Confocal direct imaging Raman microscope: Design and applications in biology

A confocal direct imaging Raman microscope (CDIRM) based on two synchronized scanning mirrors, a monochromator, and two charge-coupled device (CCD) cameras has been developed. With this system it is possible to make both Raman spectra of a small measurement volume and images of a larger sample area in one specific Raman band. The spatial resolution of the system was determined for two limiting situations: a small sphere and a thin layer. The image of a 0.282 mu m sphere appeared to have a full width at half-maximum (FWHM) of 1.2 mu m in the axial and 0.37 mu m in the lateral direction, whereas the image of a 275 nm layer showed an FWHW of 1.4 mu m in the axial direction. Confocal Raman images were made of the DNA and protein distribution in polytene chromosomes with a relatively weak Raman signal [0.1 photons/(second pixel)]. Further, a three-dimensional Raman image of the drug distribution in a phthalocyanine-incubated fixed cell is presented. These examples show that the CDIRM can be used to image samples with a weak Raman signal and that three-dimensional images of the distribution of specific molecules in a sample can be made.