Resonance Raman Microspectroscopy Research Articles

Resonance Raman and Visible Micro-Spectroscopy for the In-Vivo and In-Vitro Characterization of Anthocyanin-Based Pigments in Blue and Violet Flowers: A Comparison with HPLC-ESI- MS Analysis of the Extracts

Microanalysis techniques based on resonance Raman and reflection visible spectroscopy have been applied to the characterization of pigments responsible for the blue or violet coloration in flowers; in particular of Lobelia erinus, Campanula portenschlagiana, Cineraria, Viola tricolor, Anemone coronaria, Agapanthus, Platycodon, Salvia farinacea, Plumbago capensis, Ceratostigma plumbaginoides, Commelina communis and Salvia patens. The spectroscopic methods were applied both in vivo on the flower petals and in vitro on extracts obtained through a procedure based on SPE (solid-phase extraction) optimized for minimal quantities of vegetable raw material. Different patterns obtained for the Raman spectra have been correlated, also on the basis of density functional theory (DFT) calculations, with different schemes of substitution of the benzopyrilium nucleus of the anthocyanins and with various possible forms of copigmentation responsible for the stabilization of the blue color. The results obtained were verified by comparison with the analysis of the extracts by HPLC-ESI-MS (liquid chromatography-electrospray ionization-mass spectrometry).

Resonance Raman analysis of intracellular vitamin B12 analogs in methanogenic archaea.

Methanogenic archaea (methanogens) are microorganisms that can synthesize methane. They are found in diverse environments ranging from paddy fields to animal digestive tracts to deep-sea hydrothermal vents. Investigating their distribution and physiological activity is crucial for the detailed analysis of the dynamics of greenhouse gas generation and the search for the environmental limits of life. In methanogens, cobamide cofactors (vitamin B12 analogs) play a key role in methane synthesis and carbon fixation, thus serving as a marker compound that metabolically characterizes them. Here, we report on resonance Raman detection of cobamides in methanogenic cells without destroying cells and provide structural insights into those cobamides. We succeeded in detecting cobamides in four representative methanogens Methanosarcina mazei, Methanosarcina barkeri, Methanopyrus kandleri, and Methanocaldococcus jannaschii. The former two are mesophilic, cytochrome-containing methanogens, whereas the latter two are hyperthermophilic, non-cytochrome-containing methanogens. The 532nm-excited Raman spectra of single or multiple cells of the four species all showed resonance Raman bands of cobamides arising mainly from the corrin ring, with the most intense one at ∼1500cm-1. We envision that resonance Raman microspectroscopy could be useful for in situ, nondestructive identification of methanogenic cells that produce high levels of cobamides.

Continuous assessment of metabolic activity of mitochondria using resonance Raman microspectroscopy.

Dysfunctional mitochondrial activity can lead to a variety of different diseases. As such, there exists a need to quantify changes in mitochondria function as it relates to these specific diseased states. Here, we present the use of resonance Raman (RR) spectroscopy as a tool to determine changes in isolated mitochondrial activity. RR spectroscopy, using 532 nm as the excitation source, specifically provides information on the reduction and oxidation (RedOx) state of cytochrome c, which is determined by the activity of protein complexes in the electron transport chain (ETC). In this model, injection of the substrate succinate into the mitochondrial sample is used to drive the ETC, which causes a subsequent change in cytochrome c RedOx state. This change in RedOx state is tracked by RR spectroscopy. This tool gives real-time information on the rise and fall of the amount of reduced cytochrome c within the mitochondrial sample, providing a method for rapid assessment of mitochondrial metabolism that has broad applications in both basic science and medical research.

Chemical basis of molluscan shell colors revealed with in situ micro‐Raman spectroscopy

AbstractMolluscan shells exhibit a wide variety of color tones and patterns that are thought to increase fitness in different natural habitats. However, the chemical aspects of shell pigments have not been well understood. We studied the chemical basis of molluscan shell colors via resonance Raman microspectroscopy using a 514.5‐nm laser on six gastropod, 15 bivalve, and one cephalopod species. Polyene specific signals for C═C (ν1) or C─C (ν2) stretching bands were detected principally from the pigmented regions, and those peak positions correlated with the pigment colors. Because Raman shifts of those bands depend on the conjugation length of polyene backbones, which in turn, affect the highest occupied molecular orbital/lowest unoccupied molecular orbital energy gap, our observations suggest that the conjugation length of polyene is a determinant factor of shell color. Further, our observations and data from previous studies seemed to reveal a quantitative relationship between polyene backbone length and shell color. From an analysis of 89 polyene‐based pigments in 45 molluscan species, we found that yellow, orange/red, and purple/brown/black pigments originate from polyenes with conjugation lengths of 7–9, 9–10, and 10–12, respectively. The shell color shifts from yellow, orange, and red to purple as the polyene chain length increases as reported for free carotenoids. The biological mechanisms underlying the spatial regulations of polyene conjugation systems in molluscan shells will be the most important and intriguing issue to be addressed in the future.

