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Near‐field microscopy and fluorescence spectroscopy: application to chromosomes labelled with different fluorophores
Author(s) -
MAHIEUWILLIAME L.,
FALGAYRETTES P.,
NATIVEL L.,
GALLBORRUT P.,
COSTA L.,
SALEHZADA T.,
BISBAL C.
Publication year - 2010
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.1365-2818.2009.03326.x
Subject(s) - acridine orange , fluorescence , fluorescence microscope , microscopy , microscope , optics , optical microscope , spectroscopy , fluorescence spectroscopy , chromatin , chemistry , materials science , dna , scanning electron microscope , staining , physics , biology , genetics , biochemistry , quantum mechanics
Summary We have coupled a spectrophotometer with a scanning near‐field optical microscope to obtain, with a single scan, simultaneously scanning near‐field optical microscope fluorescence images at different wavelengths as well as topography and transmission images. Extraction of the fluorescence spectra enabled us to decompose the different wavelengths of the fluorescence signals which normally overlap. We thus obtained images of the different fluorescence emissions of acridine orange bound to single or double stranded nucleic acids in human metaphase chromosomes before and after DNAse I or RNAse A treatment. The analysis of these images allowed us to visualize some specific chromatin areas where RNA is associated with DNA showing that such a technique could be used to identify multiple components within a cell.