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 speci® c 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 m m sphere appeared to have a full width at half-maximum (FWHM)of 1.2 m m in the axial and 0.37 m m in the lateral direction, whereas the image of a 275 nm layer showed an FWHM of 1.4 m 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 hthalocyanine-incubated ® xed 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 speci® c molecules in a sample can be made