Cover Picture: J. Biophotonics 2/2013

Employing Raman microspectroscopy, biochemical fingerprint patterns of human and porcine cartilage were obtained in this study. Using this non‐contact screening tool, it was also shown that prolonged in vitro culture can lead to phenotypic changes in chondrocytes. Picture: M. Pudlas et al., pp. 206–212 in this issue)     

Use of infrared microspectroscopy to determine leaf biochemical composition of cassava in response to Bacillus subtilis CaSUT007

The objective of this study was to investigate the growth stimulating properties of Bacillus subtilis CaSUT007 applied to cassava plants using fourier transform infrared (FTIR) microspectroscopy to monitor the production of cellular components involved in plant growth and development. Cassava stakes treated with CaSUT007 or sterile distilled water were germinated in soil. After incubation for 2 months, CaSUT007 treated plants had higher growth rate and greater biomass than the control. FTIR analysis revealed that the leaves of cassava plants treated with CaSUT007 display FTIR spectra changes in the epidermis and mesophyll tissue. These changes associated with proteins, lipids, and pectins, which are related to changes in plant cell growth and development. FTIR microspectroscopy can be used as a new tool to examine the biochemical changes within the plant tissue. This technique allows us to reveal structural chemical makeup and features of different tissue types.     

Synchrotron radiation infrared microspectroscopy to assess the activity of vancomycin against endocarditis vegetation bacteria

Infrared microspectroscopy was used to show that vancomycin alters infrared spectra of endocarditis vegetation bacteria, and that vancomycin effects on bacterial biochemical contents are unevenly distributed between peripheral and central areas of bacterial masses. Infrared microspectroscopy is useful to study the activity of antibacterial agents against bacteria in tissues.     

Fourier-transform infrared microspectroscopy,a novel and rapid tool for identification of yeasts

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 micro m in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.     

Fourier-Transform Infrared Microspectroscopy, a Novel and Rapid Tool for Identification of Yeasts

Fourier-transform infrared (FT-IR) microspectroscopy was used in this study to identify yeasts. Cells were grown to microcolonies of 70 to 250 μm in diameter and transferred from the agar plate by replica stamping to an IR-transparent ZnSe carrier. IR spectra of the replicas on the carrier were recorded using an IR microscope coupled to an IR spectrometer, and identification was performed by comparison to reference spectra. The method was tested by using small model libraries comprising reference spectra of 45 strains from 9 genera and 13 species, recorded with both FT-IR microspectroscopy and FT-IR macrospectroscopy. The results show that identification by FT-IR microspectroscopy is equivalent to that achieved by FT-IR macrospectroscopy but the time-consuming isolation of the organisms prior to identification is not necessary. Therefore, this method also provides a rapid tool to analyze mixed populations. Furthermore, identification of 21 Debaryomyces hansenii and 9 Saccharomyces cerevisiae strains resulted in 92% correct identification at the strain level for S. cerevisiae and 91% for D. hansenii, which demonstrates that the resolution power of FT-IR microspectroscopy may also be used for yeast typing at the strain level.     

Fourier transform infrared microspectroscopy as a new tool for nematode studies

We report the results of a microspectroscopy study on the Fourier transform infrared (FT-IR) absorption spectra of Caenorhabditis elegans, collected from the different parts of a single intact specimen--pharynx, intestine and tail regions. The principal absorption bands were assigned to the molecular species present in C. elegans, with an excellent reproducibility for the pharynx spectrum. These results enabled us to explore if FT-IR microspectroscopy could offer a new tool for nematode identification. As an example, the discrimination among four well characterised nematode taxa is reported. The FT-IR results completely match those obtained by Blaxter and colleagues through molecular biology [Nature 392 (1998) 71].     

