Raman 18O-labeling of bacteria in visible and deep UV-ranges

Raman stable isotope labeling with ²H, ¹³C or ¹⁵N has been reported as an elegant approach to investigate cellular metabolic activity, which is of great importance to reveal the functions of microorganisms in native environments. A new strategy termed Raman ¹⁸O-labeling was developed to probe the metabolic activity of bacteria. Raman ¹⁸O-labeling refers to the combination of Raman microspectroscopy with ¹⁸O-labeling using H₂¹⁸O. At an excitation wavelength of 532 nm, the incorporation of ¹⁸O into the amide I group of proteins and DNA/RNA bases was observed in Escherichia coli cells, while for an excitation wavelength electronically resonant with DNA or aromatic amino acid absorption at 244 nm ¹⁸O assimilation was detected using chemometric tools rather than visual inspection. Raman ¹⁸O-labeling at 532 nm combined with 2D correlation analysis confirmed the assimilation of ¹⁸O in proteins and nucleic acids and revealed the growth strategy of E. coli cells; they underwent protein synthesis followed by nucleic acid synthesis. Independent cultural replicates at different incubation times corroborated the reproducibility of these results. The variations in spectral features of ¹⁸O-labeled cells revealed changes in physiological information of cells. Hence, Raman ¹⁸O-labeling could provide a powerful tool to identify metabolically active bacterial cells.     

Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies

We demonstrate a novel bio‐spectroscopic technique, “simultaneous Raman/GFP microspectroscopy”. It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti‐Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two‐component multivariate curve resolution with alternate least squares (MCR‐ALS) analysis in the anti‐Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR‐ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm^?1, previously reported as “Raman spectroscopic signature of life of yeast.”     

Effects of mobile phone emissions on human red blood cells

Raman spectroscopy was performed on GSM 900 and 1800 MHz mobile phone signal exposed red blood cells (RBCs). The observed changes in the Raman spectra of mobile signal exposed RBCs compared to unexposed control suggest reduced hemoglobin-oxygen affinity for the exposed cells. The possible mechanism may involve activation of the voltage gated membrane Ca²⁺ channels by the mobile phone emissions resulting in an increase in the levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) in cells via altered metabolic activities. Further studies carried out with fluorescent Ca²⁺ indicator confirmed increased intracellular Ca²⁺ level in the exposed cells. Since intracellular ATP level influences the shape and mechanics of RBCs, exposed cells were studied using diffraction phase microscopy and optical tweezers. Detectable changes in shape and mechanical properties were observed due to mobile signal exposure.     

Therapeutic prognosis of prostate cancer using surface-enhanced Raman scattering of patient urine and multivariate statistical analysis

Surface-enhanced Raman scattering (SERS) is highly sensitive and label-free analytical technique based on Raman spectroscopy aided by field-multiplying plasmonic nanostructures. We report the use of SERS measurements of patient urine in conjunction with biostatistical algorithms to assess the treatment response of prostate cancer (PCa) in 12 recurrent (Re) and 63 nonrecurrent (NRe) patient cohorts. Multiple Raman spectra are collected from each urine sample using monodisperse silver nanoparticles (AgNPs) for Raman signal enhancement. Genetic algorithms-partial least squares-linear discriminant analysis (GA-PLS-LDA) was employed to analyze the Raman spectra. Comprehensive GA-PLS-LDA analyses of these Raman spectral features (p = 3.50 × 10⁻¹⁶ ) yield an accuracy of 86.6%, sensitivity of 86.0%, and specificity 87.1% in differentiating the Re and NRe cohorts. Our study suggests that SERS combined with multivariate GA-PLS-LDA algorithm can potentially be used to detect and monitor the risk of PCa relapse and to aid with decision-making for optimal intermediate secondary therapy to recurred patients.     

