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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.

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We developed silicon nitride cantilevers integrating a probe tip and a wave guide that is prolonged on the silicon holder with one or two guides. A micro‐system is bonded to a photodetector. The resulting hybrid system enables us to obtain simultaneously topographic and optical near‐field images. Examples of images obtained on a longitudinal cross‐section of an optical fibre are shown.