ATR microspectroscopy with multivariate analysis segregates grades of exfoliative cervical cytology

Although cervical cancer screening in the UK has led to reductions in the incidence of invasive disease, this programme remains flawed. We set out to examine the potential of infrared (IR) microspectroscopy to allow the profiling of cellular biochemical constituents associated with disease progression. Attenuated total reflection-Fourier Transform IR (ATR) microspectroscopy was employed to interrogate spectral differences between samples of exfoliative cervical cytology collected into liquid based cytology (LBC). These were histologically characterised as normal (n = 5), low-grade (n = 5), high-grade (n = 5) or severe dyskaryosis (? carcinoma) (n = 5). Examination of resultant spectra was coupled with principal component analysis (PCA) and subsequent linear discriminant analysis (LDA). The interrogation of LBC samples using ATR microspectroscopy with PCA-LDA facilitated the discrimination of different categories of exfoliative cytology and allowed the identification of potential biomarkers of abnormality; these occurred prominently in the IR spectral region 1200 cm(-1) - 950 cm(-1) consisting of carbohydrates, phosphate, and glycogen. Shifts in the centroids of amide I (approximately 1650 cm(-1)) and II (approximately 1530 cm(-1)) absorbance bands, indicating conformational changes to the secondary structure of intracellular proteins and associated with increasing disease progression, were also noted. This work demonstrates the potential of ATR microspectroscopy coupled with multivariate analysis to be an objective alternative to routine cytology.     

Infrared Microspectroscopy Detects Protein Misfolding Cyclic Amplification (PMCA)-induced Conformational Alterations in Hamster Scrapie Progeny Seeds

The self-replicative conformation of misfolded prion proteins (PrP) is considered a major determinant for the seeding activity, infectiousness, and strain characteristics of prions in different host species. Prion-associated seeding activity, which converts cellular prion protein (PrP^C) into Proteinase K-resistant, infectious PrP particles (PrP^TSE), can be monitored in vitro by protein misfolding cyclic amplification (PMCA). Thus, PMCA has been established as a valuable analytical tool in prion research. Currently, however, it is under discussion whether prion strain characteristics are preserved during PMCA when parent seeds are amplified in PrP^C substrate from the identical host species. Here, we report on the comparative structural analysis of parent and progeny (PMCA-derived) PrP seeds by an improved approach of sensitive infrared microspectroscopy. Infrared microspectroscopy revealed that PMCA of native hamster 263K scrapie seeds in hamster PrP^C substrate caused conformational alterations in progeny seeds that were accompanied by an altered resistance to Proteinase K, higher sedimentation velocities in gradient ultracentrifugations, and a longer incubation time in animal bioassays. When these progeny seeds were propagated in hamsters, misfolded PrP from brain extracts of these animals showed mixed spectroscopic and biochemical properties from both parental and progeny seeds. Thus, strain modifications of 263K prions induced by PMCA seem to have been partially reversed when PMCA products were reinoculated into the original host species.     

Molecular monitoring of epithelial-to-mesenchymal transition in breast cancer cells by means of Raman spectroscopy

In breast cancer the presence of cells undergoing the epithelial-to-mesenchymal transition is indicative of metastasis progression. Since metabolic features of breast tumour cells are critical in cancer progression and drug resistance, we hypothesized that the lipid content of malignant cells might be a useful indirect measure of cancer progression. In this study Multivariate Curve Resolution was applied to cellular Raman spectra to assess the metabolic composition of breast cancer cells undergoing the epithelial to mesenchymal transition. Multivariate Curve Resolution analysis led to the conclusion that this transition affects the lipid profile of cells, increasing tryptophan but maintaining a low fatty acid content in comparison with highly metastatic cells. Supporting those results, a Partial Least Square-Discriminant analysis was performed to test the ability of Raman spectroscopy to discriminate the initial steps of epithelial to mesenchymal transition in breast cancer cells. We achieved a high level of sensitivity and specificity, 94% and 100%, respectively. In conclusion, Raman microspectroscopy coupled with Multivariate Curve Resolution enables deconvolution and tracking of the molecular content of cancer cells during a biochemical process, being a powerful, rapid, reagent-free and non-invasive tool for identifying metabolic features of breast cancer cell aggressiveness at first stages of malignancy.     

Cell wall architecture of the elongating maize coleoptile

The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed.     

