The last two decades have seen tremendous progress in the application of two-dimensional correlation spectroscopy (2D-COS) as a versatile analysis method for data series obtained using a large ...variety of different spectroscopic modalities, including infrared (IR) and Raman spectroscopy. The analysis technique is applicable to a series of spectra recorded under the influence of an external sample perturbation. Two-dimensional COS analysis is not only helpful to decipher correlations, which may exist between distinct spectral features, but can also be utilized to obtain the sequence of individual spectral changes. The focus of this review article is on the application of 2D-COS for analyzing spatially resolved data with special emphasis on hyperspectral imaging (HSI) study. In this review, we briefly introduce the fundamentals of the generalized 2D-COS analysis approach, discuss specific points of 2D-COS application to spatially resolved spectra and demonstrate essential aspects of data pre-processing for 2D-COS analysis of spatially resolved spectra. Based on illustrative examples, we show that 2D-COS is useful for spectral band assignment in HSI applications and demonstrate its utility for detecting subtle correlations between spectra features, or between features from different imaging modalities in the case of heterospectral (multimodal) HSI. Furthermore, a short overview on existing 2D-COS software tools is provided. It is hoped that this article represents not only a useful guideline for 2D-COS analyses of spatially resolved hyperspectral data but supports also further dissemination of the 2D-COS analysis method as a whole.
Infrared nanospectroscopy enables novel possibilities for chemical and structural analysis of nanocomposites, biomaterials or optoelectronic devices. Here we introduce hyperspectral infrared ...nanoimaging based on Fourier transform infrared nanospectroscopy with a tunable bandwidth-limited laser continuum. We describe the technical implementations and present hyperspectral infrared near-field images of about 5,000 pixel, each one covering the spectral range from 1,000 to 1,900 cm
. To verify the technique and to demonstrate its application potential, we imaged a three-component polymer blend and a melanin granule in a human hair cross-section, and demonstrate that multivariate data analysis can be applied for extracting spatially resolved chemical information. Particularly, we demonstrate that distribution and chemical interaction between the polymer components can be mapped with a spatial resolution of about 30 nm. We foresee wide application potential of hyperspectral infrared nanoimaging for valuable chemical materials characterization and quality control in various fields ranging from materials sciences to biomedicine.
Identification of microorganisms by Fourier transform infrared (FT-IR) spectroscopy is known as a promising alternative to conventional identification techniques in clinical, food, and environmental ...microbiology. In this study we demonstrate the application of FT-IR hyperspectral imaging for rapid, objective, and cost-effective diagnosis of pathogenic bacteria. The proposed method involves a relatively short cultivation step under standardized conditions, transfer of the microbial material onto suitable IR windows by a replica method, FT-IR hyperspectral imaging measurements, and image segmentation by machine learning classifiers, a hierarchy of specifically optimized artificial neural networks (ANN). For cultivation, aliquots of the initial microbial cell suspension were diluted to guarantee single-colony growth on solid agar plates. After a short incubation period when microbial microcolonies achieved diameters between 50 and 300 μm, microcolony imprints were produced by using a specifically developed stamping device which allowed spatially accurate transfer of the microcolonies’ upper cell layers onto IR-transparent CaF2 windows. Dry microcolony imprints were subsequently characterized using a mid-IR microspectroscopic imaging system equipped with a focal plane array (FPA) detector. Spectral data analysis involved preprocessing, quality tests, and the application of supervised modular ANN classifiers for hyperspectral image segmentation. The resulting easily interpretable segmentation maps suggest a taxonomic resolution below the species level.
Spatial resolution is one of the most critical measurement parameters in infrared microspectroscopy. Due to the distinct levels of morphologic heterogeneity in cells and tissues the spatial ...resolution in a given IR imaging setup strongly affects the character of the infrared spectral patterns obtained from the biomedical samples. This is particularly important when spectral data bases of reference microspectra from defined tissue structures are collected. In this paper we have also pointed out that the concept of spatial resolution in IR imaging is inseparable from the contrast. Based on infrared microspectroscopic transmittance data acquired from an USAF 1951 resolution target we have demonstrated how the spatial resolution can be determined experimentally and some numbers for the spatial resolution of popular IR imaging systems are provided. Finally, we have presented a new computational procedure which is suitable to improve the spatial resolution in IR imaging. A theoretical model of 3D-Fourier self-deconvolution (FSD) is given and advantages or pitfalls of this method are discussed. Based on synchrotron IR microspectroscopic data we have furthermore demonstrated that the technique of 3D-FSD can be successfully applied to increase the spatial resolution in a real IR imaging setup.
IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell ...functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.
Bacillus amyloliquefaciens FZB42 is a Gram-positive plant growth-promoting bacterium with an impressive capacity to synthesize nonribosomal secondary metabolites with antimicrobial activity. Here we ...report on a novel circular bacteriocin which is ribosomally synthesized by FZB42. The compound displayed high antibacterial activity against closely related Gram-positive bacteria. Transposon mutagenesis and subsequent site-specific mutagenesis combined with matrix-assisted laser desorption ionization-time of flight mass spectroscopy revealed that a cluster of six genes covering 4,490 bp was responsible for the production, modification, and export of and immunity to an antibacterial compound, here designated amylocyclicin, with a molecular mass of 6,381 Da. Peptide sequencing of the fragments obtained after tryptic digestion of the purified peptide revealed posttranslational cleavage of an N-terminal extension and head-to-tail circularization of the novel bacteriocin. Homology to other putative circular bacteriocins in related bacteria let us assume that this type of peptide is widespread among the Bacillus/Paenibacillus taxon.
