G.J. Puppels

Laser irradiation and Raman spectroscopy of single living cells and chromosomes: Sample degradation occurs with 514.5 nm but not with 660 nm laser light

In Raman spectroscopic measurements of single cells (human lymphocytes) and chromosomes, using a newly developed confocal Raman microspectrometer and a laser excitation wavelength of 514.5 nm, degradation of the biological objects was observed. In the experiments high power microscope objectives were used, focusing the laser beam into a spot approximately 0.5 micron in diameter. At the position of the laser focus a paling of the samples became visible even when the laser power on the sample was reduced to less than 1 mW. This was accompanied by a gradual decrease in the intensity of the Raman signal. With 5 mW of laser power the events became noticeable after a period of time in the order of minutes. It is shown that a number of potential mechanisms, such as excessive sample heating due to absorption of laser light, multiple photon absorption, and substrate heating are unlikely to play a role. In experiments with DNA solutions and histone protein solutions no evidence of photo damage was found using laser powers up to 25 mW. No degradation of cells and chromosomes occurs when laser light of 660 nm is used. The most plausible explanation therefore seems to be that the sample degradation is the result of photochemical reactions initiated by laser excitation at 514.5 nm of as yet unidentified sensitizer molecules or complexes present in chromosomes and cells but not in purified DNA and histone protein samples.

Raman microspectroscopic approach to the study of human granulocytes

A sensitive confocal Raman microspectrometer was employed to record spectra of nuclei and cytoplasmic regions of single living human granulocytes. Conditions were used that ensured cell viability and reproducibility of the spectra. Identical spectra were obtained from the nuclei of neutrophilic, eosinophilic, and basophilic granulocytes, which yield information about DNA and protein secondary structure and DNA-protein ratio. The cytoplasmic Raman spectra of the three cell types are very different. This was found to be mainly due to the abundant presence of peroxidases in the cytoplasmic granules of neutrophilic granulocytes (myeloperoxidase) and eosinophilic granulocytes (eosinophil peroxidase). Strong signal contributions of the active site heme group(s) of these enzymes were found. This paper illustrates the potentials and limitations for Raman spectroscopic analysis of cellular constituents and processes.

Direct imaging Raman microscope based on tunable wavelength excitation and narrow band emission detection

A new type of imaging Raman microscope is described. First the advantages and disadvantages of the two possible approaches to Raman microscopy based on signal detection by means of a charge-coupled-device camera (i.e., direct imaging and image reconstruction) are discussed. Arguments are given to show that in most cases direct imaging is to be preferred over image reconstruction, because it provides the desired information in less time. In the direct imaging Raman microscope presented in this communication, detection of scattered light occurs in a narrow interval around a fixed wavelength. Selection of the Raman wavenumber shift at which an image is recorded is established by tuning the wavelength of the exciting laser light in such a way that the wavelength of the Raman scattered light with the desired Raman shift coincides with the detected wavelength. The microscope has been incorporated in a Raman microspectrometer in a way that enables easy switching between the imaging and the multichannel spectroscopy modes of operation. Bright field, fluorescence, and Raman microscopic images can be obtained.

Intracellular carotenoid levels measured by Raman microspectroscopy: comparison of lymphocytes from lung cancer patients and healthy individuals

Most studies concerning a possible protective role of carotenoids against cancer focus on serum carotenoid levels. We have used Raman microspectroscopy to study the intracellular amounts of carotenoids in lymphocytes of lung cancer patients and of healthy individuals. Our results indicate a significant decrease of carotenoids in lung carcinoma patients compared with healthy individuals, particularly in adenocarcinoma patients. Carotenoid supplementation raised the serum concentration in 2 lung cancer patients up to normal levels, whereas intracellular content remained significantly lower. This indicates that carotenoid uptake by lymphocytes is not only dependent on serum carotenoid concentration. Our findings indicate that Raman microspectroscopy, a recently developed technique to measure intracellular levels of drugs, is also well suited to obtain quantitative data on carotenoid amounts inside cells. Int. J. Cancer 74:20–25.

Discrimination between oral cancer and healthy tissue based on water content determined by Raman spectroscopy.

