Label-free spectral confocal reflectance microscopy for ex vivo neuroimaging and neural structure visualization

Confocal microscopy is an essential technique in the field of life sciences, commonly used to study molecules in a variety of preparations, ranging from cells and tissues to entire organisms. In the field of neuroscience, it is a widely utilized tool for both anatomical-structural and functional studies. However, a lesser-known application of this microscopy method is confocal reflectance microscopy, which involves image acquisition based on reflected light from the sample. In this study, we present the use of spectral confocal reflectance microscopy (SCoRe) to explore multiple structures in the rat brain, without the use of any dyes or immunolabeling. Our results demonstrate that this technique allows for the distinction between different brain structures with high spatial resolution, enabling the observation of fibers in the cerebral cortex, corpus callosum, hippocampus, cerebellum, and other regions. These findings highlight the ability of SCoRe to provide detailed anatomical insights that are comparable to those obtained through conventional methods. Additionally, we have shown that SCoRe is compatible with samples prepared using traditional techniques, such as histochemical staining and immunofluorescence. This research emphasizes the value of SCoRe as a cost-effective and label-free method for high-resolution brain imaging, which can improve neuroscience studies and reduce long-term expenses.