A modification for the calculation of water depth profiles in oil‐treated skin by in vivo confocal Raman microscopy

In this study, an extended calculation method for the determination of the water profiles in oil‐treated skin is proposed, which is based on the calculation of the ratio between the Raman band intensities of water (3350‐3550 cm^?1) and keratin Amide I at 1650 cm^?1. The proposed method is compared with the conventional method based on the ratio of the Raman band intensities of water (3350‐3550 cm^?1) and keratin at 2930 cm^?1. The conventional method creates artifacts in the depth profiles of the water concentration in oil‐treated skin, showing a lower amount of water in the upper and intermediate layers of the stratum corneum, which is due to the superposition of oil‐ and keratin‐related Raman bands at 2930 cm^?1. The proposed extended method shows no artifacts and has the potential to determine the water depth profiles after topical application of formulations on the skin.     

Raman confocal microscopy and biophysics multiparametric characterization of the skin barrier evolution with age

Skin aging is a multifactorial phenomenon that involves alterations at the molecular, cellular and tissue levels. Our aim was to carry out a multiparametric biophysical and Raman characterization of skin barrier between individuals of different age groups (<24 and >70 years old). Our results showed a significant decrease of lipids to proteins ratio overall the thickness of the stratum corneum and higher lateral packing in the outer part of the SC for elderly. This can explain the decrease in trans epidermal water loss measured values rather than only SC thickening. Both age groups showed similar water content at SC surface while elderly presented higher water content in deep SC and viable epidermis. Mechanical measurements showed a decrease in the elasticity and an increase in the fatigability with age and were correlated with partially bound water. Highest correlation and anti-correlation values were observed for the deepest part of the SC and the viable epidermis.     

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.”     

Glycerol and natural sugar-derived complex modulate differentially stratum corneum water-binding properties and structural parameters in an in vitro Raman-desorption model

The epidermal protective functions are closely associated with skin hydration homeostasis. The understanding of different states of water binding is a rising concept in assessing topically applied formulations and their interaction within the stratum corneum (SC). In addition to global water content, primary bound water, partially bound water, and unbound water and barrier-related lipid lateral packing and protein secondary structure can be measured by Raman spectroscopy. This study aimed to establish an in vitro SC model to evaluate differences in the efficacy of a natural sugar-derived complex in combination with glycerol and a botanical extract in modulating SC water binding and structural proteins and barrier lipids. These compounds were selected due to their water-binding and soothing properties. The SC water profiles were assessed at the surface and in 8 μm SC depth. After a 12-hour hyperhydration and subsequent product incubation the measurements were performed during a 6 hours desiccation phase. The maximal water caption and the time until reaching a steady state are measured as well as water retention and resistance against water loss. Global water content, partially bound, and unbound water, as well as lipid and protein structures were assessed with confocal Raman microspectroscopy. Both the natural sugar-derived mixture and more pronounced, the same mixture with additional glycerol increased all three water-binding parameters at the surface and in 8 μm SC depth at the beginning and during the desiccation phase. Further addition of botanical extract did not result in an additional increase of the water-binding. All three formulations showed an increase in the lipid lateral packing values prevented the protein alteration as measured by β-sheets signal compared to blank. The present model is suited for screening studies comparing the specific effects of different compounds on hydration states. The natural sugar-derived mixture Aquaxyl showed evidence for an improvement of all SC hydration states, lipid and protein structure which was further enhanced by the addition of glycerol 5%. This improvement was evidenced at the surface and within the SC for all hydration-related parameters, and the lipid as well the protein structures. The addition of botanical extract phytoessence blue daisy did not show further improvement.     

Automated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins,cephalosporins, and fluoroquinolones using specific Raman marker bands

A Raman‐based, strain‐independent, semi‐automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch‐wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third‐generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug‐resistant Gram‐negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long‐term comparability of Raman measurements.     

