Raman Band Of Water Research Articles

Nucleic Acid-Rich Stress Granules Are Not Merely Crowded Condensates: A Quantitative Raman Imaging Study.

Liquid droplets, formed by intracellular liquid-liquid phase separation (LLPS), are called membraneless organelles. They provide transient enzymatic reaction fields for maintaining cellular homeostasis, although they might transform into aggregates, leading to neurodegenerative diseases. To understand the nature of intracellular droplets, it is crucial to quantify the liquid droplets inside a living cell as well as to elucidate the underlying biological mechanism. In this study, we performed near-infrared fluorescence and Raman imaging to quantify chemical components inside stress granules (SGs) formed by LLPS in living cells. The Raman images reveal that the nucleic acid concentration inside the SGs was more than 20% higher than the surrounding cytoplasm, whereas the lipid concentration was lower. Quantitative Raman intensity analysis using a water Raman band as an internal standard enables in situ concentration determination of nucleic acids in the SGs and other organelles. The intensity of the biomolecular C-H bands relative to the water band indicates that the crowding environment inside the SGs depends on the stress type; under oxidative stress, the inside of the SGs was nearly identical to the outside, whereas it was sparser in hyperosmotic stressed cells, suggesting that the high concentrations of nucleic acids play a pivotal role in maintaining the environments inside the SGs. These results demonstrate that intracellular droplets are not always highly condensed.

Applying q-Gaussians to the OH-stretching Raman bands of Water and Ice

Applying q-Gaussians to the OH-stretching Raman bands of Water and Ice

Pre-resonance effects in deep UV Raman spectra of normal and deuterated water.

We have investigated the shape of the OH/OD stretching Raman band of water as a function of the excitation wavelength in the deep UV region (200-266 nm). By analyzing the spectral profiles, we highlighted selective pre-resonance effects in the high wavenumber component of the OH/OD stretching band, associated to distorted H-bonded water configurations. A van't Hoff treatment of the temperature-dependent Raman spectra provides an estimate of the thermal energy associated to the change from ordered (ice-like) to disordered configurations that agrees with values obtained by related methods based on a two-state model of water. These results open the possibility of exploiting the observed pre-resonance deep-UV signal enhancement to investigate H-bonding properties in aqueous media.

Quantification of Water Content and Speciation in Synthetic Rhyolitic Glasses: Optimising the Analytical Method of Confocal Raman Spectrometry

Quantification of water content in silicate glasses is of vital significance in understanding magma evolution and metamorphic anataxis. Here we provide a method for the determination of total dissolved water content and water speciation in silicate melts by confocal laser Raman spectrometry based on a set of hydrous rhyolitic glasses. A series of alumino‐silicate glasses with water contents from 0.33 to 9.05% m/m were synthesised in a piston cylinder apparatus. Synchrotron‐FTIR mapping shows that these glasses have relatively homogeneous distributions of dissolved water. Total water contents of the glasses were precisely measured by TC/EA‐MS and FTIR. Both external and internal calibration were established for the quantitative analysis of water content and water speciation in the silicate glasses based on excellent linear correlation between total dissolved water content and integrated area of the water Raman band. Furthermore, by decomposing the total water Raman bands into four Gaussians components, the relative concentration of water speciation (OH groups and molecules H2Om) dissolved in the glasses was determined with a similar trend to water speciation data derived from FTIR. We suggest that the relative concentration of water speciation can be estimated in rhyolitic glasses with 4–8% m/m H2O. Our work provides an accurate method to determine total water content and a potential tool to limit the relative concentration of water speciation dissolved in silicic glasses.

Concentration Quantification of the Low-Complexity Domain of Fused in Sarcoma inside a Single Droplet and Effects of Solution Parameters.

Liquid-liquid phase separation (LLPS) is an important phenomenon in biology, and it is desirable to develop quantitative methods to analyze protein droplets generated by LLPS. This study quantified the change in protein concentration in a droplet in label-free and single-droplet conditions using Raman imaging and the Raman band of water as an intensity standard. Small changes in the protein concentration with variations in pH and salt concentration were observed, and it was shown that the concentration in the droplet decreases as the conditions become less favorable for droplet formation. The effect of exposure to 1,6-hexanediol was also examined, and this additive was found to decrease the protein concentration in the droplet. A model can be proposed in which the addition of 1,6-hexanediol reduces the protein concentration in the droplet, and the droplet disappears when the concentration falls below a certain threshold value.