Monitoring the biochemical changes occurring to human keratinocytes exposed to solar radiation by Raman spectroscopy

Solar radiation exposure is recognised to be a significant contributor to the development of skin cancer. Monitoring the simultaneous and consecutive mechanisms of interaction could provide a greater understanding of the process of photocarcinogenesis. This work presents an analysis of the biochemical and morphological changes occurring to immortalised human epithelial keratinocyte (HaCaT) cell cultures exposed to simulated solar radiation (SSR). Cell viability was monitored with the aid of the Alamar Blue assay, morphological examination was done with haematoxylin and eosin staining (H&E) and changes to the biochemical constituents (nucleic acids and proteins) as a result of the radiation insult were demonstrated through a combination of Raman microspectroscopy and multivariate analysis of spectral patterns. The spectral results suggest that SSR induces changes to the conformational structure of DNA as an immediate result of the radiation, whereas alteration in the protein signature is mostly seen as a later response.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

Time-gated interferometric detection increases Raman scattering to fluorescence signal ratio in biological samples

Attainable levels of signal-to-background ratio (SBR) in Raman spectroscopy of biological samples is limited by the presence of endogenous fluorophores. It is customary to remove the ubiquitous fluorescence background using postacquisition data processing. However, new approaches are needed to reduce background contributions and maximize the fraction of the sensor dynamical range occupied by Raman photons. Time-resolved detection using pulsed lasers and time-gated measurements can be used to address the signal-to-background problem in biological samples by limiting light detection to nonresonant interaction phenomena with relaxation time scales occurring on sub-nanosecond time scales, thereby excluding contributions from resonant phenomena such as fluorescence. A time-gated Fourier-transform spectrometer was assembled using a commercially available interferometer, a single channel single-photon avalanche diode and time tagging electronics. A time gate of 300 ps increased the signal-to-background-ratio of the 1440 cm^?1 Raman band from 36% to 69% in an olive oil sample hereby demonstrating the potential of this approach for autofluorescence suppression.      

Comparative analysis of dermal collagen and lipids in cereblon ablated mice using a multimodal nonlinear optical system

By utilizing a multimodal nonlinear optical system that combines coherent anti-Stokes Raman scattering and second harmonic generation to investigate biological characteristics of dermal tissues ex vivo, we demonstrate the potential feasibility of using this optical approach as a powerful new investigative tool for future biomedical research. For this study, our optical system was utilized for the first time to analyze lipid and collagen profiles in cereblon knockout (KO) mouse skin, and we were able to discover significant alterations in the number of carbon–carbon double bonds (wild-type vs. cereblon KO; N_CC: 0.75 vs. 0.85) of skin fatty acids in triacylglycerides as well as changes in dermal collagen fibers (25% reduction in cereblon KO). By adopting our optical system to biological studies, we provide researchers with another diagnostic approach to validate their experimental results, which will significantly advance the state of biomedical research.     

Differentiating smokers and nonsmokers based on Raman spectroscopy of oral fluid and advanced statistics for forensic applications

Raman spectroscopy has proven to be a valuable tool for analyzing various types of forensic evidence such as traces of body fluids. In this work, Raman spectroscopy was employed as a nondestructive technique for the analysis of dry traces of oral fluid to differentiate between smoker and nonsmoker donors with the aid of advanced statistical tools. A total of 32 oral fluid samples were collected from donors of differing gender, age and race and were subjected to Raman spectroscopic analysis. A genetic algorithm was used to determine eight spectral regions that contribute the most to the differentiation of smokers and nonsmokers. Thereafter, a classification model was developed based on the artificial neural network that showed 100% accuracy after external validation. The developed approach demonstrates great potential for the differentiation of smokers and nonsmokers based on the analysis of dry traces of oral fluid.     

Determination of inclusion depth in ex vivo animal tissues using surface enhanced deep Raman spectroscopy

This work presents recent developments in spatially offset and transmission Raman spectroscopy for noninvasive detection and depth prediction of a single SERS inclusion located deep inside ex vivo biological tissues. The concept exploits the differential attenuation of Raman bands brought about by their different absorption due to tissue constituents enabling to predict the inclusion depth. Four different calibration models are tested and evaluated to predict the depth of surface enhanced Raman scattering labelled nanoparticles, within an up to 40 mm slab of porcine tissue. An external measurement carried out in transmission mode, with a noninvasively built model on the analysed sample, is shown to be insensitive to variations of the overall thickness of the tissue yielding an average root‐mean‐square error of prediction of 6.7%. The results pave the way for future noninvasive deep Raman spectroscopy in vivo enabling to localise cancer biomarkers for an early diagnosis of multiple diseases.      

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm^?1 shift from 1306 cm^?1 in aqueous solution to 1314 cm^?1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding ability. In non-hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm^?1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm^?1; in water, a strong hydrogen-bonding solvent, 1306 cm^?1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm^?1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.     

