Development and application of Raman microspectroscopic and Raman imaging techniques for cell biological studies

Abstract

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

Raman spectroscopy: techniques and applications in the life sciences

“…Huang et al replaced one of the carbons (usually 12 C) in phenylalanine with 13 C, shifting the band from 1003 to 967 cm −1 . They detected no biological effects on the labeled microbes [450].…”

“…Venkata et al labeled glucose with 13 C and observed its uptake by live yeast cells [454]. The authors showed that the 13 C was incorporated into proteins (marked by the phenylalanine peak shifted from 1003 to 967 cm −1 ) localized in lipid droplets see Figure 36(a) .…”

“…[454]. 13 C is incorporated into proteins in lipid droplets over 30+ hours. (b) Interaction of Toxoplasma gondii parasites and host cells studied by Naemat et al [145].…”

“…Other important developments accelerating the progress of Raman spectroscopy include the digitization of spectra using charge-coupled devices (CCDs) [7,8], the confocal Raman microscope [9], and improved filters to remove light at the laser wavelength [10]. These inventions allowed a rapid increase in the popularity of using Raman to study biological samples in the early 1990s [11][12][13].…”

Chemotaxonomic Identification of Single Bacteria by Micro-Raman Spectroscopy: Application to Clean-Room-Relevant Biological Contaminations

“…In 1990, Puppels et al developed a confocal Raman microscope, capable of recording Raman spectra of single human cells and polytene chromosomes (42). Since then, many biological phenomena in single human cells have been studied by Puppel's group; e.g., Raman spectra of the cell nucleus and the cell cytoplasm in human white blood cells were obtained (43)(44)(45)(46). Various groups have reported the classification of bacteria by means of Raman spectroscopy (3,11,12,17,27,(31)(32)(33)(34)47).…”