Understanding the effects of culture conditions in bacterial growth: A biochemical perspective using Raman microscopy

Rapid, sensitive and label‐free methods to probe bacterial growth irrespective of the culture conditions can shed light on the mechanisms by which bacteria adapt to different environmental stimuli. Raman spectroscopy can rapidly and continuously monitor the growth of bacteria under varied conditions. In this study, the growth of Escherichia coli in Luria broth (nutrient rich conditions) and minimal media with either glucose or glycerol as carbon source (nutrient limiting conditions) is profiled using Raman spectroscopy. Moreover, the study also gives insights into the altered bacterial biochemistry upon exposure to low‐ (25°C) and high‐temperature (45°C) stress. Raman spectral measurement was performed on bulk bacteria cultured under laboratory conditions. A detailed analysis of the spectra as a function of bacterial growth reveals changes in Raman band intensities/area of biomolecules such as DNA, proteins and lipids. We also report five novel ratiometric markers (I₈₃₀/I₈₁₀, I₁₁₂₆/I₁₁₀₀, I₁₃₄₀/I₁₄₄₀, I₁₂₀₇/I₁₂₄₀ and I₁₅₈₀/I₁₄₄₀) that can identify the phase of growth, independent of the culture condition. Unsupervised multivariate methods like Principal Component Analysis also corroborate the aforementioned markers of growth. Altogether, our findings highlight the potential of Raman spectroscopy in yielding universal biochemical signatures that may be indicative of stress and aging in a growth milieu.     

Effect of hemolysis on Fourier transform infrared and Raman spectra of blood plasma

Hemolysis is a very common phenomenon and is referred as the release of intracellular components from red blood cells to the extracellular fluid. Hemolyzed samples are often rejected in clinics due to the interference of hemoglobin and intracellular components in laboratory measurements. Plasma and serum based vibrational spectroscopy studies are extensively applied to generate spectral biomarkers for various diseases. However, no studies have reported the effect of hemolysis in blood based vibrational spectroscopy studies. This study was undertaken to evaluate the effect of hemolysis on infrared and Raman spectra of blood plasma. In this study, prostate cancer plasma samples (n = 30) were divided into three groups (nonhemolyzed, mildly hemolyzed, and moderately hemolyzed) based on the degree of hemolysis and FTIR and Raman spectra were recorded using high throughput (HT)‐FTIR and HT‐Raman spectroscopy. Discrimination was observed between the infrared and Raman spectra of nonhemolyzed and hemolyzed plasma samples using principal component analysis. A classical least square fitting analysis showed differences in the weighting of pure components in nonhemolyzed and hemolyzed plasma samples. Therefore, it is worth to consider the changes in spectral features due to hemolysis when comparing the results within and between experiments.     

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.     

Profiling antibiotic resistance in Escherichia coli strains displaying differential antibiotic susceptibilities using Raman spectroscopy

The rapid identification of antibiotic resistant bacteria is important for public health. In the environment, bacteria are exposed to sub-inhibitory antibiotic concentrations which has implications in the generation of multi-drug resistant strains. To better understand these issues, Raman spectroscopy was employed coupled with partial least squares-discriminant analysis to profile Escherichia coli strains treated with sub-inhibitory concentrations of antibiotics. Clear differences were observed between cells treated with bacteriostatic (tetracycline and rifampicin) and bactericidal (ampicillin, ciprofloxacin, and ceftriaxone) antibiotics for 6 hr: First, atomic force microscopy revealed that bactericidal antibiotics cause extensive cell elongation whereas short filaments are observed with bacteriostatic antibiotics. Second, Raman spectral analysis revealed that bactericidal antibiotics lower nucleic acid to protein (I₈₁₂/I₈₃₀) and nucleic acid to lipid ratios (I₁₄₈₃/I₁₄₅₂) whereas the opposite is seen with bacteriostatic antibiotics. Third, the protein to lipid ratio (I₂₉₃₆/I₂₈₈₅ and I₂₉₃₆/I₂₈₅₀) is a Raman stress signature common to both the classes. These signatures were validated using two mutants, Δlon and ΔacrB, that exhibit relatively high and low resistance towards antibiotics, respectively. In addition, these spectral markers correlated with the emergence of phenotypic antibiotic resistance. Overall, this study demonstrates the efficacy of Raman spectroscopy to identify resistance in bacteria to sub-lethal concentrations of antibiotics.     

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.     

