In 1873, Golgi employed silver staining to visualize nervous tissue under light microscopy 3. Retrograde neuronal labeling permits study of complex neural circuits, neuropathology, and nerve regeneration 1, 2. Whole-mount brainstem explants were readily imaged to depths in excess of 450В Вµm via immersion in refractive-index matching solution. Optimal two-photon excitation of Fluoro-Gold within microscope tuning range occurred at 720В nm, while maximum lifetime contrast was observed at 760В nm with mean fluorescence lifetime of 1.4В ns. We demonstrate unprecedented imaging depth of whole-mounted murine brainstem via two-photon excitation microscopy of Fluoro-Gold labeled facial motor nuclei. Herein, we report the two-photon excitation spectrum of Fluoro-Gold between 720 and 990В nm, and its fluorescence decay rate in aqueous solution and murine brainstem 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. Two-photon excitation microscopy employs ultrafast pulsed infrared lasers to image fluorophores at high-resolution at unparalleled depths in opaque tissue. 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. Widefield fluorescence microscopy enables visualization of Fluoro-Gold, but lacks depth discrimination. If you find this project useful in your research, please consider citing our paper: Wide field-of-view volumetric imaging by a mesoscopic scanning oblique plane microscopy with switchable objective lens.Fluoro-Gold is a fluorescent neuronal tracer suitable for targeted deep imaging of the nervous system. Zemax OpticStudio 16.5 SP3 or higher version The layout for emission path: Detailed microvasculature in zebrafish larvae, mouse cortex, and neurons in C. As a result, the system is highly versatile, allowing simple switching between different objective lenses with low (10x, NA 0.3) and median NA (20x, NA 0.5). The angle of the intermediate image plane is independent of the magnification. We demonstrate a mesoscopic FOV up to ~6Г— 5Г—0.6 mm3 volumetric imaging, the largest reported FOV by OPM so far. We characterized the 3D resolutions and FOV by imaging fluorescence microspheres, and demonstrated the volumetric imaging on intact whole zebrafish larvae, mouse cortex, and multiple Caenorhabditis elegans (C. The angle of the intermediate image before the remote focusing system was increased by a demagnification under Scheimpflug condition such that the light collecting efficiency in the remote focusing system was significantly improved. We implemented an optical design of mesoscopic scanning OPM to allow using low numerical aperture (NA) objective lens. Our goal is to overcome the limitation with a new variant of OPM to achieve mesoscopic FOV. However, most reports on OPM has a limited microscopic field of view (FOV), typically within 1x1 mm2. The recent development of oblique plane microscopy (OPM) simplifies Conventional light sheet fluorescence microscopy (LSFM) design with only one single objective lens, by using off-axis excitation and remote focusing. Ray tracing and analysis for both the excitation and emission light paths of our mesoscopic OPM are provided in the Zemax files. Zemax simulation of a mesoscopic scanning oblique plane microscopy

20x 0.5na zemax file