Retrograde neuronal labeling permits study of complex neural circuits, neuropathology, and nerve regeneration [1, 2]. In 1873, Camillo Golgi used silver staining to visualize nervous tissue under light microscopy . Ramón y Cajal optimized this technique in his pioneering neuroanatomical studies underpinning the neuron doctrine [3, 4]. Weiss and Hiscoe described anterograde axonal transport in 1948 . In 1971, Kristensson and Olsson demonstrated retrograde axonal transport by injecting horseradish peroxidase (HRP) into murine gastrocnemius muscle and later observing tracer in spinal cord sections . Evans blue was the first fluorescent dye used for retrograde neuronal labeling; many others have since been reported [1, 7–12]. Fluorescent dyes allow high-fidelity neuronal labeling without resource-intensive immunohistochemical techniques.
Schmued and Fallon first described Fluoro-Gold™ (FG) in 1986, noting its intense fluorescence, specific labeling of damaged axons, and resistance to photobleaching . Hydroxystilbamidine (OHSA) is the active fluorophore in FG, an amidine similar to DAPI, True blue, and other substances that undergo retrograde transport . After axonal uptake, 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 environments .
FG can be used to label neuronal cell bodies of the central and peripheral nervous systems, and shows minimal neurotoxicity at low concentrations (2–5%) [10, 13, 15–19]. Within the peripheral nervous system, FG may be employed for labeling intact motor neurons via intramuscular injection, or axotomized motor and sensory neurons via immersion of proximal nerve stumps, using conduit reservoir or crystal application techniques [1, 9, 13, 14, 20]. Hayashi et al. demonstrated conduit reservoir delivery of FG labeled the greatest number of neurons . FG is compatible with immunohistochemistry (IHC) and tissue clearing techniques [21–23].
Optimal single-photon excitation of FG is achieved using ultraviolet light. Imaging of FG-labeled specimens is typically performed using widefield fluorescence microscopy, with standard DAPI/Hoechst filter sets yielding narrow-band 365 nm excitation and long-pass filters providing broadband detection [1, 9, 13, 14, 24, 25]. However, widefield imaging lacks depth discrimination, preventing optical sectioning and high-resolution three-dimensional (3D) imaging. Confocal microscope short-wavelength excitation lasers at 405 nm are not suitable for excitation of FG . Secondary tagging of FG by immunofluorescence may be employed to visualize FG using visible excitation lines of commerical confocal microscopes, though this approach is resource-intensive [26, 27].
Two-photon excitation microscopy (2PEM) is an alternative to confocal microscopy for volumetric imaging of biological tissues. Maria Goeppert-Mayer characterized the theoretical basis for 2PEM in 1931 and six decades later, Denk et al. first demonstrated the technique [28, 29]. Two-photon excitation (2PE) employs near-infrared (NIR) ultrafast laser pulses to achieve simultaneous absorption of two low-energy photons by a fluorophore typically excited by a single higher-energy photon. Multiphoton NIR excitation is possible for manifold fluorophores, including many which are not suitably excited using visible light [30, 31]. Use of NIR excitation in the optical window of biological tissue in 2PEM permits microscale resolution of labeled-structures in highly-scattering thick tissues . Owing to the quadratic dependence of fluorescence signal on excitation intensity in 2PEM, out-of-plane fluorescence is largely avoided, providing enhanced axial resolution compared to scanning laser confocal microscopy . Under ideal conditions, confocal microscopy can image tissues at depths up to 100 µm, while depths up to 1 mm have been reported with 2PEM [33–36]. Though the 2PE spectral properties of many fluorescent dyes have been documented, the 2PEM excitation spectra of FG has not been heretofore characterized .
Due to FG’s broad fluorescent emission spectrum, spectral overlap with other fluorophores is common and can hinder multicolor imaging. Several approaches may be employed for multicolor imaging of FG- labeled specimens. In single-photon imaging, UV excitation of FG may enable its separation from fluorophores not excited by UV light [15, 38]. Fluorescence lifetime imaging (FLIM) obtains images based on fluorophore decay rate in lieu of fluorescent intensity, yielding means to resolve fluorescent labels with overlapping absorption and emission spectra [39–41]. FLIM necessitates use of short-pulse excitation lasers, and may be implemented using single or multiphoton excitation techniques. Ultrafast pulsed lasers used for 2PEM are well-suited for FLIM; such microscope systems can readily be upgraded to enable FLIM . A priori knowledge of specific decay rates of various fluorophores enables selection of optimal candidates for multi-label experiments. Heretofore, the decay rate of FG has not been characterized.
Herein, we characterize the 2PE spectra and fluorescent lifetime distribution of FG in aqueous solution and murine facial motor nuclei. We then demonstrate the utility of 2PEM for high-throughput deep volumetric imaging of FG-labeled mammalian neurons.