Multicolor high-resolution whole-brain imaging for acquiring and comparing the brain-wide distributions of type-specific and projection-specific neurons with anatomical annotation in the same brain.

Visualizing the relationships and interactions among different biological components in the whole brain is crucial to our understanding of brain structures and functions. However, an automatic multicolor whole-brain imaging technique is still lacking. Here, we developed a multicolor wide-field large-volume tomography (multicolor WVT) to simultaneously acquire fluorescent signals in blue, green, and red channels in the whole brain. To facilitate the segmentation of brain regions and anatomical annotation, we used 4', 6-diamidino-2-phenylindole (DAPI) to provide cytoarchitecture through real-time counterstaining. We optimized the imaging planes and modes of three channels to overcome the axial chromatic aberration of the illumination path and avoid the crosstalk from DAPI to the green channel without the modification of system configuration. We also developed an automatic contour recognition algorithm based on DAPI-staining cytoarchitecture to shorten data acquisition time and reduce data redundancy. To demonstrate the potential of our system in deciphering the relationship of the multiple components of neural circuits, we acquired and quantified the brain-wide distributions of cholinergic neurons and input of ventral Caudoputamen (CP) with the anatomical annotation in the same brain. We further identified the cholinergic type of upstream neurons projecting to CP through the triple-color collocated analysis and quantified its proportions in the two brain-wide distributions. Both accounted for 0.22%, implying CP might be modulated by non-cholinergic neurons. Our method provides a new research tool for studying the different biological components in the same organ and potentially facilitates the understanding of the processing mechanism of neural circuits and other biological activities.