Two-photon imaging in living brain slices.

Two-photon excitation laser scanning microscopy (TPLSM) has become the tool of choice for high-resolution fluorescence imaging in intact neural tissues. Compared with other optical techniques, TPLSM allows high-resolution imaging and efficient detection of fluorescence signal with minimal photobleaching and phototoxicity. The advantages of TPLSM are especially pronounced in highly scattering environments such as the brain slice. Here we describe our approaches to imaging various aspects of synaptic function in living brain slices. To combine several imaging modes together with patch-clamp electrophysiological recordings we found it advantageous to custom-build an upright microscope. Our design goals were primarily experimental convenience and efficient collection of fluorescence. We describe our TPLSM imaging system and its performance in detail. We present dynamic measurements of neuronal morphology of neurons expressing green fluorescent protein (GFP) and GFP fusion proteins as well as functional imaging of calcium dynamics in individual dendritic spines. Although our microscope is a custom instrument, its key advantages can be easily implemented as a modification of commercial laser scanning microscopes.