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 photobleac...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 1999-06, Vol.18 (2), p.231-239
Main Authors: Mainen, Z.F, Maletic-Savatic, M, Shi, S.H, Hayashi, Y, Malinow, R, Svoboda, K
Format: Article
Language:English
Subjects:
Animals
Brain - physiology
Dendrites - physiology
Dendrites - ultrastructure
Dissection - methods
Equipment Design
Fluorescent Dyes
Green Fluorescent Proteins
Hippocampus - cytology
Hippocampus - physiology
In Vitro Techniques
Lasers
Luminescent Proteins - analysis
Luminescent Proteins - genetics
Microscopy, Confocal - instrumentation
Microscopy, Confocal - methods
Microscopy, Video - instrumentation
Microscopy, Video - methods
Photons
Pyramidal Cells - cytology
Pyramidal Cells - physiology
Rats
Rats, Sprague-Dawley
Recombinant Fusion Proteins - analysis
Synapses - physiology
Synapses - ultrastructure
Synaptic Transmission
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Summary: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.
ISSN:1046-2023
1095-9130
DOI:10.1006/meth.1999.0776