Analysis of type I and IV collagens by FT-IR spectroscopy and imaging for a molecular investigation of skeletal muscle connective tissue

Many muscular diseases result from abnormal organization of connective tissue and/or collagen network formation. Only a few molecular imaging techniques are able to analyze this collagen network by differentiating collagen types. In this study, FT-IR spectroscopy was used to analyze type I and IV collagens, the most important compounds of which are perimysium and endomysium, respectively. Secondary structure of collagen types was determined by curve-fitting the 1,700–1,480 cm⁻¹ spectral interval. Type I collagen could be differentiated from type IV by its higher amounts of triple helix and α-helix, but lower amounts of β-sheets (P < 0.01). FT-IR imaging was then used to determine structural features of perimysium and endomysium collagen network in bovine Flexor carpi radialis muscle. Secondary structure of proteins contained in perimysium and endomysium was found to be very close to type I and IV collagens, respectively. FT-IR spectroscopy and imaging are thus analytical tools that might be used for investigating biodistribution and assembly of collagen types in connective tissues. Figure Visible (left) and full spectral FT-IR (right) images of skeletal muscle tissue section (16 μm) exhibiting a vertical arrangement of fibers. + and × in FT-IR image show selected positions to obtain FT-IR spectra of perimysium and endomysium, respectively     

Trends in Fourier transform infrared spectroscopic imaging

Fourier transform infrared (FTIR) spectroscopic imaging is a relatively new method that has received great attention as a new field of analytical chemistry. The greatest benefit of this technique lies in the high molecular sensitivity combined with a spatial resolution down to a few micrometers. Another advantage is the ability to probe samples under native conditions, which allows new insights into samples without the need for fixation, stains, or an additional marker. Advances in instrumentation have made FTIR spectroscopic imaging the tool of choice for an increasing number of applications. The main applications are in the bioanalytical chemistry of cells and tissue, polymers, and recently as well as in homeland security. This report gives a short overview of current developments and recent applications. Figure FTIR image of a polymer blend reveals the chemical composition. Online Abstract Figure (365 KB).     

Application of XPS and ToF-SIMS for surface chemical analysis of DNA microarrays and their substrates

The chemical composition of the functional surfaces of substrates used for microarrays is one of the important parameters that determine the quality of a microarray experiment. In addition to the commonly used contact angle measurements to determine the wettability of functionalized supports, X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS) are more specific methods to elucidate details about the chemical surface constitution. XPS yields information about the atomic composition of the surface, whereas from ToF-SIMS, information on the molecular species on the surface can be concluded. Applied on printed DNA microarrays, both techniques provide impressive chemical images down to the micrometer scale and can be utilized for label-free spot detection and characterization. Detailed information about the chemical constitution of single spots of microarrays can be obtained by high-resolution XPS imaging. Figure Eye-catching image for the graphical online abstract     

Suitability of infrared spectroscopic imaging as an intraoperative tool in cerebral glioma surgery

Infrared spectroscopic imaging is a promising intraoperative tool which enables rapid, on-site diagnosis of brain tumors during neurosurgery. A classification model was recently developed using infrared spectroscopic images from thin tissue sections to grade malignant gliomas, the most frequent class of primary brain tumor. In this study the model was applied to 54 specimens from six patients with inhomogeneous gliomas composed of regions with different tumor density and morphology. The resection was controlled using neuronavigation which transfers the findings obtained by preoperative magnetic resonance imaging (MRI) into the operating field. For comparison, all specimens were independently evaluated by histopathology after hematoxylin and eosin staining. The infrared-derived grading agreed with histopathology and MRI findings for almost all specimens. With regard to histopathological assessment, sensitivities of 100% (22/22) and 93.1% (27/29) and specificities of 96.9% (31/32) and 88.0% (22/25) were achieved, depending on whether the classification was based on the predominant or maximal tumor grade, respectively, in the specimen. Altogether, in 98% (53/54) of all specimens the decision to continue or not continue tumor resection could have been made according to the infrared spectroscopic classification. This retrospective study clearly demonstrates that infrared spectroscopic imaging may help to define tumor margins intraoperatively and to detect high-grade tumor residues for achieving more radical tumor resection. MRT-guided tumor resection (left) is combined with infrared spectroscopy-based tissue classification (middle, right). With regard to histopathological assessment, sensitivities of 100% and 93.1% and specificities of 96.9% and 88.0% were achieved for 54 specimens. (TIF 47.1 KB)     

A linear modulation-based stochastic resonance algorithm applied to the detection of weak chromatographic peaks