Raman microspectroscopy, surface-enhanced Raman scattering microspectroscopy, and stable-isotope Raman microspectroscopy for biofilm characterization.

Biofilms represent the predominant form of microbial life on our planet. These aggregates of microorganisms, which are embedded in a matrix formed by extracellular polymeric substances, may colonize nearly all interfaces. Detailed knowledge of microorganisms enclosed in biofilms as well as of the chemical composition, structure, and functions of the complex biofilm matrix and their changes at different stages of the biofilm formation and under various physical and chemical conditions is relevant in different fields. Important research topics include the development and improvement of antibiotics and medical devices and the optimization of biocides, antifouling strategies, and biological wastewater treatment. Raman microspectroscopy is a capable and nondestructive tool that can provide detailed two-dimensional and three-dimensional chemical information about biofilm constituents with the spatial resolution of an optical microscope and without interference from water. However, the sensitivity of Raman microspectroscopy is rather limited, which hampers the applicability of Raman microspectroscopy especially at low biomass concentrations. Fortunately, the resonance Raman effect as well as surface-enhanced Raman scattering can help to overcome this drawback. Furthermore, the combination of Raman microspectroscopy with other microscopic techniques, mass spectrometry techniques, or particularly with stable-isotope techniques can provide comprehensive information on monospecies and multispecies biofilms. Here, an overview of different Raman microspectroscopic techniques, including resonance Raman microspectroscopy and surface-enhanced Raman scattering microspectroscopy, for in situ detection, visualization, identification, and chemical characterization of biofilms is given, and the main feasibilities and limitations of these techniques in biofilm research are presented. Future possibilities of and challenges for Raman microspectroscopy alone and in combination with other analytical techniques for characterization of complex biofilm matrices are discussed in a critical review. Graphical Abstract Applicability of Raman microspectroscopy for biofilm analysis.

Exploring the Potential of Stable Isotope (Resonance) Raman Microspectroscopy and Surface-Enhanced Raman Scattering for the Analysis of Microorganisms at Single Cell Level.

Raman microspectroscopy is a prime tool to characterize the molecular and isotopic composition of microbial cells. However, low sensitivity and long acquisition times limit a broad applicability of the method in environmental analysis. In this study, we explore the potential, the applicability, and the limitations of stable isotope Raman microspectroscopy (SIRM), resonance SIRM, and SIRM in combination with surface-enhanced Raman scattering (SERS) for the characterization of single bacterial cells. The latter two techniques have the potential to significantly increase sensitivity and decrease measurement times in SIRM, but to date, there are no (SERS-SIRM) or only a limited number (resonance SIRM) of studies in environmental microbiology. The analyzed microorganisms were grown with substrates fully labeled with the stable isotopes (13)C or (2)H and compounds with natural abundance of atomic isotopes ((12)C 98.89% or (1)H 99.9844%, designated as (12)C or (1)H, respectively). Raman bands of bacterial cell compounds in stable isotope-labeled microorganisms exhibited a characteristic red-shift in the spectra. In particular, the sharp phenylalanine band was found to be an applicable marker band for SIRM analysis of the Deltaproteobacterium strain N47 growing anaerobically on (13)C-naphthalene. The study of G. metallireducens grown with (13)C- and (2)H-acetate showed that the information on the chromophore cytochrome c obtained by resonance SIRM at 532 nm excitation wavelength can be successfully complemented by whole-organism fingerprints of bacteria cells achieved by regular SIRM after photobleaching. Furthermore, we present here for the first time the reproducible SERS analysis of microbial cells labeled with stable isotopes. Escherichia coli strain DSM 1116 cultivated with (12)C- or (13)C-glucose was used as a model organism. Silver nanoparticles synthesized in situ were applied as SERS media. We observed a reproducible red-shift of an adenine-related marker band from 733 to 720 cm(-1) in SERS spectra for (13)C-labeled cells. Additionally, Raman measurements of (12)C/(13)C-glucose and -phenylalanine mixtures were performed to elucidate the feasibility of SIRM for nondestructive quantitative and spatially resolved analysis. The performed analysis of isotopically labeled microbial cells with SERS-SIRM and resonance SIRM paves the way toward novel approaches to apply Raman microspectroscopy in environmental process studies.

