Laser Tweezers Raman Spectroscopy Research Articles

Flow cytometric and near-infrared Raman spectroscopic investigation of quality in stained, sorted, and frozen-thawed buffalo sperm

Flow cytometry and Laser Tweezers Raman spectroscopy have been used to investigate Nili-Ravi buffalo (Bubalus bubalis) sperm from different samples (fresh, stained, sorted and frozen-thawed) of the flow-sorting process to optimize sperm sex sorting procedures. During the sorting and freezing-thawing processes, the two detection methods both indicated there were differences in mitochondrial activity and membrane integrity. Moreover, a dispersive-type NIR (Near Infrared Reflection) use of the Raman system resulted in the ability to detect a variety of sperm components, including relative DNA, lipid, carbohydrates and protein contents. The use of the Raman system allowed for PCA (principal components analysis) and DFA (discriminant function analysis) of fresh, stained, sorted and frozen-thawed sperm. The methodology, therefore, allows for distinguishing sperm from different samples (fresh, stained, sorted and frozen-thawed), and demonstrated the great discriminative power of ANN (artificial neural network) classification models for the differentiating sperm from different phases of the flow-sorting process. In conclusion, the damage induced by sperm sorting and freezing-thawing procedures can be quantified, and in the present research it is demonstrated that Raman spectroscopy is a valuable technology for assessing sperm quality.

Uptake and levels of the antibiotic berberine in individual dormant and germinating Clostridium difficile and Bacillus cereus spores as measured by laser tweezers Raman spectroscopy.

Spores of Clostridium difficile and Bacillus cereus are major causes of nosocomial diarrhoea and foodborne disease. Our aim was to measure the dynamics of the uptake of the antibiotic berberine by individual germinating spores and the levels of berberine accumulated in germinated spores. Laser tweezers Raman spectroscopy (LTRS) and differential interference contrast microscopy were used to measure levels of berberine accumulated in single germinating spores and to monitor berberine uptake and germination of individual C. difficile and B. cereus spores. MIC values of berberine for C. difficile and B. cereus spores were 640 and 256 mg/L, respectively. Levels of berberine accumulated at the berberine MICs in individual germinated spores were heterogeneous, with values of 17.1 ± 5.4 and 12.7 ± 5.5 g/L for C. difficile and B. cereus spores, respectively. These values were 25-50-fold higher than the MIC values. However, berberine did not affect the germination of C. difficile and B. cereus spores, but did block germinated spores' outgrowth. Berberine uptake kinetics were similar for these two kinds of spores. After the addition of germinants, berberine began to enter germinating spores at the time (Tlag) when rapid release of the spore core's large depot of the 1:1 chelate of Ca(2+) with dipicolinic acid began, and the level of berberine taken up was maximal shortly after spore cortex lysis was completed (Tlysis). LTRS can be used to measure uptake and levels of berberine in single cells. High levels of berberine can enter spores of C. difficile and B. cereus soon after germination is initiated, thus inhibiting spore outgrowth and minimizing hazards posed by germinated spores.

Automated analysis of single cells using Laser Tweezers Raman Spectroscopy

In recent years, significant progress has been made into the label-free detection and discrimination of individual cancer cells using Laser Tweezers Raman Spectroscopy (LTRS). However, the majority of examples reported have involved manual trapping of cells, which is time consuming and may lead to different cell lines being analysed in discrete batches. A simple, low-cost microfluidic flow chamber is introduced which allows single cells to be optically trapped and analysed in an automated fashion, greatly reducing the level of operator input required. Two implementations of the flow chamber are discussed here; a basic single-channel device in which the fluid velocity is controlled manually, and a dual-channel device which permits the automated capture and analysis of multiple cell lines with no operator input. Results are presented for the discrimination of live epithelial prostate cells and lymphocytes, together with a consideration of the consequences of traditional 'batch analysis' typically used for LTRS of live cells.

Uptake of and Resistance to the Antibiotic Berberine by Individual Dormant, Germinating and Outgrowing Bacillus Spores as Monitored by Laser Tweezers Raman Spectroscopy.