Observation of liquid-liquid phase separation of ataxin-3 and quantitative evaluation of its concentration in a single droplet using Raman microscopy.

Liquid–liquid phase separation (LLPS) plays an important role in a variety of biological processes and is also associated with protein aggregation in neurodegenerative diseases. Quantification of LLPS is necessary to elucidate the mechanism of LLPS and the subsequent aggregation process. In this study, we showed that ataxin-3, which is associated with Machado–Joseph disease, exhibits LLPS in an intracellular crowding environment mimicked by biopolymers, and proposed that a single droplet formed in LLPS can be quantified using Raman microscopy in a label-free manner. We succeeded in evaluating the protein concentration and identifying the components present inside and outside a droplet using the O–H stretching band of water as an internal intensity standard. Only water and protein were detected to be present inside droplets with crowding agents remaining outside. The protein concentration in a droplet was dependent on the crowding environment, indicating that the protein concentration and intracellular environment should be considered when investigating LLPS. Raman microscopy has the potential to become a powerful technique for clarifying the chemical nature of LLPS and its relationship with protein aggregation.

Label‐Free Imaging of Intracellular Temperature by Using the O−H Stretching Raman Band of Water

AbstractWe propose a label‐free method for measuring intracellular temperature using a Raman image of a cell in the O−H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O−H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label‐free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

Label-Free Imaging of Intracellular Temperature by Using the O-H Stretching Raman Band of Water.

We propose a label-free method for measuring intracellular temperature using a Raman image of a cell in the O-H stretching band. Raman spectra of cultured cells and the medium were first measured at various temperatures using a Raman microscope and the intensity ratio of the two regions of the O-H stretching band was calculated. The intensity ratio varies linearly with temperature in both the medium and cells, and the resulting calibration lines allow simultaneous visualization of both intracellular and extracellular temperatures in a label-free manner. We applied this method to the measurement of temperature changes after the introduction of FCCP (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone) in living cells. We observed a temperature rise in the cytoplasm and succeeded in obtaining an image of the change in intracellular temperature after the FCCP treatment.

In Situ Raman Study of Liquid Water at High Pressure.

A pressure shift of Raman band of liquid water (H2O) may be an important tool for measuring residual pressures in mineral inclusions, in situ barometry in high-pressure cells, and as an indicator of pressure-induced structural transitions in H2O. However, there was no consensus as to how the broad and asymmetric water Raman band should be quantitatively described, which has led to fundamental inconsistencies between reported data. In order to overcome this issue, we measured Raman spectra of H2O in situ up to 1.2 GPa using a diamond anvil cell, and use them to test different approaches proposed for the description of the water Raman band. We found that the most physically meaningful description of water Raman band is the decomposition into a linear background and three Gaussian components, associated with differently H-bonded H2O molecules. Two of these components demonstrate a pronounced anomaly in pressure shift near 0.4 GPa, supporting ideas of structural transition in H2O at this pressure. The most convenient approach for pressure calibration is the use of "a linear background + one Gaussian" decomposition (the pressure can be measured using the formula P (GPa) = -0.0317(3)·ΔνG (cm-1), where ΔνG represents the difference between the position of water Raman band, fitted as a single Gaussian, in measured spectrum and spectrum at ambient pressure).

Raman micro-spectroscopy as a viable tool to monitor and estimate the ionic transport in epithelial cells

The typical response to the lowering of plasma Na+ concentration and blood pressure in our body involves the release of aldosterone from the adrenal glands, which triggers the reabsorption of sodium in the kidney. Although the effects of aldosterone on this physiological mechanism were extensively studied in the past decades, there are still some aspects to be fully elucidated. In the present study, we propose for the first time a new approach based on Raman spectroscopy to monitor the ionic activity in aldosterone-treated A6 renal epithelial cells. This spectroscopic technique is capable of probing the cells through their thickness in a non-destructive and nimble way. The spectroscopic variations of the Raman bands associated to the O-H stretching of water were correlated to the variations of ionic concentration in the intracellular and extracellular fluids. The increase of Na+ concentration gradients was clearly visualized in the cytosol of aldosterone-treated cells. The enhancement of the Na+ current density induced by aldosterone was estimated from the variation of the ionic chemical potential across the intracellular space. In addition, the variation of the O-H Raman bands of water was used to quantify the cell thickness, which was not affected by aldosterone.

