Nonresonant confocal Raman imaging has been used to map the DNA and the protein distributions in individual single human cells. The images are obtained on an improved homebuilt confocal Raman microscope. After statistical analysis, using singular value decomposition, the Raman images are reconstructed from the spectra covering the fingerprint region. The data are obtained at a step interval of approximately 250 nm and cover a field from 8- to 15- micro m square in size. Dwell times at each pixel are between 0.5 and 2 s, depending on the nature and the state of the cell under investigation. High quality nonresonant Raman images can only be obtained under these conditions using continuous wave high laser powers between 60 and 120 mW. We will present evidence that these laser powers can still safely be used to recover the chemical distributions in fixed cells. The developed Raman imaging method is used to image directly, i.e., without prior labeling, the nucleotide condensation and the protein distribution in the so-called nuclear fragments of apoptotic HeLa cells. In the control (nonapoptotic) HeLa cells, we show, for the first time by Raman microspectroscopy, the presence of the RNA in a cell nucleus.

Cellular imaging techniques based on vibrational spectroscopy have become powerful tools in cell biology because the molecular composition of subcellular compartments can be visualized without the need for labeling. Using high-resolution, nonresonant confocal Raman microscopy on individual cells, we demonstrate here that lipid bodies (LBs) rich in arachidonate as revealed by their Raman spectra associate with latex bead-containing phagosomes in neutrophilic granulocytes. This finding was corroborated in macrophages and in PLB-985 cells, which can be induced to differentiate into neutrophil-like cells, by selective staining of LBs and visualization by confocal fluorescence microscopy. We further show that the accumulation of LBs near phagosomes is mediated at least in part by the flavohemoprotein gp91 phox (in which ''phox'' is phagocyte oxidase), because different LB distributions around phagocytosed latex beads were observed in WT and gp91 phoxdeficient PLB-985 cells. gp91 phox , which accumulates in the phagosomal membrane, is the catalytic subunit of the leukocyte NADPH oxidase, a critical enzyme in the innate immune response. Finally, time-lapse fluorescence microscopy experiments on neutrophils revealed that the LB-phagosome association is transient, similar to the ''kiss-and-run'' behavior displayed by endosomes involved in phagosome maturation. Because arachidonic acid (AA) has been shown to be involved in NADPH oxidase activation and phagosome maturation in neutrophils and macrophages, respectively, the findings reported here suggest that LBs may provide a reservoir of AA for local activation of these essential leukocyte functions.innate immunity ͉ NADPH oxidase ͉ Raman microscopy ͉ phagocytes

A confocal Raman microscope is used to study the protein distribution inside biological cells. It is shown that high quality Raman imaging of the protein distribution can be obtained using confocal nonresonant Raman imaging ( exc ϭ 647.1 nm). The results are shown for two different human cell types. Perpheral blood lymphocytes are used as an example of the fully maturated cells with a low level of nuclear transcription. Human eye lens epithelial cells are used as an example of cells with a high level of nuclear activity. The protein distribution in both cell types is completely different. The nuclear distribution of the protein largely varies in the peripheral blood lymphocyte cells, while proteins are more homogenously distributed over the nuclear space in the eye lens epithelial cells. The imaging time is ϳ20 min for a field of view of 10 ϫ 10 m 2 . The size of the sampling volume is 1.4 fL using a full width at halfmaximum criterion along the z axis and a 1/e 2 criterion in the xy plane. The results presented here indicate that Raman imaging is particularly of interest in the study of cellular processes, like phagocytosis, apoptosis, chromatin compaction, and cellular differentiation, which are accompanied by relatively large-scale redistributions of the materials.

We describe a straightforward approach to the covalent immobilization of cytophilic proteins by microcontact printing, which can be used to pattern cells on substrates. Cytophilic proteins are printed in micropatterns on reactive self-assembled monolayers by using imine chemistry. An aldehyde-terminated monolayer on glass or on gold was obtained by the reaction between an amino-terminated monolayer and terephthaldialdehyde. The aldehyde monolayer was employed as a substrate for the direct microcontact printing of bioengineered, collagen-like proteins by using an oxidized poly(dimethylsiloxane) (PDMS) stamp. After immobilization of the proteins into adhesive "islands", the remaining areas were blocked with amino-poly(ethylene glycol), which forms a layer that is resistant to cell adhesion. Human malignant carcinoma (HeLa) cells were seeded and incubated onto the patterned substrate. It was found that these cells adhere to and spread selectively on the protein islands, and avoid the poly(ethylene glycol) (PEG) zones. These findings illustrate the importance of microcontact printing as a method for positioning proteins at surfaces and demonstrate the scope of controlled surface chemistry to direct cell adhesion.