Berberine, an alkaloid originally extracted from the plant Coptis chinensis and other herb plants, has been used as a pharmacological substance for many years. The therapeutic effect of berberine has been attributed to its interaction with nucleic acids and blocking cell division. However, levels of berberine entering individual microbial cells minimal for growth inhibition and its effects on bacterial spores have not been determined. In this work the kinetics and levels of berberine accumulation by individual dormant and germinated spores were measured by laser tweezers Raman spectroscopy and differential interference and fluorescence microscopy, and effects of berberine on spore germination and outgrowth and spore and growing cell viability were determined. The major conclusions from this work are that: (1) colony formation from B. subtilis spores was blocked ~ 99% by 25 μg/mL berberine plus 20 μg/mL INF55 (a multidrug resistance pump inhibitor); (2) 200 μg/mL berberine had no effect on B. subtilis spore germination with L-valine, but spore outgrowth was completely blocked; (3) berberine levels accumulated in single spores germinating with ≥ 25 μg/mL berberine were > 10 mg/mL; (4) fluorescence microscopy showed that germinated spores accumulated high-levels of berberine primarily in the spore core, while dormant spores accumulated very low berberine levels primarily in spore coats; and (5) during germination, uptake of berberine began at the time of commitment (T1) and reached a maximum after the completion of CaDPA release (Trelease) and spore cortex lysis (Tlysis).

Raman Spectra Analysis for Single Mitochondrias after Apoptosis Process of Yeast Cells Stressed by Acetic Acid

Abstract Laser tweezers Raman spectroscopy (LTRS) as a non-invasive tool for mitochondria analysis, combined with oxygen electrode and ultraviolet spectrophotometric method, was used to investigate the Raman spectra of mitochondria of yeast cells in vitro and in vivo after induced with acetic acid. The results showed that when the mitochondria of yeast cells in vivo was induced by acetic acid, the spectral peaks of the nucleic acid (1081 and 1301 cm −1 ), proteins (872, 1604, 1445 and 1657 cm −1 ), lipids (1125, 1301, 1445 and 1657 cm −1 ), cytochrome c (750 and 1125 cm −1 ) and mitochondria respiration (1604 cm −1 ) were significantly decreased as a function of the duration of the acetic acid stress, and the obtained physiological and biochemical indexes of respiration rate, phosphorous/oxygen (P/O) ration and cytochrome c content were similar to those by conventional method. Furthermore, when the mitochondria of yeast cells in vitro was induced by acetic acid, the spectral peaks of the nucleic acid (1081 and 1301 cm −1 ), proteins (872, 1604, 1445 and 1657 cm −1 ), lipids (1125, 1301, 1445 and 1657 cm −1 ), cytochrome c (750 and 1125 cm −1 ) and mitochondria respiration (1604 cm −1 ) were significantly decreased as the function of the duration of the acetic acid stress, and the obtained physiological and biochemical indexes of respiration rate, P/O ration and cytochrome c content were similar to those by conventional method. The results indicated that acetic acid could penetrate into the cell interior and directly impacted the mitochondria possibly, resulting in the release of inclusions from the mitochondria, subsequently, causing the apoptosis of yeast cells via mitochondrial pathway-induced apoptosis.

A rapidly modulated multifocal detection scheme for parallel acquisition of Raman spectra from a 2-D focal array.

We report the development of a rapidly modulated multifocal detection scheme that enables full Raman spectra (~500-2000 cm(-1)) from a 2-D focal array to be acquired simultaneously. A spatial light modulator splits a laser beam to generate an m × n multifocal array. Raman signals generated within each focus are projected simultaneously into a spectrometer and imaged onto a TE-cooled CCD camera. A shuttering system using different masks is constructed to collect the superimposed Raman spectra of different multifocal patterns. The individual Raman spectrum from each focus is then retrieved from the superimposed spectra with no crosstalk using a postacquisition data processing algorithm. This system is expected to significantly improve the speed of current Raman-based instruments such as laser tweezers Raman spectroscopy and hyperspectral Raman imaging.