FTIR-spectral analysis of two photosynthetic H2-producing strains and their extracellular polymeric substances

The Fourier transform infrared (FTIR) spectra of the cells of two photosynthetic H₂-producing strains, Rhodoblastus acidophilus and Rhodobacter capsulatus, as well as their extracellular polymeric substances (EPS), were evaluated. The FTIR spectra of R. capsulatus and its EPS during its cultivation were also recorded. The main peaks in the spectra, including 1,080 cm⁻¹ (carbohydrates), 1,250 cm⁻¹ (nucleic acids), 2,830–2,930 cm⁻¹ (lipids), 1,660–1,535 cm⁻¹ (Amide I and II of proteins), were observed. The relative heights of these peaks in the spectra of the two strains were different, showing the difference in contents of various components in the cells or EPS. The ratios among the main components in the EPS obtained from the FTIR spectra were in good agreement with those from a conventional quantitative chemical analysis. As an easy, rapid, and direct technique, the FTIR spectroscopy could be used to characterize the components and their relative contents of EPS of photosynthetic bacteria.An erratum to this article can be found at     

Supervised learning methods for the recognition of melanoma cell lines through the analysis of their Raman spectra

Malignant melanoma is an aggressive form of skin cancer, which develops from the genetic mutations of melanocytes – the most frequent involving BRAF and NRAS genes. The choice and the effectiveness of the therapeutic approach depend on tumour mutation; therefore, its assessment is of paramount importance. Current methods for mutation analysis are destructive and take a long time; instead, Raman spectroscopy could provide a fast, label-free and non-destructive alternative. In this study, confocal Raman microscopy has been used for examining three in vitro melanoma cell lines, harbouring different molecular profiles and, in particular, specific BRAF and NRAS driver mutations. The molecular information obtained from Raman spectra has served for developing two alternative classification algorithms based on linear discriminant analysis and artificial neural network. Both methods provide high accuracy (≥90%) in discriminating all cell types, suggesting that Raman spectroscopy may be an effective tool for detecting molecular differences between melanoma mutations.     

The AC conductivity of human sweat ducts as the dominant factor in the sub-THz reflection coefficient of skin

The helical nature of human sweat ducts, combined with the morphological and dielectric properties of skin, suggests electromagnetic activity in the sub-THz frequency band. A detailed electromagnetic simulation model of the skin, with embedded sweat ducts, was created. The model includes realistic dielectric properties based on the measured water content of each layer of skin, derived from Raman Spectroscopy. The model was verified by comparing it to measurements of the reflection coefficient of the palms of 13 volunteers in the frequency band 350–410 GHz. They were subjected to a measurement protocol intended to induce mental stress, thereby also activating the sweat glands. The Galvanic Skin Response was concurrently measured. Using the simulation model the optimal ac-conductivity for each measurement was found. The range of variation for all subjects was found to be from 100 S/m to a maximum value of 6000 S/m with averages of 1000 S/m. These are one order of magnitude increase from the accepted values for water at these frequencies (~100 s/m at 100 GHz). Considering the known biochemical mechanism for inducing perspiration, we conclude that these ac-conductivity levels are probably valid, even though the real time measurements of sweat ac-conductivity levels inside the duct are inaccessible.     

Measurement of cartilage sub-component distributions through the surface by Raman spectroscopy-based multivariate analysis

Articular cartilage posesses unique material properties due to a complex depth-dependent composition of sub-components. Raman spectroscopy has proven valuable in quantifying this composition through cartilage cross-sections. However, cross-sectioning requires tissue destruction and is not practical in situ. In this work, Raman spectroscopy-based multivariate curve resolution (MCR) was employed in porcine cartilage samples (n = 12) to measure collagen, glycosaminoglycan, and water distributions through the surface for the first time; these were compared against cross-section standards. Through the surface Raman measurements proved reliable in predicting composition distribution up to a depth of approximately 0.5 mm. A fructose-based optical clearing agent (OCA) was also used in an attempt to further improve depth of resolution of this measurement method. However, it did not; mainly due to a high-spectral overlap with the Raman spectra of main cartilage sub-components. This measurement technique potentially could be used in situ, to better understand the etiology of joint diseases such as osteoarthritis (OA).     

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.

