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Five‐colour in vivo imaging of neurons in Caenorhabditis elegans
Author(s) -
HUTTER H.
Publication year - 2004
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.0022-2720.2004.01367.x
Subject(s) - green fluorescent protein , caenorhabditis elegans , confocal , fluorescence , in vivo , yellow fluorescent protein , axon , confocal microscopy , biophysics , resolution (logic) , microscope , biology , optics , physics , microbiology and biotechnology , computer science , genetics , artificial intelligence , gene
Summary In the last few years variants of the ‘green fluorescent protein’ (GFP) with different spectral properties have been generated. This has greatly increased the number of possible applications for these fluorochromes in cell biology. The significant overlap of the excitation and emission spectra of the different GFP variants imposes constraints on the number of variants that can be used simultaneously in a single sample. In particular, the two brightest variants, GFP and YFP, are difficult to separate spectrally. This study shows that GFP and YFP can be readily separated with little spectral overlap (cross‐talk) with the use of a confocal microscope equipped with an acusto‐optical beam splitter and freely adjustable emission windows. Under optimal recording conditions cross‐talk is less than 10%. Together with two other fluorescent proteins and the lipophilic dye DiD a total of five different colours can now be used simultaneously to label in vivo distinct anatomical structures such as neurons and their processes. Spatial resolution of the confocal microscope is sufficient to resolve the relative position of labelled axons within a single axon bundle. The use of five distinct marker dyes allows the in vivo analysis of the Caenorhabditis elegans nervous system at unprecedented resolution and richness in detail at the light microscopic level.