A simple stochastic resonance algorithm based on linear modulation was developed to amplify and detect weak chromatographic peaks. The output chromatographic peak is often distorted when using the traditional stochastic resonance algorithm due to the presence of high levels of noise. In the new algorithm, a linear modulated double-well potential is introduced to correct for the distortion of the output peak. Method parameter selection is convenient and intuitive for linear modulation. In order to achieve a better signal-to-noise ratio for the output signal, the performance of two-layer stochastic resonance was evaluated by comparing it with wavelet-based stochastic resonance. The proposed algorithm was applied to the quantitative analysis of dimethyl sulfide and the determination of chloramphenicol residues in milk, and the good linearity of the method demonstrated that it is an effective tool for detecting weak chromatographic peaks. Figure The linear modulation-based stochastic resonance algorithm (LSRA) improved the output chromatographic peak of chloramphenicol     

Nuclear magnetic resonance of mass-limited samples using small RF coils

Figure Schematic diagram of a typical arrangement used for hyphenating chemical microseparations (e.g. capillary HPLC, CE, or CEC) with microcoil NMR detection     

Impedimetric genosensors for the detection of DNA hybridization

Impedance spectroscopy is proposed as the transduction principle for detecting the hybridization of DNA complementary strands. In our experiments, different DNA oligonucleotides were used as model gene substances. The gene probe is first immobilized on a graphite-epoxy composite working electrode based genosensor. Detection principle is based on changes of impedance spectra of a redox marker, the ferro/ferricyanide couple, after hybridization with target DNA. Resistance offered to the electrochemical reaction serves as the working signal, allowing for an unlabelled gene assay.      

A synchrotron FTIR microspectroscopy investigation of fungal hyphae grown under optimal and stressed conditions

Synchrotron FTIR can provide high spatial resolution (<10 μm pixel size) in situ biochemical analyses of intact biotissues, an area of increasing importance in the post-genomic era, as gene functions and gene networks are coming under direct scrutiny. With this technique, we can simultaneously assess multiple aspects of cell biochemistry and cytoplasmic composition. In this paper, we report the first results of our synchrotron FTIR examination of hyphae of three important fungal model systems, each with sequenced genomes and a wealth of research: Aspergillus, Neurospora, and Rhizopus. We have analyzed the FTIR maps of Aspergillus nidulans cells containing the hypA1 allele, a well-characterized single-gene temperature-sensitive morphogenetic mutation. The hypA1 cells resemble wildtype at 28 °C but have growth defects at 42 °C. We have also investigated Neurospora and Rhizopus cultures grown in media with optimal or elevated pH. Significant differences between the spectra of the three fungi are likely related to differences in composition and structure. In addition, high spatial resolution synchrotron FTIR spectroscopy provides an outstanding method for monitoring subtle subcellular changes that accompany environmental stress. Figure Photomicrographs and FTIR spectra acquired along Rhizopus hyphae grown at pH 6.5 (a) and pH 8.5 (b). Scale bars 50 μm     

Application of high-resolution EFTEM SI in an AEM

In this work we show how energy-filtered imaging can be used to obtain spectrum images of electron energy-loss spectrometric data. Focus is placed on improved energy resolution within these data sets. Using two multilayer samples (GaN/AlN and InP/InAs), we demonstrate the advantages of spectrum-imaging and its extended mapping capabilities. Plasmon-ratio maps are used to quickly create high-contrast material maps with high signal-to-noise ratio, ratio-contrast plots are used to gain optimum settings for the ratio maps, and plasmon-position maps are used to map small shifts of the energy position of bulk plasmon peaks. Figure Scheme of EELS SI and derived plasman-position map     

Colorimetric detection of immunoglobulin G by use of functionalized gold nanoparticles on polyethylenimine film

A method based on use of functionalized gold nanoparticles on polyethylenimine film has been developed for colorimetric detection of immunoglobulin G (IgG). The immunogold nanoparticles were immobilized on quartz slides by recognition between antibody and antigen, with the antigen chemically adsorbed on the polyethylenimine film. By measurement of the UV–visible spectra of the immobilized immunogold, detection of h-IgG was achieved. The detection limit for h-IgG by use of this method can be as low as 0.01 μg mL⁻¹. This method is quite promising for numerous applications in immunoassay. Figure     

Study of the dynamics of surface molecules by time-resolved sum-frequency generation spectroscopy

Sum-frequency generation (SFG) is a nonlinear laser-spectroscopy technique suitable for analysis of adsorbed molecules. The sub-monolayer sensitivity of SFG spectroscopy enables vibrational spectra to be obtained with high specificity for a variety of molecules on a range of surfaces, including metals, oxides, and semiconductors. The use of ultra-short laser pulses on time-scales of picoseconds also makes time-resolved measurements possible; this can reveal ultrafast transient changes in molecular arrangements. This article reviews recent time-resolved SFG spectroscopy studies revealing site-hopping of adsorbed CO on metal surfaces and the dynamics of energy relaxation at water/metal interfaces. Time-resolved sum frequency generation spectroscopy at surfaces with non-resonant laser pulse irradiation     