     

Impact of optical clearing on ex vivo human skin optical properties characterized by spatially resolved multimodal spectroscopy

A spatially resolved multimodal spectroscopic device was used on a two-layered “hybrid” model made of ex vivo skin and fluorescent gel to investigate the effect of skin optical clearing on the depth sensitivity of optical spectroscopy. Time kinetics of fluorescence and diffuse reflectance spectra were acquired in four experimental conditions: with optical clearing agent (OCA) 1 made of polyethylene glycol 400 (PEG-400), propylene glycol and sucrose; with OCA 2 made of PEG-400 and dimethyl sulfoxide (DMSO); with saline solution as control and a “dry” condition. An increase in the gel fluorescence back reflected intensity was measured after optical clearing. Effect of OCA 2 turned out to be stronger than that of OCA 1, possibly due to DMSO impact on the stratum corneum keratin conformation. Complementary experimental results showed increased light transmittance through the skin and confirmed that the improvement in the depth sensitivity of the multimodal spectroscopic approach is related not only to the dehydration and refractive indices matching due to optical clearing, but also to the mechanical compression of tissues caused by the application of the spectroscopic probe.     

In vivo detection of human cutaneous beta‐carotene using computational optical clearing

The content of dermal beta‐carotene can be a good indicator showing the body health. Because, it is involved in production of vitamin A maintaining healthy skin and mucous membranes. Also, it reduces the risk of cardiovascular diseases and its antioxidant capacity prevents the formation of cancerous cells. In this work, we use Raman spectroscopy and a low‐cost diffuse reflectance spectroscopy (DRS) to detect the dermal beta‐carotene spectra. We apply computational optical clearing (OC) method to in vivo evaluation the concentration of this chromophore. The results show that Raman spectroscopy is a good tool for in vitro detection of carotenoids but is not able to clearly discriminate the individual carotenoids in skin tissue in vivo. The results also show that using OC enhances the ability of low‐cost diffuse reflection spectroscopy for in vivo detection of dermal beta‐carotene in humans. This method can be used as a low‐cost and portable device to screening the concentration of chromophores such as melanin and carotenoid molecules for oncological studies.     

拉曼光谱分析技术在资源微生物发酵性能评价中的应用

微生物发酵过程是细胞新陈代谢进行物质转化的过程,为了提高目标产物的转化率,需要对微生物发酵动态特性进行实时分析,以便实时优化发酵过程。拉曼光谱(Raman spectroscopy)量化测试作为一种有应用前景的在线过程分析技术,可以在避免微生物污染的条件下,实现精准监测,进而用于优化控制微生物发酵过程。【目的】以运动发酵单胞菌(Zymomonas mobilis)为例,建立微生物发酵过程中葡萄糖、木糖、乙醇和乳酸浓度拉曼光谱预测模型,并进行准确性验证。【方法】采用浸入式在线拉曼探头,收集运动发酵单胞菌发酵过程中多个组分的拉曼光谱,采用偏最小二乘法对光谱信号进行预处理和多元数据分析,结合离线色谱分析数据,对拉曼光谱进行建模分析和浓度预测。【结果】针对运动发酵单胞菌,首先实现拉曼分析仪对单一产品乙醇发酵过程的精准检测,其次基于多元变量分析,建立葡萄糖、乙醇和乳酸浓度变化的预测模型,实现对发酵过程中各成分浓度变化的准确有效分析。【结论】成功建立了一种评价资源微生物尤其是工业菌株发酵液多种组分的拉曼光谱分析方法。该方法为运动发酵单胞菌等工业菌株利用多组分底物工业化生产不同产物的实时检测,以及其他微生物尤其工业菌株的选育和过程优化提供了新方法。     

Estimation of the content of lipids composing endothelial lipid droplets based on Raman imaging

Lipid droplets (LDs) are dynamic organelles involved in intracellular lipid metabolism, and the biogenesis of LDs in endothelium is triggered by the excess of lipids in the environment. In this paper we present the methodology aimed to define the composition of endothelial LDs formed upon stimulation with oleic acid (OA) in two models: endothelial cells cultured in vitro and in isolated blood vessel ex vivo. The biochemical composition of LDs was determined using Raman imaging, followed by the lipid unsaturation calibration analysis and modelling of spectral bands based on individual spectra of selected lipids. Among LDs formed in response to OA in vitro or ex vivo conditions there were two types of LDs; those with more unsaturated (average number of CC bonds equalled 1.40) or saturated (average number of CC bonds equalled 0.95) lipids. The modelling of endothelial LDs composition revealed the OA represented a major component of LDs (80.6–91.3%) with an important content of arachidonic acid (8.7–19.4%). In conclusion, endothelial LDs consist of exogenous oleic acid uptaken from the extracellular space, and the endogenous arachidonic acid released from plasma membranes.     

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.