Two-photon imaging in living brain slices

Mainen, Z. F., Maletic-Savatic, M., Shi, S. H., Hayashi, Y., Malinow, R., Svoboda, K. (June 1999) Two-photon imaging in living brain slices. Methods, 18 (2). pp. 231-39. ISSN 1046-2023 (Print)

DOI: 10.1006/meth.1999.0776


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.

Item Type: Paper
Uncontrolled Keywords: Animals Brain/ physiology Dendrites/physiology/ultrastructure Dissection/methods Equipment Design Fluorescent Dyes Green Fluorescent Proteins Hippocampus/cytology/ physiology In Vitro Lasers Luminescent Proteins/analysis/genetics Microscopy, Confocal/instrumentation/ methods Microscopy, Video/instrumentation/ methods Photons Pyramidal Cells/cytology/ physiology Rats Rats, Sprague-Dawley Recombinant Fusion Proteins/analysis Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Synapses/physiology/ultrastructure Synaptic Transmission
Subjects: organs, tissues, organelles, cell types and functions > organs types and functions > brain
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > green fluorescent protein
organs, tissues, organelles, cell types and functions > tissues types and functions > hippocampus
Investigative techniques and equipment > optical devices > lasers
organs, tissues, organelles, cell types and functions > cell types and functions > cell functions > synaptic transmission
CSHL Authors:
Communities: CSHL labs > Enikopolov lab
CSHL labs > Mainen lab
CSHL labs > Malinow lab
CSHL labs > Svoboda lab
Depositing User: Kathleen Darby
Date: June 1999
Date Deposited: 23 Apr 2014 20:17
Last Modified: 23 Apr 2014 20:17
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