Dynamic confocal imaging in acute brain slices and organotypic slice cultures using a spectral confocal microscope with single photon excitation.

Abstract

Confocal imaging in living brain slices allows the resolution of submicrometre structures of nerve cells, glia and brain vessels. Imaging living brain slices is in many respects different from conventional fixed histological preparations for which confocal microscopes were designed originally. Several problems (i.e. mechanical and thermal drift, and autofluorescence) resulting from the optical and structural properties of brain slices are discussed. Fluorescent indicators may be used to monitor numerous intracellular parameters such as pH and Ca(2+) concentration, but not all of them are equally suitable for this type of work. Genetically engineered fluorescent proteins can be used to visualise the fine dendritic structure of neurones or track particular intracellular structures and proteins. They have also been used to generate indicators for Ca(2+), cAMP and other molecules. While conventional chemical indicators can be either loaded into neurones via patch pipettes or as membrane-permeable esters, protein indicators can be expressed in various types of cells using adenoviral vectors. Adenoviral transgenesis can be performed in vitro in both acute slices and organotypic slice cultures. Organotypic slice cultures give excellent optical access to neurones loaded with either conventional fluorescent indicators or transfected with adenovirus to express fluorescent proteins. They are most suitable for experiments where both conventional and genetically engineered indicators are combined. Single photon imaging in brain slices is limited to the superficial layers (approximately<or=50 microm), while multiphoton excitation has a much greater depth of penetration. However, the overall optical resolution achievable in single photon mode is at least as good as when using multiphoton excitation.