Raman spectroscopy of human skin for kidney failure detection

The object of this paper is in vivo study of skin spectral-characteristics in patients with kidney failure by conventional Raman spectroscopy in near infrared region. The experimental dataset was subjected to discriminant analysis with the projection on latent structures (PLS-DA). Application of Raman spectroscopy to investigate the forearm skin in 85 adult patients with kidney failure (90 spectra) and 40 healthy adult volunteers (80 spectra) has yielded the accuracy of 0.96, sensitivity of 0.94 and specificity of 0.99 in terms of identifying the target subjects with kidney failure. The autofluorescence analysis in the near infrared region identified the patients with kidney failure among healthy volunteers of the same age group with specificity, sensitivity, and accuracy of 0.91, 0.84, and 0.88, respectively. When classifying subjects by the presence of kidney failure using the PLS-DA method, the most informative Raman spectral bands are 1315 to 1330, 1450 to 1460, 1700 to 1800 cm⁻¹. In general, the performed study demonstrates that for in vivo skin analysis, the conventional Raman spectroscopy can provide the basis for cost-effective and accurate detection of kidney failure and associated metabolic changes in the skin.     

Blood plasma resonance Raman spectroscopy combined with multivariate analysis for esophageal cancer detection

We herein report a novel, reliable and inexpensive method for detecting esophageal cancer using blood plasma resonance Raman spectroscopy combined with multivariate analysis methods. The blood plasma samples were divided into late stage cancer group (n = 164), early stage cancer group (n = 35) and normal group (n = 135) based on clinical pathological diagnosis. Using a specially designed quartz capillary tube as sample holder, we obtained higher quality resonance Raman spectra of blood plasma than existing method. The study demonstrated that the carotenoids levels in blood plasma were reduced in esophageal cancer patients. The area under the receiver operating characteristic curve (and 95% confidence interval) calculated by wavenumber selection and principal component analysis combined with linear discriminant analysis (PC-LDA) algorithm were 0.894 (0.858-0.929), 0.901 (0.841-0.960) and 0.871 (0.799-0.942) for differentiating late cancer from normal, late cancer from early cancer, and early cancer from normal respectively. The contribution from the two carotenoids wavenumber regions of 1155 and 1515 cm⁻¹ were more than 84.2%. The results show that the plasma carotenoids could be a potential biomarker for screening esophageal cancer using resonance Raman spectroscopy combined with wavenumber selection and PC-LDA algorithms.      

On the origin of the 1602 cm–1 Raman band of yeasts; contribution of ergosterol

The 1602 cm^–1 Raman signature, which we call the “Raman spectroscopic signature of life” in yeasts, is a marker Raman band for cell metabolic activity. Despite the established fact that its intensity sensitively reflects the metabolic status of the cell, its molecular origin remained unclear. In this work, we propose ergosterol as the major contributor of the 1602 cm^–1 Raman signature. The theoretical isotope shift calculation for ergosterol agreed with previous observations. Furthermore, experiments showed that the Raman spectrum of ergosterol corresponds very well with the depleting spectral component in yeast that behaves together with the 1602 cm^–1 signature when the cells are under stress. This work implies that the 1602 cm^–1 Raman signature could serve as an intrinsic ergosterol marker in yeasts for the study of sterol metabolism in vivo and in a label‐free manner, which could not be done by any other techniques at the current stage. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Simultaneous fingerprint and high‐wavenumber fiber‐optic Raman spectroscopy enhances real‐time in vivo diagnosis of adenomatous polyps during colonoscopy