Fourier transform infrared microspectroscopy reveals unique phenotypes for human embryonic and induced pluripotent stem cell lines and their progeny

Fourier transform infrared (FTIR) microspectroscopy was employed to elucidate the macromolecular phenotype of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and their differentiated progeny. Undifferentiated hESCs and hiPSC lines were found to be not clearly distinguishable from each other. However, although both hESC and hiPSC variants appeared to undergo similar changes during differentiation in terms of cell surface antigens, the derived cell types from all cell lines could be discriminated using FTIR spectroscopy. We foresee a possible future role for FTIR microspectroscopy as a powerful and objective investigative and quality control tool in regenerative medicine. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Inflammatory bowel diseases as an intermediate stage between normal and cancer: a FTIR-microspectroscopy approach

Elucidation of the evolution of inflammatory bowel disease (IBD) to cancer by clinical symptoms and histopathology of biopsies is important. Fourier transform infrared microspectroscopy (FTIR-MSP) has shown promise as a diagnostic tool for distinction of normal and cancer cells and tissues. In the present work, FTIR-MSP is used to evaluate IBD cases and to study the IR spectral characteristic with respect to cancer and normal tissues from formalin-fixed colonic biopsies from patients. Specific regions of the spectra were analyzed by statistical tools to study variations in metabolites that signified changes between the two pathological conditions: IBD and cancer. IBD tissues can be grouped with cancer or normal tissue using certain parameters such as phosphate content and RNA/DNA ratio as calculated from the spectra and show intermediate levels with regard to these metabolites. Further classification of the spectra by cluster analysis indicated which cases of Crohn's disease (3 of 10 cases) or ulcerative colitis (7 of 10 cases) were more likely to progress to cancer. The study exhibits that FTIR-MSP can detect gross biochemical changes in morphologically identical IBD and cancer tissues and suggest which cases of IBD may require further evaluation for carcinogenesis.     

A Multiscale Vibrational Spectroscopic Approach for Identification and Biochemical Characterization of Pollen

Background

Analysis of pollen grains reveals valuable information on biology, ecology, forensics, climate change, insect migration, food sources and aeroallergens. Vibrational (infrared and Raman) spectroscopies offer chemical characterization of pollen via identifiable spectral features without any sample pretreatment. We have compared the level of chemical information that can be obtained by different multiscale vibrational spectroscopic techniques.

Methodology

Pollen from 15 different species of Pinales (conifers) were measured by seven infrared and Raman methodologies. In order to obtain infrared spectra, both reflectance and transmission measurements were performed on ground and intact pollen grains (bulk measurements), in addition, infrared spectra were obtained by microspectroscopy of multigrain and single pollen grain measurements. For Raman microspectroscopy measurements, spectra were obtained from the same pollen grains by focusing two different substructures of pollen grain. The spectral data from the seven methodologies were integrated into one data model by the Consensus Principal Component Analysis, in order to obtain the relations between the molecular signatures traced by different techniques.

Results

The vibrational spectroscopy enabled biochemical characterization of pollen and detection of phylogenetic variation. The spectral differences were clearly connected to specific chemical constituents, such as lipids, carbohydrates, carotenoids and sporopollenins. The extensive differences between pollen of Cedrus and the rest of Pinaceae family were unambiguously connected with molecular composition of sporopollenins in pollen grain wall, while pollen of Picea has apparently higher concentration of carotenoids than the rest of the family. It is shown that vibrational methodologies have great potential for systematic collection of data on ecosystems and that the obtained phylogenetic variation can be well explained by the biochemical composition of pollen. Out of the seven tested methodologies, the best taxonomical differentiation of pollen was obtained by infrared measurements on bulk samples, as well as by Raman microspectroscopy measurements of the corpus region of the pollen grain. Raman microspectroscopy measurements indicate that measurement area, as well as the depth of focus, can have crucial influence on the obtained data.     

In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.     

