Raman microspectroscopy is a technique that can be used to obtainudinformation about the chemical composition of a very small measurementudvolume (0.5 fl) in a (biological) sample. Molecules present in the sampleudcan be identified based on their scattering characteristics and no specialudtreatment or preparation of the samples is necessary. Therefore, biologicaludsamples can be measured under physiological conditions and reactions inudliving cells can be monitored.udWe have developed a Confocal Direct Imaging Raman Microscopeud(CDIRM) which enables the measurement of both Raman microspectra ofuda small measurement volume and of Raman images which show theuddistribution of a specific compound over the sample. The CDIRM is theudfirst example in literature of a confocal microscope which is based uponuddirect imaging. All currently used confocal Raman microscopes work withudimage reconstruction. Direct imaging has several advantages among whichudthe shorter measurement times that can be used in most applications.udIn chapter 2 the design of the system has been discussed and its mainsudcharacteristics, like resolution and image quality have been described. Theudresolution of the set up as determined with a 0.282 μm sphere appeared toudbe 0.37 μm in the lateral direction and 1.2 μm in the axial direction (FulludWidth at Half Maximum (FWHM)). The resolution for a 275 nm layer wasuddetermined to be 1.4 μm in the axial direction. We have demonstrated thatudhigh resolution Raman images of biological samples can be made with theudCDIRM. Raman images have been measured of the DNA and proteinuddistribution in a polytene chromosome. These images illustrate theudcapability of our system to make Raman images of a sample with audrelatively weak Raman signal: only 0.1 photons/(second×pixel) wereuddetected. Further, we have shown that our system can be used to make 3-uddimensional Raman images of biological samples. 3-dimensional images ofudthe distribution of a drug in a living cell and of cholesterol in an eye lensudslice have been presented.udRaman microspectroscopy is one of the few techniques that enables theudmonitoring of processes in single living cells, without chemical treatmentudof the sample which might disturb the cellular system. In chapter 3 Ramanudmeasurements on single activated human neutrophilic and eosinophilicudgranulocytes have been shown. The granulocytes were activated byudaddition of the soluble activator Phorbol Myristate Acetate or byudopsonized particles. Raman spectra were measured in the cytoplasm andudthe phagosome of activated granulocytes. The resulting spectra wereudyud123udcompared with spectra of the native cells and clear differences could beudrecognized. The results indicated an intracellular reduction of bothudMyeloperoxidase and cytochrome b558, two heme-proteins which areudknown to play a role in the human immune system.udAn important advantage of Raman imaging compared to fluorescenceudimaging is that no extrinsic labels have to be introduced to distinguishudspecific molecules. However, in samples with a low concentration of weakudRaman scattering molecules it can be advantageous to introduce extrinsicudlabels. These Raman labels should bind specifically to the molecules ofudinterest and have a relatively large Raman scattering cross section. Inudcertain applications it can be preferable to use such Raman labels insteadudof fluorescent labels, because of their much narrower bandwidth, whichudallows the detection of many more different labels in a limited wavelengthudrange and because they do not bleach. In chapter 4 two examples ofudextrinsic Raman labeling have been demonstrated: the use of theudcholesterol specific label filipin for visualizing the cholesterol distributionudin an eye lens and the application of antibody coated polystyrene spheresudto distinguish different phenotypes of human leukocytes. Further, auddiscussion is given about which molecules and structures can be used inudthe development of other suitable Raman labels.
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