A genuine biophysical method, Fourier transform-infrared (FT-IR) spectroscopy has become a versatile research tool in biochemistry and biomedicine. Topical applications in microbiology and prion ...research are impressive illustrations of the vigorous evolution of the technique. FT-IR spectroscopy has established itself as a powerful method for the rapid differentiation and identification of microorganisms, thereby contributing to both clinical medicine and the prevention of bioterrorism. It has also led to considerable progress in various other fields of basic research, not least in prion sciences. In this field, FT-IR spectroscopy has been increasingly applied as a tool for elucidating structural features of the pathological prion protein, and also to study the molecular changes induced by prions in neuronal tissue and blood. This article sets out to give a review of current examples of the analytical potential of FT-IR spectroscopy in microbiology and prion research.
Summary
Nearly 1400 Bacillus strains growing in the plant rhizosphere were sampled from different sites on the Qinghai–Tibetan Plateau. Forty‐five of the isolates, selected due to their biocontrol ...activity, were genome‐sequenced and their taxonomic identification revealed that they were representatives of the Bacillus subtilis species complex (20) and the Bacillus cereus group (9). Majority of the remaining strains were found closely related to Bacillus pumilus, but their average nucleotide identity based on BLAST and electronic DNA/DNA hybridization values excluded closer taxonomic identification. A total of 45 different gene clusters involved in synthesis of secondary metabolites were detected by mining the genomes of the 45 selected strains. Except eight mesophilic strains, the 37 remaining strains were found either cold‐adapted or psychrophilic, able to propagate at 10°C and below (Bacillus wiedmannii NMSL88 and Bacillus sp. RJGP41). Pot experiments performed at 10°C with winter wheat seedlings revealed that cold‐adapted representatives of B. pumilus, B. safensis and B. atrophaeus promoted growth of the seedlings under cold conditions, suggesting that these bacilli isolated from a cold environment are promising candidates for developing of bioformulations useful for application in sustainable agriculture under environmental conditions unfavourable for the mesophilic bacteria presently in use.
MALDI-TOF mass spectrometry (MALDI-TOF MS) is increasingly used as a reliable technique for species identification of bacterial pathogens. In this study we investigated the question of whether ...MALDI-TOF MS can be used for accurate sub-differentiation of strains and isolates of two important nosocomial pathogens Enterococcus faecium and Staphylococcus aureus. For this purpose, a selection of 112 pre-characterized E. faecium isolates (clonal complexes CC2, CC5, CC9, CC17, CC22, CC25, CC26, CC92 altogether 52 multilocus sequence types) and 59 diverse S. aureus isolates (mostly methicillin resistant; CC5, CC8, CC22, CC30, CC45, CC398) were studied using a combination of MALDI-TOF MS and advanced methods of spectral data analysis. The strategy of MS data evaluation included manual peak inspection on the basis of pseudo gel views, unsupervised hierarchical cluster analysis and supervised artificial neural network (ANN) analysis. We were capable of differentiating patterns of hospital-associated E. faecium isolates (CC17) from other strains of E. faecium with 87% accuracy, but failed to identify lineage-specific biomarker peaks. For S. aureus pattern analyses we were able to confirm a number of signals described in previous studies, but often failed to identify biomarkers that would allow a consistent and reliable identification of phylogenetic lineages, clonal complexes or sequence types. Hence, the discriminatory power of MALDI-TOF MS was found to be insufficient for reliably sub-differentiating E. faecium and S. aureus isolates to the level of distinct clones or clonal complexes, such as assessed by MLST. Further, a comparison between peak patterns of susceptible and resistant isolates did not identify statistically relevant marker peaks linked to glycopeptide resistance determinants (vanA, vanB) in E. faecium, or the methicillin resistance determinant (mecA) in S. aureus.
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•MALDI TOF MS of strains and isolates from E. faecium and S. aureus•Analysis of MALDI data carried out by gel views, cluster analysis and ANNs•Differentiation of hospital-associated and other isolates of E. faecium is possible•No lineage-specific biomarker peaks for E. faecium were found•In S. aureus no MALDI biomarkers found for phylogenetic lineages, CC, or ST•No biomarkers detected for methicillin and/or vancomycin resistance•Discriminatory power of MALDI insufficient for typing of S. aureus and E. faecium
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•Anosteocytic bone material is more water permeable than osteocytic bone material.•In osteocytic zebrafish bone, water flow appears to be confined to the lacunar-canalicular ...network.•Anosteocytic medaka bone is water permeable and containes far less proteoglycans.•3D correlation of neutron and X-ray tomographies with micron resolution reveals water diffusion across the bone matrix.
Vertebrate bones are made of a nanocomposite consisting of water, mineral and organics. Water helps bone material withstand mechanical stress and participates in sensation of external loads. Water diffusion across vertebrae of medaka (bone material lacking osteocytes) and zebrafish (bone material containing osteocytes) was compared using neutron tomography. Samples were measured both wet and following immersion in deuterated-water (D2O). By quantifying H+ exchange and mutual alignment with X-ray µCT scans, the amount of water expelled from complete vertebra was determined. The findings revealed that anosteocytic bone material is almost twice as amenable to D2O diffusion and H2O exchange, and that unexpectedly, far more water is retained in osteocytic zebrafish bone. Diffusion in osteocytic bones (only 33 % – 39 % water expelled) is therefore restricted as compared to anosteocytic bone (∼ 60 % of water expelled), presumably because water flow is confined to the lacunar-canalicular network (LCN) open-pore system. Histology and Raman spectroscopy showed that anosteocytic bone contains less proteoglycans than osteocytic bone. These findings identify a previously unknown functional difference between the two bone materials. Therefore, this study proposes that osteocytic bone retains water, aided by non-collagenous proteins, which contribute to its poroelastic mechano-transduction of water flow confined inside the LCN porosity.
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