Structural changes at the cellular and subcellular level in the cerebral cortex of mice visualized by means of trans-cranial multi photon in-vivo microscopy

Abstract

Neural cell structural change is associated with numerous processes in the normal and diseased brain. We report about a multi photon laser scanning microscope setup that permits visualization of such neuronal and glial structural dynamics in living mice and recent observations performed by means of this instrument. The modular system consists of a modified industrial standard CSLM and is to a large degree composed of commercially available components. From a technical point of view, two developments are essential: Firstly, a multifunctional stage adapted to both laser scanning microscopic observation and pre-observational surgery has been designed and built. Secondly, the essential component of the detection unit is a state-of-the-art photomultiplier tube installed in a Peltier cooled thermal box shielding the detector from both room temperature and distortions caused by external electromagnetic fields. An electro-optical modulator is used to accurately adjust the power of the applied laser beam and to blank the beam when no data are sampled, e.g. in the dead time intervals between adjacent pixels. Photo bleaching and thermal damage are thus kept on a minimum level. In transgenic mice expressing fluorescent proteins in neuronal or glial cells, details such as spines and astrocytic endfeet can be imaged trans-cranially to at least 80 microns below the brain surface. Individual cells and cell compartments can be visualized repeatedly over extensive periods of time.