We report a novel fluorescence imaging approach to imaging nonfluorescence-labeled biological tissue samples. The method was demonstrated by imaging neurons in Golgi-Cox-stained and epoxy-resin-embedded samples through the excitation of the background fluorescence of the specimens. The dark neurons stood out clearly against background fluorescence in the images, enabling the tracing of a single dendritic spine using both confocal and wide-field fluorescence microscopy. The results suggest that the reported fluorescence imaging method would provide an effective alternative solution to image nonfluorescence-labeled samples, and it allows tracing the dendritic spine structure of neurons.
Imaging brain circuits is the basis for us to understand brain function and dysfunction. However, imaging axon at micrometer resolution while tracing the centimeter-scale axon projection across the whole-brain is still challenging. Here, we developed a fluorescence micro-optical sectioning tomography (fMOST) imaging system based on confocal fluorescence imaging scheme that can obtain whole brain image stack for visualizing brain circuits at neurite level. We use confocal detection to remove fluorescence background to clearly see one single neurite and use acoustical optical deflector (AOD), an inertia-free beam scanner to realize fast and prolonged stable imaging. We had acquired several complete datasets of whole-mouse brain at a one-micron voxel resolution. Based on these datasets, the uninterrupted tracing of brain-wide, long-distance axonal projections was demonstrated for the first time using a systematic reconstruction and annotation pipeline. Our method is believed to open an avenue to exploring both local and long-distance neural circuits that are related to brain functions and brain diseases down to the neurite level.
A recently reported micro-optical sectioning tomography system has great potential to draw the neuronal circuits of large brain volume with submicron resolution by combining fine mechanic sectioning with simultaneous optical imaging. However, sectioning the fluorescence sample sometimes induces tears between adjacent tiles and causes difficulties in continuous fiber tracing from fluorescence imaging. A confocal detection to recover the interruptions of the nerve fiber is introduced. With a 50-μm-width confocal slit, the signal-to-background ratio is increased 16- to 49-fold more than that without the slit, which effectively improves the detectability of the signal in the interruptions and enables continuous tracing of the neuronal circuits.
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