and decay properties of retrograde neuronal tracer Fluoro-Gold. Scientific reports. 2021 Sep 10;11(1):18053