Photonic gratings of the metal–organic framework {Fe(bpac)[Pt(CN)4]} with synergetic spin transition and host–guest properties

Surface-relief photonic gratings of the spin-crossover metal-organic framework {Fe(bpac)[Pt(CN)4]} (bpac = bis(4-pyridyl)acetylene) were elaborated by the combination of a sequential assembly process and lithographic methods. Optical diffraction, surface plasmon resonance spectroscopy and Raman micro-spectroscopy were used to investigate the temperature dependence of the spin state of the iron(II) ions and the concomitant change of the refractive index of the grating material. The refractive index change associated with the high spin ((5)T) to low spin ((1)A) transition was found to be as high as Δn = 0.08 ± 0.005, which was attributed to the pronounced mass density difference between the two spin states. While the grating thickness (15-90 nm) had no influence on the spin-crossover properties of the gratings, the adsorption of aromatic guest molecules was found to have a substantial effect both on the spin transition temperature and the completeness of the transition.

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of thealgae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. Thedifferences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm2 area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.

Quantitative analysis of the redox states of cytochromes in a living L929 (NCTC) cell by resonance Raman microspectroscopy

Raman spectra and images of a living L929 (NCTC) cell have been measured with 532 nm excitation. Both reduced and oxidized forms of cytochromes b and c (cyt b and cyt c) have been observed in situ without any pretreatment. The redox states of cyts b and c have been assessed quantitatively with a spectral analysis. It has been found that reduced cyt c is more abundant than oxidized cyt c, while oxidized cyt b is slightly more abundant than reduced cyt b in a living cell.

Resonance-Raman microspectroscopy for quality assurance of dye-sensitized NiOx films with respect to dye desorption kinetics in water

Resonance Raman microspectroscopy is used to investigate dye-sensitized NiO(x) nanoparticle films to be used as photocathodes in tandem dye-sensitized solar cells. It is shown that rR microspectroscopy has potential for applications in quality assurance in such systems and also in integrated dye-sensitized solar cell modules. Here, ruthenium dye-sensitized NiO(x) nanoparticle layers were produced both as single and double NiO(x) films using a one or two-step deposition process, respectively. The distribution of the sensitizer on the surfaces was investigated by rR microspectroscopy. The chemical images obtained from rR microspectroscopy yield complementary information to bright field microscope pictures and provide detailed insight into the sensitization pattern e.g. in the vicinity of surface vacancies and other inhomogeneities. Furthermore, based on the mapping results the dye desorption kinetics upon addition of water has been analysed. Desorption on the single NiO(x) film is faster and more efficient than on the double film. These changes are attributed to binding sites on the NiO(x) surface that are passivated with regard to water penetration. This passivation is introduced by the second synthesis step in building the second film of NiO(x) on the glass substrate. Both findings highlight the potential of rR microspectroscopy for quality assurance of dye-sensitized solar cell electrodes.

Resonance Raman and EPR spectroscopy studies of untreated spring wheat leaves

Leaves are the structures in plants that have evolved primarily to absorb sunlight and to convert this energy into carbohydrates, which is the essence of the photosynthesis process. In the current study Raman and electron paramagnetic resonance (EPR) spectroscopic methods were applied to investigate the drought stress occurring in the fresh wheat leaves immediately after cutting them off from the rest of the plant. Resonance Raman microspectroscopy mapping allowed us to visualize the changes in concentrations of carotenoids, playing an important role in the photosynthetic membrane, as well as quinones and carbohydrates in the selected areas in plant tissues. EPR spectroscopy was used to detect free radicals in the leaf tissue directly. Besides these very reactive, short lifetime species (monodehydroascorbate–ascorbate radical (MDA)), the long-living semiquinone and carbohydrate radicals have been also detected.

