Abstract
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.
Highlights
Retrograde neuronal labeling permits study of complex neural circuits, neuropathology, and nerve regeneration[1,2]
Confocal microscopy can image tissues at depths up to 100 μm, while depths up to 1 mm have been reported with 2PEM35–38
The 2PE fluorescent spectral and decay properties for FG in aqueous solution (2% w/v in distilled water) and murine brainstem tissue fixed with a phosphate-buffered formalin solution were determined using a commercial multiphoton microscope, powered by a tunable mode-locked Ti:Sapphire laser between 720–990 nm (Fig. 1A–C)
Summary
Retrograde neuronal labeling permits study of complex neural circuits, neuropathology, and nerve regeneration[1,2]. Evans blue was the first fluorescent dye used for retrograde neuronal labeling; many others have since been r eported[1,7,8,9,10,11,12]. Fluorescent dyes allow high-fidelity neuronal labeling without resource-intensive immunohistochemical techniques. FG accumulates in acidic lysosomes and endosomes and is transported to the cell body where it labels cytoplasm and dendrites[13,14] It produces a broad fluorescence emission spectrum, with an intense yellow peak at neutral pH that is blue-shifted in acidic e nvironments[13]. Use of NIR excitation in the optical window of biological tissue in 2PEM permits microscale resolution of labeled structures in highly-scattering thick tissues[34]. Though the 2PE spectral properties of many fluorescent dyes have been documented, the 2PEM excitation spectra of FG have not been heretofore characterized[39]
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