Superpixel Raman spectroscopy for rapid skin cancer margin assessment

Spontaneous Raman micro‐spectroscopy has been demonstrated great potential in delineating tumor margins; however, it is limited by slow acquisition speed. We describe a superpixel acquisition approach that can expedite acquisition between ~×100 and ×10 000, as compared to point‐by‐point scanning by trading off spatial resolution. We present the first demonstration of superpixel acquisition on rapid discrimination of basal cell carcinoma tumor from eight patients undergoing Mohs micrographic surgery. Results have been demonstrated high discriminant power for tumor vs normal skin based on the biochemical differences between nucleus, collagen, keratin and ceramide. We further perform raster‐scanned superpixel Raman imaging on positive and negative margin samples. Our results indicate superpixel acquisition can facilitate the use of Raman microspectroscopy as a rapid and specific tool for tumor margin assessment.     

Molecular chemistry imaging to reveal structural features of various plant feed tissues

Synchrotron Fourier transform infrared (FTIR) microspectroscopy as a rapid, direct, and non-destructive analytical technique can explore molecular chemical features of the micro-structure of biological samples. However, the application of this synchrotron technology to feed science and feed chemistry is extremely rare. This article reviews that with synchrotron FTIR microspectroscopy, the molecular chemistry of various feed tissues could be imaged. These images revealed spatial intensity and distribution of chemical functional groups in various feeds tissues within cellular dimensions. Such information can be used for plant breeding program for selecting superior variety of plant for targeted feed purposes and for prediction of feed quality and nutritive value. The final purpose of this article shows that Synchrotron FTIR microspectroscopy can be used for biological structure study.     

Carotenoids Co-Localize with Hydroxyapatite,Cholesterol, and Other Lipids in Calcified Stenotic Aortic Valves. Ex Vivo Raman Maps Compared to Histological Patterns

Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.Key words: Valve calcification, stenosis, carotenoids, lipids, Raman microspectroscopy     

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/c mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and Nos2 ^−/− mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnfα and Ifnγ in sera are not significantly affected, Nos2 ^−/− mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.     

Biochemical,biophysical, and genetic changes of porcine trophoblast‐derived stem‐like cells during differentiation as evaluated using Raman microspectroscopy,Atomic force microscopy,and quantitative polymerase chain reaction

Porcine trophoblast‐derived stem‐like cells grown into serum medium start to differentiate and become senescent within 30 days. However, trophoblast‐derived cells, cultured in vitro in a defined and non‐serum medium, have the regenerative properties, such as indefinite passage and foreign DNA receptivity, similar to stem cells. To evaluate the biochemical, biophysical, and genetic changes of the terminal differentiation of trophoblast derived cells, Raman microspectroscopy, atomic force microscopy, and qPCR were applied. It was found that Raman spectral intensities of characteristic peaks, cell morphology, and Young's modulus can be used to distinguish differentiated and undifferentiated trophoblast cells. In addition, 17 cytoskeleton and extracellular matrix‐related genes were significantly impacted by medium type (non‐serum versus serum). Our findings suggest that Raman microspectroscopy and atomic force microscopy—both considered as label‐free, non‐invasive techniques—can be applied to distinguish differentiated trophoblast cells, and cellular biochemical information and biophysical properties can be indicative of cellular differences during cell differentiation. In addition, most of cytoskeleton‐related genes exhibit similar pattern to that of Young's modulus during trophoblast cell differentiation, indicating the potential connection between cytoskeleton‐related genes and cellular stiffness. genesis 53:749–761, 2015. © 2015 Wiley Periodicals, Inc.     

Raman Microspectroscopy Analysis in the Treatment of Acanthamoeba Keratitis

Acanthamoeba keratitis is a rare but serious corneal disease, often observed in contact lens wearers. Clinical treatment of infected patients frequently involves the use of polyhexamethylene biguanide (PHMB), a polymer used as a disinfectant and antiseptic, which is toxic also for the epithelial cells of the cornea. Prompt and effective diagnostic tools are hence highly desiderable for both starting early therapy and timely suspension of the treatment. In this work we use Raman microspectroscopy to analyse in vitro a single Acanthamoeba cell in cystic phase. In particular, we investigate the effect of PHMB at the single-cell level, providing useful information on both the underlying biochemical mechanism and the time frame for Acanthamoeba eradication in ocular infections. Furthermore, we demonstrate that Raman spectroscopy, in conjunction with standard multivariate analysis methods, allows discriminating between live and dead Acanthamoebas, which is fundamental to optimizing patients’ treatment.