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Application of photoconversion technique for correlated confocal and ultrastructural studies in organotypic slice cultures
Author(s) -
Nikonenko Irina,
Boda Bernadett,
Alberi Stefano,
Muller Dominique
Publication year - 2005
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20239
Subject(s) - ultrastructure , electron microscope , dendritic spine , confocal microscopy , confocal , axon , transmission electron microscopy , biophysics , staining , biology , microscopy , fluorescence , microbiology and biotechnology , chemistry , hippocampal formation , anatomy , materials science , nanotechnology , neuroscience , optics , physics , genetics
Photoconversion of fluorescent staining into stable diaminobenzidine (DAB) precipitate is widely used for neuroanatomical and developmental studies. An important advantage of the approach is to make correlations between light and electron microscopy analyses possible, the DAB reaction product formed during photoconversion being electron dense. By combining a photoconversion approach with biolistic transfection of neurons in organotypic hippocampal slice cultures, we describe here a methodology that allowed us to study at the electron microscopy level the fine details of cells expressing specific genes of interest. The same approach has also been used to analyze the ultrastructural characteristics of specific cells such as neurons recorded with patch clamp techniques. This approach revealed particularly useful for studies of dendritic arborisation, dendritic spines, and axon varicosities of identified cells, as precise morphometric parameters of these structures can only be obtained by electron microscopy. The techniques used for fluorescent staining and photoconversion of these different cell structures and the results obtained by electron microscopic analyses are described. Microsc. Res. Tech. 68:90–96, 2005. © 2005 Wiley‐Liss, Inc.