Proof of principle of a generalized fuzzy Hough transform approach to peak alignment of one-dimensional <Superscript>1</Superscript>H NMR data

In metabolic profiling, multivariate data analysis techniques are used to interpret one-dimensional (1D) ¹H NMR data. Multivariate data analysis techniques require that peaks are characterised by the same variables in every spectrum. This location constraint is essential for correct comparison of the intensities of several NMR spectra. However, variations in physicochemical factors can cause the locations of the peaks to shift. The location prerequisite may thus not be met, and so, to solve this problem, alignment methods have been developed. However, current state-of-the-art algorithms for data alignment cannot resolve the inherent problems encountered when analysing NMR data of biological origin, because they are unable to align peaks when the spatial order of the peaks changes—a commonly occurring phenomenon. In this paper a new algorithm is proposed, based on the Hough transform operating on an image representation of the NMR dataset that is capable of correctly aligning peaks when existing methods fail. The proposed algorithm was compared with current state-of-the-art algorithms operating on a selected plasma dataset to demonstrate its potential. A urine dataset was also processed using the algorithm as a further demonstration. The method is capable of successfully aligning the plasma data but further development is needed to address more challenging applications, for example urine data. Figure Traces of NMR peaks visualizing the Generalized Fuzzy Hough Transform (GFHT) method for elucidating peak correspondence between samples. The spectra are sorted according to one shift sensitive peak and reveals that other peaks exhibit a similar shift pattern. This pattern(s) can now be searched for using the GFHT. The red and black spectra in the figure are the most shifting spectra (top and bottom), by following the GFHT traces peak correspondence is easily established although peaks change spatial location Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detection of bacteria by surface-enhanced Raman spectroscopy

The detection and identification of dilute bacterial samples by surface-enhanced Raman spectroscopy has been explored by mixing aqueous suspensions of bacteria with a suspension of nanocolloidal silver particles. An estimate of the detection limit of E. coli was obtained by varying the concentration of bacteria. By correcting the Raman spectra for the broad librational OH band of water, reproducible spectra were obtained for E. coli concentrations as low as approximately 10³ cfu/mL. To aid in the assignment of Raman bands, spectra for E. coli in D₂O are also reported. Figure Light scattering apparatus used to detect bacteria     

Determination of total <Emphasis Type="Italic">trans</Emphasis> fats and oils by infrared spectroscopy for regulatory compliance

The mandatory requirement in many countries to declare the amount of trans fat present in food products and dietary supplements has led to a need for sensitive and accurate methodologies for the rapid quantitation of total trans fats and oils. Capillary gas chromatography (GC) and infrared spectroscopy (IR) are the two methods most commonly used to identify and quantify trans fatty acids for food labeling purposes (see the article by Delmonte and Rader in this ABC issue for a detailed presentation of GC methodology). The present article provides a comprehensive review of the IR technique and the current attenuated total reflection (ATR) Fourier-transform (FT) IR methodologies for the rapid determination of total trans fats and oils. This review also addresses potential sources of interferences and inaccuracies in FTIR determinations, particularly those done at low trans levels. Recent observations have shown that the presence of saturated fats caused interferences in the FTIR spectra observed for trans triacylglycerols. The recognition and resolution of previously unresolved quantitative issues improved the accuracy and sensitivity of the FTIR methodology. Once validated, it is anticipated that the new negative second-derivative ATR-FTIR procedure will make IR spectroscopy more suitable than ever, and a rapid alternative and/or complementary method to GC, for the rapid determination of total trans fats for regulatory compliance. Figure Infrared light bouncing inside an internal reflection crystal     

Electrochemical determination of pyrophosphate at nanomolar levels using a gold electrode covered with a cysteine nanofilm and based on competitive coordination of Cu(II) ion to cysteine and pyrophosphate

We are presenting an electrochemical method for the determination of pyrophosphate ions (PPi) that is based on the competitive coordination of Cu(II) ion to a nanofilm of cysteine (Cys) and dissolved PPi. Cys was immobilized on the surface of a gold electrode by self-assembly. The Cys-modified gold electrode was loaded with Cu(II) ion which is released from the surface on addition of a sample containing PPi. The sensor shows an unprecedented electrochemical response to PPi, and the reduction peak currents is linearly related to the logarithm of the concentration of PPi in the 100 nM to 10 mM range (with an R² or 0.982). The limit of detection is ~10 nM which is lower than the detection limits hitherto reported for PPi. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and common anions give a much weaker response. The method demonstrated here is simple, effective, highly sensitive, hardly interfered, and does not require the addition of a reagent. The method was applied to the determination of PPi in (spiked) serum samples.