Rapid resonance Raman microspectroscopy to probe carbon dioxide fixation by single cells in microbial communities

Photosynthetic microorganisms play crucial roles in aquatic ecosystems and are the major primary producers in global marine ecosystems. The discovery of new bacteria and microalgae that play key roles in CO(2) fixation is hampered by the lack of methods to identify hitherto-unculturable microorganisms. To overcome this problem we studied single microbial cells using stable-isotope probing (SIP) together with resonance Raman (RR) microspectroscopy of carotenoids, the light-absorbing pigments present in most photosynthetic microorganisms. We show that fixation of (13)CO(2) into carotenoids produces a red shift in single-cell RR (SCRR) spectra and that this SCRR-SIP technique is sufficiently sensitive to detect as little as 10% of (13)C incorporation. Mass spectrometry (MS) analysis of labelled cellular proteins verifies that the red shift in carotenoid SCRR spectra acts as a reporter of the (13)C content of single cells. Millisecond Raman imaging of cells in mixed cultures and natural seawater samples was used to identify cells actively fixing CO(2), demonstrating that the SCRR-SIP is a noninvasive method for the rapid and quantitative detection of CO(2) fixation at the single cell level in a microbial community. The SCRR-SIP technique may provide a direct method for screening environmental samples, and could help to reveal the ecophysiology of hitherto-unculturable microorganisms, linking microbial species to their ecological function in the natural environment.

Of microparticles and bacteria identification – (resonance) Raman micro-spectroscopy as a tool for biofilm analysis

Confocal resonance Raman microscopy is a powerful tool for the non-invasive analysis of complex biological aggregates without preparation and prior knowledge of the samples. We present the capabilities of confocal resonance Raman microscopy with a spatial resolution of 350 nm2 × 2.0 μm and excitation times of 1 s and less per recorded spectrum. Granules sampled from two sequencing batch reactors (SBR) for anaerobic ammonium oxidization (anammox) were regularly mapped in vivo for three months after SBR startup. Uncultured microorganisms and mineral particles were tracked throughout operation and identified in situ by their (resonance) Raman spectra. Co-existing microcolonies of Nitrosomonae formed the outer layer of anammox granules. Polymorph TiO2 microparticles were found embedded in the outer layer of granules overgrown with purple bacteria, indicating bacterial response to the variant toxicity of the mineral phase.

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Raman spectroscopy is often plagued by a strong fluorescent background, particularly for biological samples. If a sample is excited with a train of ultrafast pulses, a system that can temporally separate spectrally overlapping signals on a picosecond timescale can isolate promptly arriving Raman scattered light from late-arriving fluorescence light. Here we discuss the construction and operation of a complex nonlinear optical system that uses all-optical switching in the form of a low-power optical Kerr gate to isolate Raman and fluorescence signals. A single 808 nm laser with 2.4 W of average power and 80 MHz repetition rate is split, with approximately 200 mW of 808 nm light being converted to < 5 mW of 404 nm light sent to the sample to excite Raman scattering. The remaining unconverted 808 nm light is then sent to a nonlinear medium where it acts as the pump for the all-optical shutter. The shutter opens and closes in 800 fs with a peak efficiency of approximately 5%. Using this system we are able to successfully separate Raman and fluorescence signals at an 80 MHz repetition rate using pulse energies and average powers that remain biologically safe. Because the system has no spare capacity in terms of optical power, we detail several design and alignment considerations that aid in maximizing the throughput of the system. We also discuss our protocol for obtaining the spatial and temporal overlap of the signal and pump beams within the Kerr medium, as well as a detailed protocol for spectral acquisition. Finally, we report a few representative results of Raman spectra obtained in the presence of strong fluorescence using our time-gating system.

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Rejection of Fluorescence Background in Resonance and Spontaneous Raman Microspectroscopy

Hierarchical Cluster Analysis (HCA) of Microorganisms: An Assessment of Algorithms for Resonance Raman Spectra

Resonance Raman microspectroscopy in combination with hierarchical cluster analysis (HCA) is one of the most promising tools for the rapid examination of complex biological and medical samples. HCA is a ready, computerized tool for examining large sets of data for common characteristics, and a multitude of algorithms for this purpose have been developed over the years. However, resonance Raman spectra obtained from complex biological samples may originate from different chromophores as well as from a common chromophore found in different host environments, i.e., bacteria. Therefore, algorithms applied to resonance Raman spectra must handle data of high intrinsic similarity, i.e., spectra originating from a common chromophore, and data with highly dissimilar features, i.e., spectra from different chromophores, in the same unsupervised analysis. We examined the performance of eight widely used algorithms for hierarchical cluster analysis in clustering resonance Raman spectra of bacteria: Single-Linkage (Nearest-Neighbor), Complete-Linkage (Farthest-Neighbor), Average-Linkage, Weighted-Average-Linkage, Centroid, Median, and the Ward algorithm. Algorithm performance was evaluated by comparing the results of clustering a set of high-quality reference spectra with the results obtained when clustering a set of spectra recorded from single cells. References were formed by averaging 100 spectra of individual cells. While all algorithms returned highly similar results when clustering the reference spectra, their performance differed significantly when applied to single spectra. The best-performing algorithm, Weighted-Average-Linkage, correctly grouped single spectra with a reliability of above 95% while the spectral distances between the clusters deviated less than 10% from the results obtained with reference spectra. In contrast, the algorithm performing worst showed no similarity to the reference clustering at all. The widely used Ward algorithm deviated up to 30% from the reference in the spectral distances and returned a different spectral relation between bacteria expressing the same chromophore.

