Christian Matthäus

Raman and Infrared Microspectral Imaging of Mitotic Cells

We report the first ever Raman and infrared microspectroscopic images of human cells at different stages of mitosis. These spectroscopic methods monitor the distribution of condensed nuclear chromatin, and other biochemical components, utilizing inherent protein and DNA spectral markers, and, therefore, do not require the use of any stains. In conjunction with previously reported data from the G1, S, and G2 phases of the cell cycle, the complete cell division cycle has now been mapped by spectroscopic methods. Although the results reported here do not offer new insights into the distribution of biochemical components during mitosis, the recognition of cell division without the use of stains, and the possibility of doing so on living cells, may be useful for an automatic, spectroscopic determination of the proliferation rates of cells and tissues. Spectral images were constructed by plotting spectral intensities of DNA or protein versus the coordinates from which spectra were recorded. We found that both Raman and infrared intensities depend on the overall chromatin density variation among the individual subphases of mitosis.

New ways of imaging uptake and intracellular fate of liposomal drug carrier systems inside individual cells, based on Raman microscopy.

Recent developments, combining Raman spectroscopy with optical microscopy, provide a new noninvasive technique to assess and image cellular processes. Of particular interest are the uptake mechanisms of various cytologically active compounds. In order to distinguish the species of interest from their cellular environment spectroscopically, compounds may be labeled with deuterium. Here, we apply Raman microspectroscopy to follow the uptake of liposomal drug carrier systems that have been introduced to deliver biologically active compounds to their site of action within human breast adenocarcinoma MCF-7 cells. The distribution patterns of liposomes and liposomes surface-modified with a cell-penetrating peptide (TAT-peptide, TATp) have been imaged over time. The spectroscopic information obtained provides a clear evidence for variable rates, as well as different efficiencies of liposome uptake depending on their surface properties. Depending on the experimental setup, the technique may be applied to fixed or living cell organisms.

Chapter 10: Infrared and Raman microscopy in cell biology.

This chapter presents novel microscopic methods to monitor cell biological processes of live or fixed cells without the use of any dye, stains, or other contrast agent. These methods are based on spectral techniques that detect inherent spectroscopic properties of biochemical constituents of cells, or parts thereof. Two different modalities have been developed for this task. One of them is infrared micro-spectroscopy, in which an average snapshot of a cells biochemical composition is collected at a spatial resolution of typically 25 mum. This technique, which is extremely sensitive and can collect such a snapshot in fractions of a second, is particularly suited for studying gross biochemical changes. The other technique, Raman microscopy (also known as Raman micro-spectroscopy), is ideally suited to study variations of cellular composition on the scale of subcellular organelles, since its spatial resolution is as good as that of fluorescence microscopy. Both techniques exhibit the fingerprint sensitivity of vibrational spectroscopy toward biochemical composition, and can be used to follow a variety of cellular processes.

Label-Free Raman Spectral Imaging of Intracellular Delivery and Degradation of Polymeric Nanoparticle Systems

Novel optical imaging methods, such as Raman microspectroscopy, have been gaining recognition in their ability to obtain noninvasively the distribution of biochemical components of a sample. Raman spectroscopy in combination with optical microscopy provides a label-free method to assess and image cellular processes, without the use of extrinsic fluorescent dyes. The submicrometer resolution of the confocal Raman instrumentation allows us to image cellular organelles on the scale of conventional microscopy. We used the technique to monitor subcellular degradation patterns of two biodegradable nanocarrier systems-poly(epsilon-caprolactone) (PCL) and poly(lactic-co-glycolic acid) (PLGA). Our results suggest that both drug-delivery systems eventually are incorporated into Golgi-associated vesicles of late endosomes. These processes were monitored via the decrease of the molecule-characteristic peaks of PCL and PLGA. As the catabolic pathways proceed, shifts and variations in peak intensities and intensity ratios in the rendered Raman spectra unequivocally delineate their degradation patterns.

Phenalenone-type phytoalexins mediate resistance of banana plants (Musa spp.) to the burrowing nematode Radopholus similis

Significance The ongoing decline of banana yields caused by pathogens and the use of toxic chemicals to manage them has attracted considerable attention because of the importance of bananas as a major staple food for more than 400 million people. We demonstrate that secondary metabolites (phenylphenalenones) of Musa are the reason for differences in cultivar resistance, and detected the phenylphenalenone anigorufone in greater concentrations in lesions in roots of a nematode-resistant cultivar than in those of a susceptible one. An in vitro bioassay identified anigorufone as the most active nematostatic and nematocidal compound. We discovered that large lipid–anigorufone complex droplets are formed in the bodies of Radopholus similis exposed to anigorufone, resulting in the nematode being killed. The global yield of bananas—one of the most important food crops—is severely hampered by parasites, such as nematodes, which cause yield losses up to 75%. Plant–nematode interactions of two banana cultivars differing in susceptibility to Radopholus similis were investigated by combining the conventional and spatially resolved analytical techniques 1H NMR spectroscopy, matrix-free UV-laser desorption/ionization mass spectrometric imaging, and Raman microspectroscopy. This innovative combination of analytical techniques was applied to isolate, identify, and locate the banana-specific type of phytoalexins, phenylphenalenones, in the R. similis-caused lesions of the plants. The striking antinematode activity of the phenylphenalenone anigorufone, its ingestion by the nematode, and its subsequent localization in lipid droplets within the nematode is reported. The importance of varying local concentrations of these specialized metabolites in infected plant tissues, their involvement in the plant’s defense system, and derived strategies for improving banana resistance are highlighted.