Schematic illustration of the pyrophosphate sensing process.

     

Towards single biomolecule handling and characterization by MEMS

Applications of microelectromechanical systems (MEMS) technology are widespread in both industrial and research fields providing miniaturized smart tools. In this review, we focus on MEMS applications aiming at manipulations and characterization of biomaterials at the single molecule level. Four topics are discussed in detail to show the advantages and impact of MEMS tools for biomolecular manipulations. They include the microthermodevice for rapid temperature alternation in real-time microscopic observation, a microchannel with microelectrodes for isolating and immobilizing a DNA molecule, and microtweezers to manipulate a bundle of DNA molecules directly for analyzing its conductivity. The feasibilities of each device have been shown by conducting specific biological experiments. Therefore, the development of MEMS devices for single molecule analysis holds promise to overcome the disadvantages of the conventional technique for biological experiments and acts as a powerful strategy in molecular biology. Figure Towards single bio molecular handling and characterization by MEMS     

Time-resolved fluorescence spectroscopy and imaging of proteinaceous binders used in paintings

The differentiation of proteins commonly found as binding media in paintings is presented based on spectrally resolved and time-resolved laser-induced fluorescence (LIF) and total emission spectroscopy. Proteins from eggs and animal glue were analysed with pulsed laser excitation at 248 nm (KrF excimer) and 355 nm (third harmonic of Nd:YAG) for spectrally resolved measurements, and at 337 nm (N₂) and 405 nm (N₂ pumped dye laser) for spectrally resolved lifetime measurements and fluorescence lifetime imaging (FLIM). Total emission spectra of binding media are used for the interpretation of LIF spectra. Time-resolved techniques become decisive with excitation at longer wavelengths as fluorescence lifetime permits the discrimination amongst binding media, despite minimal spectral differences; spectrally resolved measurements of fluorescence lifetime have maximum differences between the binding media examined using excitation at 337 nm, with maximum observed fluorescence at 410 nm. FLIM, which measures the average lifetime of the emissions detected, can also differentiate between media, is non-invasive and is potentially advantageous for the analysis of paintings. Figure The fluorescence of solid ox glue extracted from collagen can be visualised in this Total Fluorescence Spectrum; three different peaks from multiple fluorophores are present and allow the discrimination between collagen- and non-collagen proteinaceous binding media found in paintings     

Fourier transform infrared spectroscopy as a tool to study structural properties of cytochromes P450 (CYPs)

Cytochrome P450 proteins (CYPs) are a big class of heme proteins which are involved in various metabolic processes of living organisms. CYPs are the terminal catalytically active components of monooxygenase systems where the substrate binds and is hydroxylated. In order to be functionally competent, the protein structures of CYPs possess specific properties that must be explored in order to understand structure–function relationships and mechanistic aspects. Fourier transform infrared spectroscopy (FTIR) is one tool that is used to study these structural properties. The application of FTIR spectroscopy to the secondary structures of CYP proteins, protein unfolding, protein–protein interactions and the structure and dynamics of the CYP heme pocket is reviewed. A comparison with other thiolate heme proteins (nitric oxide synthase and chloroperoxidase) is also included. Figure The protein secondary structure, protein unfolding, redox-partner protein–protein interaction, structural changes induced by the reduction of the heme iron, and the structure and dynamics of the active site of cytochromes P450 (CYP) can be studied using Fourier transform infrared spectroscopy (FTIR). FTIR spectroscopy is a good approach for gaining a deeper insight into structure–function relationships in CYPs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Investigation of sample-purification procedures for MALDI-based proteomic studies

Purification methods for proteomics samples are of crucial concern for improving the quality of the sample delivered to the mass spectrometer. They constitute the link between the mass spectrometer and protein processing and peptide isolation steps that usually require solvents, buffers, or detergents completely incompatible with MS-analysis conditions. This work describes three new clean-up procedures using synthetic membranes and polymer media and compares them with standard procedures. The efficiency of each of the purification procedures was studied via application to four standards and two membrane proteins. This work highlights the importance of versatility in sample preparation, especially for MS-based proteomic investigations. Figure PMF spectra obtained after MALDI-TOF measurements of bovine mitochondrial complex III (A) and complex IV (B) in-solution digests, with and without purification     

Determination of the titanium content of human transferrin by inductively-coupled plasma-atomic-emission spectroscopy

We report a simple method that combines dialysis, as a purification method, with the multielement capability of ICP to determine the titanium-to-transferrin mole ratio at physiological pH, under buffer conditions. The method, by means of which titanium and transferrin are determined simultaneously, enabled us to assess the binding capacities of different titanocene complexes. Figure Titanocene dichloride