Combination of alkaline and enzymatic treatments as a process for upgrading sisal paper-grade pulp to dissolving-grade pulp

A sequence of treatments consisting of an initial xylanase treatment followed by cold alkaline extraction and a final endoglucanase treatment was investigated as a process for upgrading non-wood paper-grade pulps to dissolving-grade pulps for viscose production. Five commercial dried bleached non-wood soda/AQ paper pulps, from flax, hemp, sisal, abaca, and jute, were studied for this purpose. Commercial dried bleached eucalyptus dissolving pulp was used as reference sample. Sisal pulp showed the highest improvement in Fock’s reactivity, reaching levels nearly as high or even higher than that of eucalyptus dissolving pulp (65%), and a low hemicellulose content (3–4%) when was subjected to this sequence of treatments. The viscosity, however, decreased considerably. A uniform and narrow molecular weight distribution was observed by size exclusion chromatography. 13C nuclear magnetic resonance spectroscopy and Raman microspectroscopy revealed that the cellulose structure consisted of cellulose I.

Analysis of the cytochrome distribution via linear and nonlinear Raman spectroscopy

The cytochrome distribution in hyphal tip cells of Schizophyllum commune was visualized using resonance Raman mapping and CARS microscopy. For comparison, resonance Raman mapping and CARS imaging of cytochrome was also performed during branch formation and in completely developed central hyphae. Cytochrome, as an essential component of the electron transport chain in mitochondria, plays an important role in providing energy to actively growing mycelia. It could be shown that mitochondria at the growing hyphal tips and at branching regions are more active, i.e. contain more cytochrome, as compared to those in older parts of the hyphae. This finding is compatible with the idea of high energy consumption for biosynthesis and intracellular transport at the growing tip, while older hyphae have lower needs for ATP generated via the respiratory chain in mitochondria. To the best of our knowledge this is the first study reporting about the localization and distribution of cytochrome, as an indirect mitochondria localization within S. commune or other basidiomycetous fungi, by means of resonance Raman microspectroscopy and CARS microscopy. These Raman methods bear the potential of label-free in vivo mitochondria localization and investigation.

Morphology-sensitive Raman modes of the malaria pigment hemozoin

Resonance Raman spectroscopy was applied for investigating the malaria pigment hemozoin, which is an important target structure of antimalarial drugs. Morphology-sensitive low wavenumber modes of hemozoin were selectively enhanced with help of excitation wavelengths at lambda = 633 nm and lambda = 647 nm. The assignment of the most prominent bands in the Raman spectra at 343 cm(-1) and 368 cm(-1) was assisted by DFT calculations of the hemozoin dimer. The mode at 343 cm(-1) in the Raman spectrum of hemozoin is strongly enhanced with lambda(exc.) = 647 nm and is represented by a combined, symmetric doming mode of the two hematin units in the hemozoin dimer. The enhancement of this vibration is stronger in the resonance Raman spectrum of hemozoin compared with less crystalline beta-hematin. The selective resonance enhancement of the morphology-sensitive Raman modes of hemozoin is caused by absorption bands in the UV-VIS-NIR spectrum. This absorption spectrum of the crystalline malaria pigment hemozoin shows a strong band at 655 nm. Another broad absorption band at 870 nm is the reason for the strong relative resonance enhancement of the mode at 1372 cm(-1) in the Raman spectrum of crystalline hemozoin with lambda(exc.) = 830 nm. In conclusion, resonance Raman micro-spectroscopy with lambda(exc.) = 647 nm was shown to have great potential as an analytical tool to probe the morphology of hematin samples.

A SERRS‐Active Bead/Microelectromagnet System for Small‐Scale Sensitive Molecular Identification and Quantitation

How do you catch and identify a molecule in a very dilute solution? The question is important for the detection of trace amounts of such molecules as explosives, drugs, and biomolecules. Here we use a specifically designed microelectromagnet and surface-enhanced resonance Raman microspectroscopy (see figure) to provide an answer to this question, advancing a technique that has advantages over other current technologies.