Inelastic hyperspectral lidar for profiling aquatic ecosystems

AbstractMonitoring the aquatic environment and the life of free‐floating organisms remains on the borderline of our technical capabilities. Therefore, our insights into aquatic habitats, such as, abundance and behavior of organisms are limited. In order to improve our understanding of aquatic life, we have developed a low‐cost inelastic hyperspectral lidar with unlimited focal depth and enough sensitivity and spatiotemporal resolution to detect and resolve position and behavior of individual submillimeter organisms. In this work, we demonstrate elastic as well as molecular ranging by using the water Raman band, and by observing fluorescence from chlorophyll and from dye‐tagged organisms. We present an aquatic laser‐diode‐based inelastic light detection and ranging (lidar) system with unprecedented sensitivity, spatiotemporal resolution and number of spectral bands. Our system offers new opportunities for quantitative in situ studies of aquatic organisms, and has the potential to considerably advance our understanding of biological life in aquatic systems. image

Bimodal dependence of light scattering/fluctuations on the concentration of aqueous solutions

Two concentration ranges (from 10−5 to 10−9 and from 10−13 to 10−18 M) corresponding to enhanced fluctuations of Rayleigh and Raman scattering of second-harmonic (527 nm) pulses of YVO4:Nd3+ laser are found for aqueous solutions of antioxidant potassium phenosan. A correlation is revealed between the rise in elastic Rayleigh scattering intensity and its fluctuations and the shift of the center of OH Raman band of water toward the ice component characteristic frequency (3200 cm−1). The development of phase-equilibrium instabilities is analyzed based on the model of fluctuations of the number of hydrogen bonds on the assumption of formation/destruction of ordered hydration layer of phenosan molecules in water.

Two-wavelength Raman imaging for non-intrusive monitoring of transient temperature in microfluidic devices

The present study proposes a non-intrusive visualization technique based on two-wavelength Raman imaging for in-situ monitoring of the unsteady temperature field in microfluidic systems. The measurement principle relies on the contrasting temperature dependencies of hydrogen-bonded and non-hydrogen-bonded OH stretching modes of the water Raman band, whose intensities were simultaneously captured by two cameras equipped with corresponding bandpass filters. The temperature distributions were then determined from the intensity ratio of the simultaneously-obtained Raman images, which enables compensation for temporal fluctuation and spatial inhomogeneity of the excitation laser intensity. A calibration experiment exhibited a linear relationship between the temperature and the intensity ratio in the range 293–343 K and least-regression analysis gave an uncertainty of 1.43 K at 95% confidence level. By applying the calibration data, time series temperature distributions were quantitatively visualized in a Y-shaped milli-channel at a spatial resolution of 6.0 × 6.0 µm2 with an acquisition time of 16.5 s. The measurement result clearly exhibited the temporal evolution of the temperature field and was compared with the values obtained by thermocouples. This paper therefore demonstrates the viability of employing the two-wavelength Raman imaging technique for temperature measurements in microfluidic devices.

Molecular component distribution imaging of living cells by multivariate curve resolution analysis of space-resolved Raman spectra

Label-free Raman microspectroscopy combined with a multivariate curve resolution (MCR) analysis can be a powerful tool for studying a wide range of biomedical molecular systems. The MCR with the alternating least squares (MCR-ALS) technique, which retrieves the pure component spectra from complicatedly overlapped spectra, has been successfully applied to in vivo and molecular-level analysis of living cells. The principles of the MCR-ALS analysis are reviewed with a model system of titanium oxide crystal polymorphs, followed by two examples of in vivo Raman imaging studies of living yeast cells, fission yeast, and budding yeast. Due to the non-negative matrix factorization algorithm used in the MCR-ALS analysis, the spectral information derived from this technique is just ready for physical and/or chemical interpretations. The corresponding concentration profiles provide the molecular component distribution images (MCDIs) that are vitally important for elucidating life at the molecular level, as stated by Schroedinger in his famous book, "What is life?" Without any a priori knowledge about spectral profiles, time- and space-resolved Raman measurements of a dividing fission yeast cell with the MCR-ALS elucidate the dynamic changes of major cellular components (lipids, proteins, and polysaccharides) during the cell cycle. The MCR-ALS technique also resolves broadly overlapped OH stretch Raman bands of water, clearly indicating the existence of organelle-specific water structures in a living budding yeast cell.

