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• W1567707754 abstract "Raman microspectroscopy is a technique that can be used to obtaininformation about the chemical composition of a very small measurementvolume (0.5 fl) in a (biological) sample. Molecules present in the samplecan be identified based on their scattering characteristics and no specialtreatment or preparation of the samples is necessary. Therefore, biologicalsamples can be measured under physiological conditions and reactions inliving cells can be monitored.We have developed a Confocal Direct Imaging Raman Microscope(CDIRM) which enables the measurement of both Raman microspectra ofa small measurement volume and of Raman images which show thedistribution of a specific compound over the sample. The CDIRM is thefirst example in literature of a confocal microscope which is based upondirect imaging. All currently used confocal Raman microscopes work withimage reconstruction. Direct imaging has several advantages among whichthe shorter measurement times that can be used in most applications.In chapter 2 the design of the system has been discussed and its mainscharacteristics, like resolution and image quality have been described. Theresolution of the set up as determined with a 0.282 μm sphere appeared tobe 0.37 μm in the lateral direction and 1.2 μm in the axial direction (FullWidth at Half Maximum (FWHM)). The resolution for a 275 nm layer wasdetermined to be 1.4 μm in the axial direction. We have demonstrated thathigh resolution Raman images of biological samples can be made with theCDIRM. Raman images have been measured of the DNA and proteindistribution in a polytene chromosome. These images illustrate thecapability of our system to make Raman images of a sample with arelatively weak Raman signal: only 0.1 photons/(second×pixel) weredetected. Further, we have shown that our system can be used to make 3-dimensional Raman images of biological samples. 3-dimensional images ofthe distribution of a drug in a living cell and of cholesterol in an eye lensslice have been presented.Raman microspectroscopy is one of the few techniques that enables themonitoring of processes in single living cells, without chemical treatmentof the sample which might disturb the cellular system. In chapter 3 Ramanmeasurements on single activated human neutrophilic and eosinophilicgranulocytes have been shown. The granulocytes were activated byaddition of the soluble activator Phorbol Myristate Acetate or byopsonized particles. Raman spectra were measured in the cytoplasm andthe phagosome of activated granulocytes. The resulting spectra werey123compared with spectra of the native cells and clear differences could berecognized. The results indicated an intracellular reduction of bothMyeloperoxidase and cytochrome b558, two heme-proteins which areknown to play a role in the human immune system.An important advantage of Raman imaging compared to fluorescenceimaging is that no extrinsic labels have to be introduced to distinguishspecific molecules. However, in samples with a low concentration of weakRaman scattering molecules it can be advantageous to introduce extrinsiclabels. These Raman labels should bind specifically to the molecules ofinterest and have a relatively large Raman scattering cross section. Incertain applications it can be preferable to use such Raman labels insteadof fluorescent labels, because of their much narrower bandwidth, whichallows the detection of many more different labels in a limited wavelengthrange and because they do not bleach. In chapter 4 two examples ofextrinsic Raman labeling have been demonstrated: the use of thecholesterol specific label filipin for visualizing the cholesterol distributionin an eye lens and the application of antibody coated polystyrene spheresto distinguish different phenotypes of human leukocytes. Further, adiscussion is given about which molecules and structures can be used inthe development of other suitable Raman labels." @default.
• W1567707754 created "2016-06-24" @default.
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• W1567707754 date "1997-10-03" @default.
• W1567707754 modified "2023-09-26" @default.
• W1567707754 title "Towards Chemical Imaging of Living Cells: Design and Application of a Confocal Raman Microscope" @default.
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