Tumor-positive resection margins are a major problem in oral cancer surgery. High-wavenumber Raman spectroscopy is a reliable technique to determine the water content of tissues, which may contribute to differentiate between tumor and healthy tissue. The aim of this study was to examine the use of Raman spectroscopy to differentiate tumor from surrounding healthy tissue in oral squamous cell carcinoma. From 14 patients undergoing tongue resection for squamous cell carcinoma, the water content was determined at 170 locations on freshly excised tongue specimens using the Raman bands of the OH-stretching vibrations (3350-3550 cm(-1)) and of the CH-stretching vibrations (2910-2965 cm(-1)). The results were correlated with histopathological assessment of hematoxylin and eosin stained thin tissue sections obtained from the Raman measurement locations. The water content values from squamous cell carcinoma measurements were significantly higher than from surrounding healthy tissue (p-value < 0.0001). Tumor tissue could be detected with a sensitivity of 99% and a specificity of 92% using a cutoff water content value of 69%. Because the Raman measurements are fast and can be carried out on freshly excised tissue without any tissue preparation, this finding signifies an important step toward the development of an intraoperative tool for tumor resection guidance with the aim of enabling oncological radical surgery and improvement of patient outcome.

Spectroscopy of Biological Molecules: New Directions

Preface. Section I: Proteins and Peptides. Section II: Chromophores and chromophoric proteins. Section III: Nucleic Acids. Section IV: Carbohydrates. Section V: Lipids and Biomembranes. Section VI: Theoretical methods. Section VII: Biocomplex systems. Section VIII: Biomedical Applications. Section IX: Analytical applications and Biotechnology. Section X: Methods and Techniques.

RESONANCE RAMAN MICROSPECTROSCOPIC CHARACTERIZATION OF EOSINOPHIL PEROXIDASE IN HUMAN EOSINOPHILIC GRANULOCYTES

A resonance Raman microspectroscopic study is presented of eosinophil peroxidase (EPO) in human eosinophilic granulocytes. Experiments were carried out at the single cell level with laser excitation in Soret-, Qv-, and charge transfer absorption bands of the active site heme of the enzyme. The Raman signal obtained from the cells was almost exclusively due to EPO. Methods were developed to determine depolarization ratios and excitation profiles of Raman bands of EPO in situ. A number of Raman band assignments based on earlier experiments with isolated EPO have been revised. The results show that in agreement with literature on isolated eosinophil peroxidase, the prosthetic group of the enzyme in the (unactivated) cells is a high spin, 6-coordinated, ferric protoporphyrin IX. The core size of the heme is about 2.04 A. The proximal and distal axial ligands are most likely a histidine with the strong imidazolate character typical for peroxidases, and a weakly bound water molecule, respectively. The data furthermore indicate that the central iron is displaced from the plane of the heme ring. The unusual low wavenumber Raman spectrum of EPO, strongly resembling that of lactoperoxidase, intestinal peroxidase and myeloperoxidase, suggests that these mammalian peroxidases are closely related, and characterized by, as yet unspecified, interactions between the peripheral substituents and the protein, different from those found in other protoheme proteins.

Development and application of Raman Microspectroscopic and Raman Imaging Techniques for Cell Biological Studies

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.

A high‐throughput Raman notch filter set

A chevron‐type Raman notch filter (RNF) set is described. lt combines a high signal throughput (up to 90% around 1600 cm−1 and ≳80% between and 700 and 2700 cm−1) with a laser line suppression of 108–109. The filter set can be used to replace the first two dispersion stages in triple‐stage Raman monochromators commonly employed in multichannel detection systems. This yields a gain in intensity of the detected Raman signal of a factor of 4. It is shown that in Raman spectrometers with a backscatter geometry, the filter set can also be used to optically couple the microscope and the spectrometer. This leads to a further increase in signal intensity of a factor of 3–4 as compared to the situation where a beam splitter is used. Additional advantages of the RNF set are the fact that signal throughput is almost polarization independent over a large spectral interval and that it offers the possibility to simultaneously record Stokes and anti‐Stokes spectra.

Confocal Raman microspectroscopy in biology: Applications and future developments

Abstract Recent progress and future developments of Raman microspectroscopy are discussed. Its great potential for cell biological investigations is illustrated with some examples.