Towards Chemical Imaging of Living Cells: Design and Application of a Confocal Raman Microscope

Raman microspectroscopy is a technique that can be used to obtain information about the chemical composition of a very small measurement volume (0.5 fl) in a (biological) sample. Molecules present in the sample can be identified based on their scattering characteristics and no special treatment or preparation of the samples is necessary. Therefore, biological samples can be measured under physiological conditions and reactions in living cells can be monitored. We have developed a Confocal Direct Imaging Raman Microscope (CDIRM) which enables the measurement of both Raman microspectra of a small measurement volume and of Raman images which show the distribution of a specific compound over the sample. The CDIRM is the first example in literature of a confocal microscope which is based upon direct imaging. All currently used confocal Raman microscopes work with image reconstruction. Direct imaging has several advantages among which the shorter measurement times that can be used in most applications. In chapter 2 the design of the system has been discussed and its mains characteristics, like resolution and image quality have been described. The resolution of the set up as determined with a 0.282 μm sphere appeared to be 0.37 μm in the lateral direction and 1.2 μm in the axial direction (Full Width at Half Maximum (FWHM)). The resolution for a 275 nm layer was determined to be 1.4 μm in the axial direction. We have demonstrated that high resolution Raman images of biological samples can be made with the CDIRM. Raman images have been measured of the DNA and protein distribution in a polytene chromosome. These images illustrate the capability of our system to make Raman images of a sample with a relatively weak Raman signal: only 0.1 photons/(second×pixel) were detected. Further, we have shown that our system can be used to make 3- dimensional Raman images of biological samples. 3-dimensional images of the distribution of a drug in a living cell and of cholesterol in an eye lens slice have been presented. Raman microspectroscopy is one of the few techniques that enables the monitoring of processes in single living cells, without chemical treatment of the sample which might disturb the cellular system. In chapter 3 Raman measurements on single activated human neutrophilic and eosinophilic granulocytes have been shown. The granulocytes were activated by addition of the soluble activator Phorbol Myristate Acetate or by opsonized particles. Raman spectra were measured in the cytoplasm and the phagosome of activated granulocytes. The resulting spectra were y 123 compared with spectra of the native cells and clear differences could be recognized. The results indicated an intracellular reduction of both Myeloperoxidase and cytochrome b558, two heme-proteins which are known to play a role in the human immune system. An important advantage of Raman imaging compared to fluorescence imaging is that no extrinsic labels have to be introduced to distinguish specific molecules. However, in samples with a low concentration of weak Raman scattering molecules it can be advantageous to introduce extrinsic labels. These Raman labels should bind specifically to the molecules of interest and have a relatively large Raman scattering cross section. In certain applications it can be preferable to use such Raman labels instead of fluorescent labels, because of their much narrower bandwidth, which allows the detection of many more different labels in a limited wavelength range and because they do not bleach. In chapter 4 two examples of extrinsic Raman labeling have been demonstrated: the use of the cholesterol specific label filipin for visualizing the cholesterol distribution in an eye lens and the application of antibody coated polystyrene spheres to distinguish different phenotypes of human leukocytes. Further, a discussion is given about which molecules and structures can be used in the development of other suitable Raman labels.