Detection of Recombinant Protein Expression of Formate Dehydrogenase in Single Living Escherichia Coli Cell by Laser Tweezers Raman Spectroscopy

Detection of Recombinant Protein Expression of Formate Dehydrogenase in Single Living Escherichia Coli Cell by Laser Tweezers Raman Spectroscopy

Monitoring Trehalose Uptake and Conversion by Single Bacteria using Laser Tweezers Raman Spectroscopy

Having the ability to monitor metabolic activity at the scale of single bacterial cells noninvasively would enable us to follow changes in the distribution of activity in bacterial systems which is of major importance for topics such as integration of metabolism and development, metabolic engineering, microbial activity and drug resistance, cell-cell interactions, and quorum sensing. Here, we used laser tweezers Raman spectroscopy to monitor the in vivo real-time uptake and conversion of trehalose by single bacterial cells. This approach can be used for the quantitative determination of sugar uptake by a single bacterium and its metabolic response to the sugar application with time. We show that uptake of trehalose can be quantified in single living bacterial cells held in place by an optical trap while simultaneously collecting Raman spectra upon application of sugar to the medium. This technique yields real-time chemical information in a label-free manner, thus eliminating the limitations of toxicity of the isotopic probes common in studying transport processes. It can substitute the laborious and time-consuming analytical evaluation. Although the single-cell Raman spectroscopy method demonstrated here is focused on the study of trehalose uptake by Sinorhizobium meliloti, the demonstrated approach is applicable to many different organisms and carbohydrates in general.

Optical tweezers for medical diagnostics

Laser trapping by optical tweezers makes possible the spectroscopic analysis of single cells. Use of optical tweezers in conjunction with Raman spectroscopy has allowed cells to be identified as either healthy or cancerous. This combined technique is known as laser tweezers Raman spectroscopy (LTRS), or Raman tweezers. The Raman spectra of cells are complex, since the technique probes nucleic acids, proteins, and lipids; but statistical analysis of these spectra makes possible differentiation of different classes of cells. In this article the recent development of LTRS is described along with two illustrative examples for potential application in cancer diagnostics. Techniques to expand the uses of LTRS and to improve the speed of LTRS are also suggested.

Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: Application for sickle cell disease

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell. However, sickle RBCs deoxygenated more readily than normal RBCs when subjected to the same optical forces. Conversely, cord blood RBCs were able to maintain their oxygenation better than normal RBCs. These results suggest that differences in the chemical or mechanical properties of fetal, normal, and sickle cells affect the degree to which applied mechanical forces can deoxygenate the cell. Populations of normal, sickle, and cord RBCs were identified and discriminated based on this mechanochemical phenomenon. This study demonstrates the potential application of laser tweezers Raman spectroscopy as a single-cell, label-free analytical tool to characterize the functional (e.g., mechanical deformability, oxygen binding) properties of normal and diseased RBCs.

Single-cell discrimination based on optical tweezers Raman spectroscopy

The ability to discriminate between single cells in a label-free and noninvasive fashion is important for the classification of cells, and for the identification of similar cells from different origins. In this paper, we present the Raman spectroscopy-based identification of different types of single cells in aqueous media, and discrimination between the same types of cells from different donors using a novel Laser Tweezers Raman Spectroscopy (LTRS) technique, which combines laser trapping and micro-Raman spectroscopy. First, we measured the spectra of individual living human erythrocytes, i.e. red blood cells, and leucocytes (U937 cancer cells). High-quality Raman spectra with low fluorescence were obtained using a home-LTRS apparatus and 20 cells were measured for each cell type. The smoothing, baseline subtraction, and normalization of the data were followed by a principal components analysis (PCA). The PCA loading plots showed that the two different types of cells could be completely separated based only on the first component (PC1) (i.e. the peaks at 1300 cm−1); the discrimination accuracy could therefore reach 100%. More than 50 spectra were taken for each erythrocyte obtained from the four healthy volunteers. The average discrimination accuracy was 84.5% for two random individuals taken from the four volunteers, according to the first and second PCs. This work demonstrates that LTRS is a powerful tool for the accurate identification and discrimination of single cells, and it has the potential to be applied for the highly sensitive identification of cells in clinical diagnosis and medical jurisprudence.

Recent advances in laser tweezers Raman spectroscopy (LTRS) for label‐free analysis of single cells

Laser tweezers Raman spectroscopy (LTRS), a technique that integrates optical tweezers with confocal Raman spectroscopy, is a variation of micro-Raman spectroscopy that enables the manipulation and biochemical analysis of single biological particles in suspension. This article provides an overview of the LTRS method, with an emphasis on highlighting recent advances over the past several years in the development of the technology and several new biological and biomedical applications that have been demonstrated. A perspective on the future developments of this powerful cytometric technology will also be presented.