Noninvasive imaging of intracellular lipid metabolism in macrophages by Raman microscopy in combination with stable isotopic labeling.

Monocyte-derived macrophages play a key role in atherogenesis because their transformation into foam cells is responsible for deposition of lipids in plaques within arterial walls. The appearance of cytosolic lipid droplets is a hallmark of macrophage foam cell formation, and the molecular basics involved in this process are not well understood. Of particular interest is the intracellular fate of different individual lipid species, such as fatty acids or cholesterol. Here, we utilize Raman microscopy to image the metabolism of such lipids and to trace their subsequent storage patterns. The combination of microscopic information with Raman spectroscopy provides a powerful molecular imaging method, which allows visualization at the diffraction limit of the employed laser light and biochemical characterization through associated spectral information. In order to distinguish the molecules of interest from other naturally occurring lipids spectroscopically, deuterium labels were introduced. Intracellular distribution and metabolic changes were observed for serum albumin-complexed palmitic and oleic acid and cholesterol and quantitatively evaluated by monitoring the increase in CD scattering intensities at 0.5, 1, 3, 6, 24, 30, and 36 h. This approach may also allow for investigating the cellular trafficking of other molecules, such as nutrients, metabolites, and drugs.

Fluorescence-based fixative and vital staining of lipid droplets in Caenorhabditis elegans reveal fat stores using microscopy and flow cytometry approaches

The proportions of body fat and fat-free mass are determining factors of adiposity-associated diseases. Work in Caenorhabditis elegans has revealed evolutionarily conserved pathways of fat metabolism. Nevertheless, analysis of body composition and fat distribution in the nematodes has only been partially unraveled because of methodological difficulties. We characterized metabolic C. elegans mutants by using novel and feasible BODIPY 493/503-based fat staining and flow cytometry approaches. Fixative as well as vital BODIPY staining procedures visualize major fat stores, preserve native lipid droplet morphology, and allow quantification of fat content per body volume of individual worms. Colocalization studies using coherent anti-Stokes Raman scattering microscopy, Raman microspectroscopy, and imaging of lysosome-related organelles as well as biochemical measurement confirm our approaches. We found that the fat-to-volume ratio of dietary restriction, TGF-β, and germline mutants are specific for each strain. In contrast, the proportion of fat-free mass is constant between the mutants, although their volumes differ by a factor of 3. Our approaches enable sensitive, accurate, and high-throughput assessment of adiposity in large C. elegans populations at a single-worm level.

Shedding New Light on the Molecular Architecture of Oocytes Using a Combination of Synchrotron Fourier Transform-Infrared and Raman Spectroscopic Mapping

Synchrotron Fourier transform-infrared (FT-IR) and Raman microspectroscopy were applied to investigate changes in the molecular architecture of mouse oocytes and demonstrate the overall morphology of the maturing oocyte. Here we show that differences were identified between immature mouse oocytes at the germinal vesicle (GV) and mature metaphase II (MII) stage when using this technology, without the introduction of any extrinsic markers, labels, or dyes. GV mouse oocytes were found to have a small, centrally located lipid deposit and another larger polar deposit of similar composition. MII oocytes have very large, centrally located lipid deposits. Each lipid deposit for both cell types contains an inner and outer lipid environment that differs in composition. To assess interoocyte variability, line scans were recorded across the diameter of the oocytes and compared from three independent trials (GV, n = 91; MII, n = 172), and the data were analyzed with principal component analysis (PCA). The average spectra and PCA loading plots show distinct and reproducible changes in the CH stretching region that can be used as molecular maturation markers. The method paves the way for developing an independent assay to assess oocyte status during maturation providing new insights into lipid distribution at the single cell level.

Characterization of atherosclerotic plaque depositions by Raman and FTIR imaging

Spectroscopy-based imaging techniques can provide useful biochemical information about tissue samples. Here, we employ Raman and Fourier transform infrared (IR) imaging to characterize composition and constitution of atherosclerotic plaques of rabbits, fed with a high cholesterol diet. The results were compared with conventional light microscopy after staining with hematoxylin eosin, and elastica van Gieson. The spectral unmixing algorithm vertex component analysis was applied for data analysis and image reconstruction. IR microscopy allowed for differentiation between lipids and proteins in plaques of full aortic cross sections. Raman microscopy further discriminated cholesterol esters, cholesterol and triglycerides. FTIR and Raman images were recorded at a resolution near 20 micrometer per pixel for a large field of view. High resolution Raman images at 1 micrometer per pixel revealed structural details at selected regions of interest. The intima-media and the lipid-protein ratio were determined in five specimens for quantitation. These results correlate well with histopathology. The described method is a promising tool for easy and fast molecular imaging of atherosclerosis.

In Vivo Characterization of Atherosclerotic Plaque Depositions by Raman-Probe Spectroscopy and in Vitro Coherent Anti-Stokes Raman Scattering Microscopic Imaging on a Rabbit Model

Visualization as well as characterization of inner arterial plaque depositions is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable visual information but cannot deliver information about the chemical composition of individual plaques. Here, we employ Raman-probe spectroscopy to characterize the plaque compositions of arterial walls on a rabbit model in vivo, using a miniaturized filtered probe with one excitation and 12 collection fibers integrated in a 1 mm sleeve. Rabbits were treated with a cholesterol-enriched diet. The methodology can improve the efficiency of animal experiments and shows great potential for applications in cardiovascular research. In order to further characterize the plaque depositions visually, coherent anti-Stokes Raman scattering (CARS) microscopy images have been acquired and are compared with the Raman-probe results.