A new type of imaging Raman microscope is described. First the advantages and disadvantages of the two possible approaches to Raman microscopy based on signal detection by means of a charge-coupleddevice camera (i.e., direct imaging and image reconstruction) are discussed. Arguments are given to show that in most cases direct imaging is to be preferred over image reconstruction, because it provides the desired information in less time. In the direct imaging Raman microscope presented in this communication, detection of scattered light occurs in a narrow interval around a fixed wavelength. Selection of the Raman wavenumber shift at which an image is recorded is established by tuning the wavelength of the exciting laser light in such a way that the wavelength of the Raman scattered light with the desired Raman shift coincides with the detected wavelength. The microscope has been incorporated in a Raman microspectrometer in a way that enables easy switching between the imaging and the multichannel spectroscopy modes of operation. Bright field, fluorescence, and Raman microscopic images can be obtained.

Raman spectroscopy is being used to study biological molecules for some three decades now. Thanks to continuing advances in instrumentation more and more applications have become feasible in which molecules are studied in situ, and this has enabled Raman spectroscopy to enter the realms of biomedicine and cell biology [ 1-5]. Here we will describe some of the recent work carried out in our laboratory, concerning studies of human white blood cells and further instrumentational developments. 1. Carotenoids in lymphocytes Lymphocytes are white blood cells, responsible for the body's defense against virus infected cells and tumor cells. Epidemiological and experimental investigations have indicated that that there may exist an inverse relationship between blood plasma carotenoid levels, and the risk of developing cancer, especially lung cancer [6,7]. Recently we studied, combining fluorescence activated cell sorting and Raman microspectroscopy, the presence and subcellular location of carotenoids in lymphocyte subtypes, specifically in Thelper/inducer-lymphocytes (CD4+), Tcytotoxic/suppressor-lymphocytes (CD8+), T,//~-lymphocytes (TCR~,/8+), Natural Killer cells (CD16+) and B-lymphocytes (CD19+) [8]. The carotenoids appeared to be very specifically distributed among the different lymphocyte subtypes, which all have their own specific function in the immune system. The highest concentration of carotenoids (-10-3M) was found in the Gall body of Thelper/inducer-lymphocytes and Tcytotoxic/suppressor-lymphocytes. For comparison, the carotenoid concentration in blood plasma is of the order of 10-6M. In T~,/8-1ymphocytes, Natural Killer cells and Tcvtotoxic/suppressor lymphocytes the Golgi-complex appeared to be rich in carotenoids (-10-4 M). B-lymphocytes appeared to contain little or no carotenoids (i.e. < 10-SM), although they also possess a well-developed Golgi-complex. This very cell typespecific subcellular accumulation of carotenoids, which suggests a specific role for carotenoids in the immunesystem, has prompted further studies, in which lymphocyte carotenoid levels in lung cancer patients are compared with those of healthy individuals. Figure 1 shows a typical result. In comparison with the healthy donor the Gall bodies in the lymphocytes of the small cell lung carcinoma patient contain very little carotenoids. Comparison of blood plasma (measured by HPLC) and lymphocyte total carotenoid levels indicates that there does not exist