Deep imaging of living mouse brain utilizing novel laser photonics technologies

Abstract

For the field of neuroscience, laser-scanning florescence microscopy utilizing two-(or multi-) photon excitation process (two-(multi-)photon excitation laser scanning microscopy, two-(multi-)photon microscopy) has become widely used as an essential tool for biological and medical research including cancer, and immune researches. Especially, “in vivo” two-photon microscopy has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. To break the limit of this penetration depth, we introduced a novel light source based on a semiconductor laser. The light source successfully visualized not only cortex layer V pyramidal neurons spreading to all cortex layers at a superior S/N ratio, but visualize hippocampal CA1 neurons in young adult mice. In addition, we developed liquid crystal devices to convert linearly polarized beams (LP) to vector beams. A liquid device generated a vector beam called higher-order radially polarized (HRP) beam, that enabled us to identify individual fluorescent beads of which diameter is 170 nm; smaller than classical PSF width. HRP beam also visualized finer structures of microtubules in fixed cells. Here, we will discuss these improvements and future application on the basis of our recent data.