C.J. de Grauw

Line-Scan Raman Microspectrometry for Biological Applications

A high-resolution confocal line-scan Raman microscope was developed for the study of biological samples such as cells and chromosomes. With the illumination of a line on the sample, all the spectral information of the line is recorded. The line-scan microscope was attained by the introduction of one scanning mirror, a slit, and two cylindrical lenses in a standard confocal Raman microscope. The resolution, for an ideal sample, is 0.5 μm in the lateral direction and 3.5 μm in the axial direction. Line-scan experiments were performed to demonstrate the applicability of the instrument for biological samples. Line-scan images of calcium phosphate bone implant coatings and of the banding pattern of polytene chromosomes are presented.

Applications of Micro-Raman Imaging in Biomedical Research

Recent results are presented of the application of imaging micro-Raman spectrometers in cellular biophysics and biomedical research. Various micro-Raman spectrometers have been developed that are now routinely applied in these fields. Results are presented that were obtained with a linescan Raman microspectrometer and with a Raman imaging microscope. Applications of Raman linescan spectrometry concern the investigation of polytene chromosomes obtained from the salivary gland of Chironomus thummi thummi. The distribution of DNA and proteins was investigated in bands and interbands. In a second example of Raman linescan spectrometry, bone implants were investigated. These bone implants were coated with thin layers of materials that improve biocompatibility. The density, crystallinity and protein distribution can be investigated. Information from Raman imaging may help in selecting the proper materials for maximum biocompatibility. Raman imaging microscopy is used whenever two-dimensional spatial information is required on the distribution of molecules or molecular components. Raman images are presented of filipine and phenylalanine in human eye-lenses in and around radial shades. Raman imaging is a particularly important tool for the study of the distribution of non-fluorescent drugs inside living cells. This is illustrated for the non-fluorescent drug cobalt octacarboxyphthalocyanine.

Axial resolution of confocal Raman microscopes: Gaussian beam theory and practice

A straightforward and transparent model, based on Gaussian beam optics, for the axial r0 resolution of a confocal microscope is presented. A confocal Raman microscope was used to determine the axial confocality in practice. The axial response of a thin planar object was measured for three different objectives, two pinhole sizes and a slit. The results show that, in the case of a confocal configuration, the response calculated with the model provides a good prediction of the axial resolution of the confocal microscope.

Confocal Raman Microspectroscopy of Polytene Chromosomes, Granulocytes and K 562 Cells

Functioning biological cells consist of many different (macro)molecules, present in specific cellular organelles, which all have to act in perfect register to ensure that the cells fulfill their function properly and can reproduce. Because of the size of the cell, which is usually in the order of ten micrometer, and the many coupled reactions and interactions that take place inside a cell it is not always possible to design satisfactory model systems for a whole cell or even for organelles. This is the reason for the continuing development of methods which allow studies of living whole cells and of intact organelles. Fluorescence microscopy and fluorescent (immuno)labeling are very powerfull techniques which however cannot always provide the information that is needed because the fluorescent markers may not be available or the labeling may disturb the system. Application of Raman scattering would have the advantage of the high molecular specificity which is present in Ramanspectra. For this reason we are developing Confocal Raman Micro(spectro)scopy and are applying it in studies on biological cells and cellular organelles.

Line Scan-Raman Spectroscopy and Atomic Force Microscopy of Chomosomal Banding Patterns

The objects of our study are the banding patterns of polytene chromosomes. These natural banding patterns are readily visible under an optical microscope. Salivary gland chromosomes from Chironomus thummi thummi, which were physiologically isolated, and squashed (fixed) chromosomes from Drosophila were studied. Measurements are performed on a Confocal Raman Microspectrometer (CRM) and an Atomic Force Microscope (AFM).

Raman Spectroscopy and Atomic Force Microscopy of Polytene Chromosomes

In recent years much progress was made in the development of new and quantitative microscopes. Technological renewal took place both in the components used — which now include sensitive noise free CCD detectors, better filters and image formation in the computer following point by point (scanning) data collection — and in the detection configuration — as e.g. use of confocal detection. As a consequence Raman microscopy (RM) could be revived(1-2) and measurements on living cells became quite well possible(3) Also completely new non-optical principles were introduced as the use of a scanning tip in Atomic Force Microscopy (AFM)(4) which even allows detection of single atoms, provided the sample studied is flat.