Fluorescent labeling has opened up the possibility of clarifying the complex distribution and circuit wiring of specific neural circuits for particular functions. To acquire the brain-wide fluorescently labeled neural wiring, we have previously developed the fluorescence micro-optical sectioning tomography imaging system. This employs simultaneous mechanical sectioning and confocal imaging of the slices, and is capable of acquiring the image dataset of a centimeter-sized whole-mouse brain at a voxel resolution of 1 μm. We analyze the key optical considerations for the use of an acousto-optical deflector (AOD) scanner-based confocal detection scheme in this system. As a result, the influence of confocal detection, the imaging site during sectioning, and AOD fast scan mode on signal-to-background noise ratio are described. It is shown that mechanical sectioning to separate the slice and optical sectioning by confocal detection should be combined to maximize background suppression in simultaneous fast scan imaging while sectioning system setup.
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.