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry. Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material. For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. Measurement of quantum yield in powder samples is performed following these steps: Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations. Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements. Reference and Sample measurement using direct excitation and indirect excitation. Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation. Corrected quantum yield calculation. Chromaticity coordinates calculation using Report Generator program. The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this application, as follows: High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate. Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. Measurement of quantum yield over a wide wavelength range from 240 to 800 nm. Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample. Large selection of calculated parameters provided by dedicated and easy to use software. During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry. Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material. For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. Measurement of quantum yield in powder samples is performed following these steps: 1. Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations. 2. Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements. 3. Reference and Sample measurement using direct excitation and indirect excitation. 4. Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation. 5. Corrected quantum yield calculation. 6. Chromaticity coordinates calculation using Report Generator program. The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this application, as follows: High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate. Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. Measurement of quantum yield over a wide wavelength range from 240 to 800 nm. Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample. Large selection of calculated parameters provided by dedicated and easy to use software. During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

Spectroscopic characterization of aqueous microdroplets containing inorganic salts

We have developed and studied methods to characterize the time-varying composition of liquid microdroplets, under controlled changes to environmental conditions, using Raman tweezers. This work has focussed on measurements of inorganic salts, such as nitrate and sulfate anions, which comprise a major fraction of the dissolved solutes in atmospheric aerosols. The experimental Raman intensities for the anions of inorganic salts in optically tweezed droplets were found to be in good agreement with theoretical estimates of photon scattering. The detection limit for sodium nitrate salt in a single particle was found to be ~4 pg. The mass of an inorganic salt in the droplet can be estimated from the Raman intensity of the anion bands using a calibration curve which is independent of droplet volume. The volume of the droplet, and concentration of solute, can be found directly from the spacing of morphology dependent resonances in the Raman band of water, or indirectly from the integrated-intensity of the Raman band for the solvent. The later strategy eliminates the uncertainty in the collection efficiency of Raman-scattered light related to varying particle sizes.

Decomposition of water Raman stretching band with a combination of optimization methods

In this study, an investigation of the behaviour of stretching bands of CH and OH groups of water–ethanol solutions at alcohol concentrations ranging from 0 to 96% by volume has been performed. A new approach to decomposition of the wide structureless water Raman band into spectral components based on modern mathematical methods of solution of inverse multi-parameter problems–combination of Genetic Algorithm and the method of Generalized Reduced Gradient–has been demonstrated. Application of this approach to decomposition of Raman stretching bands of water–ethanol solutions allowed obtaining new interesting results practically without a priori information. The behaviour of resolved spectral components of Raman stretching OH band in binary mixture with rising ethanol concentration is in a good agreement with the concept of clathrate-like structure of water–ethanol solutions. The results presented in this paper confirm existence of essential structural rearrangement in water–ethanol solutions at ethanol concentrations 20–30% by volume.

High Wavenumber Raman Spectroscopy for in Vivo Detection of Cervical Dysplasia

Raman spectroscopy is a vibrational spectroscopic technique capable of optically probing the biomolecular changes associated with neoplastic transformation. The purpose of this study was to apply near-infrared (NIR) Raman spectroscopy in the high wavenumber (HW) region (2800-3700 cm(-1)) for in vivo detection of cervical dysplasia. A rapid-acquisition NIR Raman spectroscopy system associated with a ball-lens fiber-optic Raman probe was developed for in vivo spectroscopic measurements at 785 nm excitation. A total of 92 in vivo HW Raman spectra (46 normal, 46 dysplasia) were acquired from 46 patients with Pap smear abnormalities of the cervix. Significant difference in Raman intensities of prominent Raman bands at 2850 and 2885 cm(-1) (CH(2) stretching of lipids), 2940 cm(-1) (CH(3) stretching of proteins), and the broad Raman band of water (peaking at 3400 cm(-1) in the 3100-3700 cm(-1) range) were observed in normal and dysplasia cervical tissue. The diagnostic algorithms based on principal components analysis and linear discriminant analysis together with the leave-one-patient-out cross-validation method on in vivo HW Raman spectra yielded a diagnostic sensitivity of 93.5% and specificity of 97.8% for dysplasia tissue identification. This study demonstrates for the first time that HW Raman spectroscopy has the potential for the noninvasive, in vivo diagnosis and detection of precancer of the cervix.

Identification of inorganic salts and determination of their concentrations in aqueous solutions based on the valence Raman band of water using artificial neural networks

The characteristic features of the valence Raman band of water in the solutions of electrolytes are revealed. These features allow the noncontact recognition of the type of salt and the determination of its concentration in aqueous solutions using artificial neural networks.