Vibrational microspectroscopy has become a powerful tool in cellular biology because detailed information about the chemical composition of subcellular, femtoliter volumes can be obtained. Moreover, biological imaging techniques based on this type of spectroscopy avoid labeling methods and artifacts arising from them because the required contrast is generated from endogenous molecules. Here, we report the visualization, by confocal Raman microscopy, of the intracellular distribution of two enzymes important in the immune response of granulocytes (i.e., the NADPH oxidase subunit cytochrome b558 (cyt b558) in neutrophils and eosinophil peroxidase (EPO) in eosinophils). We excited these leukocytes with 413.1 nm laser light, allowing the Raman scattering signal from the heme-containing enzymes to be dramatically enhanced by resonance. In neutrophils, there is a nonnegligible contribution from the hemoprotein myeloperoxidase to the resonance Raman signal. The effect of photobleaching of the Raman signal at 413.1 nm excitation on the reconstructed Raman images is discussed. We also show how singular value decomposition can significantly reduce the noise that is present in the raw spectral data. Stimulation of the neutrophils, either by phagocytosis or by phorbol 12-myristate 13-acetate (PMA), resulted in intracellular redistributions of cyt b558. Spectra extracted from Raman images of PMA-activated neutrophils displayed a significantly higher level of cyt b558 reduction than spectra from resting neutrophils, indicating that cyt b558 reduction is linked with NADPH oxidase activation. The versatility of resonance Raman microscopy on leukocytes is further demonstrated by the visualization of EPO in single eosinophils. In conclusion, high-resolution cellular imaging based on resonance Raman spectroscopy enables the label-free visualization of the intracellular distribution of cyt b558 in neutrophils and EPO in eosinophils, two crucial enzymes in leukocyte innate immunity.

Protein glycation causes loss-of-function through a process that has been associated with several diabetic-related diseases. Additionally, glycation has been hypothesized as a promoter of protein aggregation, which could explain the observed link between hyperglycaemia and the development of several aggregating diseases. Despite its relevance in a range of diseases, the mechanism through which glycation induces aggregation remains unknown. Here we describe the molecular basis of how glycation is linked to aggregation by applying a variety of complementary techniques to study the nonenzymatic glycation of hen lysozyme with ribose (ribosylation) as the reducing carbohydrate. Ribosylation involves a chemical multistep conversion that induces chemical modifications on lysine side chains without altering the protein structure, but changing the protein charge and enlarging its hydrophobic surface. These features trigger lysozyme native-like aggregation by forming small oligomers that evolve into bigger insoluble particles. Moreover, lysozyme incubated with ribose reduces the viability of SH-SY5Y neuroblastoma cells. Our new insights contribute toward a better understanding of the link between glycation and aggregation.

This report describes a feasibility study concerning the use of a visible diode laser for two important fluorescence applications in a flow cytometer. With a 3 mW 635 nm diode laser, we performed immunofluorescence measurements using the fluorophore allophycocyanin (APC). We have measured CD8 positive lymphocytes with a two-step labeling procedure and the resulting histograms showed good separation between the negative cells and the dim and the bright fluorescent subpopulations. As a second fluorescence application, we chose DNA analysis with the recently developed DNA/ RNA stains TOTO-3 and TO-PRO-3. In our setup TO-PRO-3 yielded the best results with a CV of 3.4%. Our results indicate that a few milliwatts of 635 nm light from a visible diode laser is sufficient to do single color immunofluorescence measurements with allophycocyanin and DNA analysis with TO-PRO-3. The major advantages of using a diode laser in a flow cytometer are the small size, the low price, the high efficiency, and the long lifetime. 0 1994 Wiley-Liss, Inc.

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. © 1997 Wiley‐Liss, Inc.