Description and performance of a highly sensitive confocal Raman microspectrometer

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Abstract
A confocal Raman microspectrometer was developed for the study of small biological objects such as single living cells and metaphase and polytene chromosomes. It employs a confocal detection scheme, well known from confocal fluorescence microscopes, in order to avoid signal contributions from the environment of the samples. The resolution is 0.45 ± 0.05 μm in the lateral direction and 1.3 ± 0.1 μm in the axial direction. The laser excitation wavelength is 660 nm. At this wavelength biological samples do not degrade in the laser radiation as was the case when laser radiation of 514.5 nm was used. The signal throughput from the sample position to the detector was optimized to the extent that in the spectral region around a 1000 cm−1 Raman shift 15% of the Raman scattered light collected by the microscope objective is detected. For signal detection a liquid nitrogen‐cooled slow‐scan CCD camera is used. Laser powers of 5–10 mW suffice to obtain high‐quality Raman spectra, with signal integration times of the order of minutes. As an example, spectra obtained from the nucleus and the cytoplasm of an intact human lymphocyte are shown.