Colorectal cancer can be prevented if detected early (e.g., precancerous polyps‐adenoma). Endoscopic differential diagnosis of hyperplastic polyps (that have little or no risk of malignant transformation) and adenomas (that have prominent malignant latency) remains an unambiguous clinical challenge. Raman spectroscopy is an optical vibrational technique capable of probing biomolecular changes of tissue associated with neoplastic transformation. This work aims to apply a fiber‐optic simultaneous fingerprint (FP) and high wavenumber (HW) Raman spectroscopy technique for real‐time in vivo assessment of adenomatous polyps during clinical colonoscopy. We have developed a fiber‐optic Raman endoscopic technique capable of simultaneously acquiring both the FP (i.e., 800–1800 cm^–1) and HW (i.e., 2800–3600 cm^–1) Raman spectra from colorectal tissue subsurface (<200 µm) for real‐time assessment of colorectal carcinogenesis. In vivo FP/HW Raman spectra were acquired from 50 patients with 17 colorectal polyps during clinical colonoscopy. Prominent Raman spectral differences (p < 0.001) were found between hyperplastic (n = 118 spectra), adenoma (n = 184 spectra) that could be attributed to changes in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations. Simultaneous FP/HW Raman endoscopy provides a diagnostic sensitivity of 90.9% and specificity of 83.3% for differentiating adenoma from hyperplastic polyps, which is superior to either the FP or HW Raman technique alone. This study shows that simultaneous FP/HW Raman spectroscopy technique has the potential to be a clinically powerful tool for improving early diagnosis of adenomatous polyps in vivo during colonoscopic examination.

     

The molecular interaction of a protein in highly concentrated solution investigated by Raman spectroscopy

We used Raman spectroscopy to investigate the structure and interactions of lysozyme molecules in solution over a wide range of concentrations (2.5–300 mg ml^?1). No changes in the amide‐I band were observed as the concentration was increased, but the width of the Trp band at 1555 cm^?1 and the ratios of the intensities of the Tyr bands at 856 and 837 cm^?1, the Trp bands at 870 and 877 cm^?1, and the bands at 2940 (CH stretching) and 3420 cm^?1 (OH stretching) changed as the concentration was changed. These results reveal that although the distance between lysozyme molecules changed by more than an order of magnitude over the tested concentration range, the secondary structure of the protein did not change. The changes in the molecular interactions occurred in a stepwise process as the order of magnitude of the distance between molecules changed. These results suggest that Raman bands can be used as markers to investigate the behavior of high‐concentration solutions of proteins and that the use of Raman spectroscopy will lead to progress in our understanding not only of the basic science of protein behavior under concentrated (i.e., crowded) conditions but also of practical processes involving proteins, such as in the field of biopharmaceuticals. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 237–246, 2015.     

Erythrocyte heme‑oxygenation status indicated as a risk factor in prehypertension by Raman spectroscopy

Raman spectroscopy of erythrocytes provides detailed information about the structure and status of heme moiety, which can be used to provide new insights into molecular pathogenesis of several diseases. In this study, we present the first Raman spectroscopy investigations of the effect of hemoglobin oxygenation in the context of hypertensive disease. The experimental data was subjected to Logistic Regression, which indicated heme?oxygenation status as an important risk factor alongside other clinical parameters. The 1605/1621?cm^?1 band ratio was selected as an optimal Raman metric for risk assessment and along with other band ratios (1583, 1639, 1310?cm^?1) related to heme status and when combined with clinical data via logistic regression gave an Area Under the Curve (AUC) >0.95 for prehypertension risk prediction. The work demonstrates the feasibility of Raman spectroscopy to distinguish between prehypertensive and normotensive states. Simultaneously, it is implied that the etiology of the high blood pressure progression may be connected with the changes in hemoglobin oxygenation.     

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.     

Biochemical impact of solar radiation exposure on human keratinocytes monitored by Raman spectroscopy; effects of cell culture environment

Understanding and amelioration of the effects of solar radiation exposure are critical in preventing the occurrence of skin cancer. Towards this end, many studies have been conducted in 2D cell culture models under simplified and unrealistic conditions. 3D culture models better capture the complexity of in vivo physiology, although the effects of the 3D extracellular matrix have not been well studied. Monitoring the instantaneous and resultant cellular responses to exposure, and the influence of the 3D environment, could provide an enhanced understanding of the fundamental processes of photocarcinogenesis. This work presents an analysis of the biochemical impacts of simulated solar radiation (SSR) occurring in immortalised human epithelial keratinocytes (HaCaT), in a 3D skin model, compared to 2D culture. Cell viability was monitored using the Alamar Blue colorimetric assay (AB), and the impact of the radiation exposure, at the level of the biomolecular constituents (nucleic acids and proteins), were evaluated through the combination of Raman microspectroscopy and multivariate statistical analysis. The results suggest that SSR exposure induces alterations of the conformational structure of DNA as an immediate impact, whereas changes in the protein signature are primarily seen as a subsequent response.