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Free keywords:
Laser-Rastermikroskopie, Infrarotmikroskopie, In vivo, Zellpopulation, Neocortex, Gehirn, Laser-Scanning Microscopy , Brain , Cell Population
Abstract:
Two-photon microscopy has enabled high-resolution imaging of single cells in the brain of anaesthetized animals. Here we developed two-photon microscopy towards imaging of cell populations in the neocortex of awake behaving rodents. For this purpose, we developed two miniature two-photon microscopes based on fluorescence excitation through a hollow-core photonic crystal fiber and a coherent fiber-bundle, respectively. In addition, we demonstrate their applicability to in vivo imaging. Furthermore, as meaningful biological application critically depends on fluorescence labeling, we developed staining methods for three different cell populations. In particular, we used Sindbisand Lentiviral gene transfer into neurons for targeted expression of fluorescent indicators. We discovered a method for specific staining of astroglia in vivo, and we employed transgenic fluorescent protein expression to label microglia. Using a standard two-photon microscope, we show that in the adult brain neurons and astroglia show overall stable morphologies. In contrast, microglia displayed continuous structural changes, and responded rapidly to local injury. Furthermore, we uncovered the distinctive calcium dynamics underlying neuronal and astroglial cell signaling in vivo. Taken together, these advances in miniaturization and fluorescence labeling promise to enable optical studies of network activity during behavior.
