Gero Bergner

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.

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.

Quantitative CARS Microscopic Detection of Analytes and Their Isotopomers in a Two‐Channel Microfluidic Chip

Raman microspectroscopy provides label-free vibrational contrast at submicron spatial resolution without the need for sample preparation, and has therefore become an indispensable characterization method in various disciplines, including analytical, life, and materials sciences. The technique is particularly useful for spatially resolved quantification of the concentrations of chemical constituents in a sample, and in situations where labeling of low-molecular-weight compounds by fluorescent labels is not possible or not desired. Coherent anti-Stokes Raman scattering (CARS) microscopy benefits from significantly faster acquisition rates than conventional Raman microspectroscopy. The combination of this nonlinear Raman technique withmicrofluidics for reactionmonitoring and cytometry has been introduced recently. Unfortunately, CARS is not background-free: the signal generation leads to both a coherent excitation of molecular vibrations (nuclear motions) and an intrinsic non-Ramanresonant background (electronic response). This chemically nonspecific background constitutes a severe limitation for CARS detection and the quantification of analytes at low concentrations. Multiplex CARS microspectroscopy with subsequent CARS band-shape analysis allows extraction of

Immuno-surface-enhanced coherent anti-stokes Raman scattering microscopy: immunohistochemistry with target-specific metallic nanoprobes and nonlinear Raman microscopy.

Immunohistochemistry (IHC) is one of the most widely used staining techniques for diagnostic purposes. The selective localization of target proteins in tissue specimens by conventional IHC is achieved with dye- or enzyme-labeled antibodies in combination with light microscopy. In this contribution, we demonstrate the proof-of-principle for IHC based on surface-enhanced coherent Raman scattering for contrast generation. Specifically, antibody-labeled metallic nanoshells in conjunction with surface-enhanced coherent anti-Stokes Raman scattering (SECARS) microscopy are employed for the selective, sensitive, and rapid localization of the basal cell protein p63 in normal prostate tissue. Negative control experiments were performed in order to confirm the selective binding of the target-specific metal nanoprobes and to disentangle the role of plasmonic (metal) and molecular (Raman reporter) resonances in this plasmon-assisted four-wave mixing technique.

Characterization of Atherosclerotic Plaque-Depositions by Infrared, Raman and CARS Microscopy

Atherosclerotic plaques are mainly composed of proteoglycans, triglycerides, cholesterol, cholesterolester and crystalline calcium. From histopathological characterizations it is known that the composition of these atherosclerotic plaques can vary to a great extent, due to different risk factors as smoking, hyperlipedemia, or genetic background ect. The individual plaque components can be spectroscopically easily identified. Furthermore, spectroscopic imaging technologies offer the possibility to study the plaque compositions in a more quantitative manner than traditional staining techniques. Here, we compare the potential of IR, Raman and CARS microscopy to characterize the constitution of atherosclerotic plaques as well as the structure of the surrounding tissue. For data analysis and image reconstruction spectral decomposition algorithms such as vertex component analysis (VCA) were introduced. The results are in good agreement with the histopathology. Aim of the study is to correlate the compositional characteristics of atherosclerotic plaques with individual disease patterns.

Monitoring intra-cellular lipid metabolism in macrophages by Raman- and CARS-microscopy

Monocyte-derived macrophages play a key role in lipid metabolism in vessel wall tissues. Macrophages can take up lipids by various mechanisms. As phagocytes, macrophages are important for the decomposition of lipid plaques within arterial walls that contribute to arteriosclerosis. Of special interest are uptake dynamics and intra-cellular fate of different individual types of lipids as, for example, fatty acids, triglycerides or free and esterified cholesterol. Here we utilize Raman microscopy to image the metabolism of such lipids and follow subsequent storage or degradation patterns. The combination of optical microscopy with Raman spectroscopy allows visualization at the diffraction limit of the employed laser light and biochemical characterization through the associated spectral information. Relatively long measuring times, due to the weakness of Raman scattering can be overcome by non-linear effects such as coherent anti-Stokes Raman scattering (CARS). With this contribution we introduce first results to monitor the incorporation of lipid components into individual cells employing Raman and CARS microscopy.

Optimal control enhances contrast in coherent anti-Stokes Raman scattering images

Coherent anti-Stokes Raman scattering (CARS) has become an established microscopy tool in biophysics, biomedicine, and chemistry for rapid imaging of living cells.1 Unlike fluorescence, white-light, and phase-contrast microscopies, for example, CARS provides chemical contrast by looking at the intrinsic vibrational structure of analytes (molecules of interest). Consequently, it works on unstained, unlabeled—i.e., native—samples with imaging speeds of up to video rates. A particularly desirable area of application is visualizing drugs or metabolites (byproducts of drugs) in complex samples such as mammalian and plant tissue. But in this context CARS microscopy remains challenging because the technique is insufficiently sensitive to detect low concentrations of target materials as with drugs.2 Solving this problem is key to more widespread use of CARS, which is currently largely restricted to detecting lipid (fat) content in biological samples due to the high density of carbon-hydrogen groups contained in these molecules.3 Here, we report our recent work on combining spatial-light modulators for controlling incident light fields in microfluidic chips. The latter serve as the basis for performing microspectroscopic experiments on controlled and reproducible samples. Consequently, the focus of this study is on developing and validating methods. Our experiments aim at establishing the detection limits of model analytes in water-based solutions using CARS image contrast.4 Unlike previous efforts, the experiments reported here use an image-contrast parameter for direct feedback in a self-learning algorithm. CARS-imaging systems are typically combined with a microchannel flow system to quantify the relative contributions of Figure 1. Coherent anti-Stokes Raman scattering (CARS) intensity profiles for a two-channel chip. High intensities indicate higher concentration of deuterated toluene. C7D8: Deuterated toluene. C7H8: Toluene.

Two Channel Microfluidic CARS for Quantifying Pure Vibrational Contrast of Model Analytes

We discuss the combination of a CARS-imaging system with microfluidics. Such system is a versatile tool to quantify the relative contributions of resonant and non-resonant scattering at the CARS frequency. We will show that the twochannel microfluidic chip employed in combination with deuterated isotopomers as an internal standard allows for fast and quantitative detection of organic molecules by CARS microscopy. The experimental design enables the simultaneous measurement of both the chemically relevant Raman-resonant signal and the non-Raman-resonant background.

Optimal control of coherent anti-Stokes Raman scattering image contrast

Optimal control of coherent anti-Stokes Raman scattering (CARS) image contrast is reported. The setup combines an evolutionary strategy and a closed-loop feedback with a liquid-crystal spatial modulator to control the spectrum of the Stokes pulse within a CARS scheme to optimize the vibrational contrast of CARS images. The CARS excitation spectrum is optimized for image contrast at a pre-determined wavenumber position. The optimization feedback uses an image-contrast parameter generated from the image itself as the experimentally imposed fitness parameter. This strategy allows for enhancing the image contrast by a factor of up to 2.6.