Klaus Becker (Vienna University of Technology)
Optical microscopy is one method used to visualize how neurons are connected by their axons and dendrites. However, most microscopy techniques generate two-dimensional images, which are unsuitable for understanding the three dimensional morphology of large-range neural connections. Especially, axons often extend over long distances and rarely lie in a single twodimensional plane. This limitation can be addressed using three-dimensional microscopy techniques. One such method, known as light sheet microscopy, involves scanning a transparent specimen with a thin sheet of laser light. The resulting scan can then be reconstructed and visualized in three dimensions on a computer. For this technique to be effective, the brain tissue must be rendered transparent, and fluorescent dyes are used to label the neurons. Our research has focused on developing new methods for making brain tissue transparent (clearing) and fluorescently labeling neurons. Using mice and rats as model organisms, we developed improved tissue-clearing methods. This process involved extensive testing of different chemical treatments, ultimately resulting in a completely transparent rat brain. We then applied light sheet microscopy to rat brain specimens with pre-labeled fluorescent neurons, enabling three-dimensional visualization of dendrites and the paths of long axon projections. Our innovative protocol is also compatible with other fluorescent labeling techniques, such as antibody labeling, which facilitates the identification of specific proteins within the cleared brain.