Raman Spectroscopic Analysis of Single Red Blood Cells in Vivo Rat

Raman spectroscopy was used to study red blood cells (RBCs) and blood in vein capillary, in artery capillary in vivo and in vitro with the use of optical tweezers. Results showed that, (1) We could get Raman spectroscopy of blood in vivo; (2) We could clearly observe that different blood spectra in. oxygenated and deoxygenated state in artery and vein capillary; (3) By studying Raman spectroscopy of single RBC, we concluded that the hemoglobin concentration in capillary was larger than that in vitro, and oxygenated Raman peak of RBC in artery capillary was More obvious than those in vein capillary; (4) By comparing Raman spectrum in vein with that in artery, we found that that pH value in artery capillary is higher than that in vein capillary, and by studying the change of the band at 1604 cm(-1), we found that the band change was in connection with the circumstance pH value around RBCs. These findings make it possible to study conformational changes within single cells in vivo. The experiments also have shown that laser tweezers Raman spectroscopy can provide a strong tool for research in vivo.

Laser Tweezers Raman Spectroscopy As a Novel Red Blood Cell Functional Assay for Sickle Cell Disease

Abstract 4847The number of individuals with sickle cell disease (SCD) in the U.S. is estimated to be over 70,000, and the average life span of a SCD patient is 39 years. Despite the high incidence of SCD in the U.S. and its significant negative effects on patient health, there are currently only limited treatment options for this disease. Also, although a single mutation in the globin gene is known to cause SCD, there is significant clinical diversity. As technology advances, many studies, including genome-wide analyses and proteomics, have been conducted to understand the disease mechanism and find better treatments. However, none has yet led to the development of novel therapies. We need a practical approach to study red blood cells (RBCs) to increase our knowledge of this heterogeneous disease.Laser tweezers Raman spectroscopy (LTRS) is a label-free, laser-based method for the biochemical analysis of single living cells. Based on the phenomenon of inelastic light scattering of photons by molecular bonds, Raman spectroscopy can provide detailed information, in the form of a Raman spectrum, about the molecular structure and conformations inside single living cells. Moreover, the integration of laser tweezers enables 1) simple, convenient analysis of single suspension cells such as RBCs and 2) a method to impart a mechanical force on a cell (via the optical forces) and simultaneously monitor the biochemical response of that cell to the applied force. Previous studies using optical tweezers have shown different RBC elasticity between samples from SCD patients treated with hydroxyurea and those that were not treated with hydroxyurea. Raman spectroscopy has been applied to both normal and pathological RBCs in previous studies, but the results were limited to the identification of characteristic signatures. Our integration of the two methods, Raman spectroscopy and laser tweezers, is a new biophotonic approach for studying the intricate relationship between the biochemical and mechanical properties of a single cell that was not previously possible and is not easily carried out with existing methods. As such, we believe that this recent emerging single cell-based technology can be developed and used as an RBC functional assay.We have previously shown that LTRS can detect a force dependent oxygenation state transition in individual RBCs trapped at different laser powers, presumably due to the optical forces stretching the cell and inducing changes in the hemoglobin-hemoglobin and hemoglobin-membrane interactions that change the oxygen content of the cell. We have also shown characteristic Raman signatures indicating different oxygen content in different RBC samples at specific applied forces. Signatures were different among RBCs from cord, normal adult, and SCD patient blood, suggesting laser tweezers Raman spectroscopy can be used to measure the different force dependent oxygen content in a single RBC. We are currently validating our system to determine whether Raman spectroscopy can be used to identify and distinguish gene-corrected RBCs from normal and sickle RBCs. Our preliminary results showed distinct Raman fingerprints associated with the membrane structure of RBCs. Spectral fingerprints were observed to be different between normal and sickle RBCs, and gene-corrected RBCs were observed to have Raman profiles similar to those of normal RBCs. We speculate that the signature overlap between normal and gene-corrected samples is due to partial gene correction. These results are encouraging and strongly suggest that Raman spectroscopy can not only identify molecular structures but also be used as a functional assay. Disclosures:No relevant conflicts of interest to declare.

Protective Effects of Melatonin against Oxidative Stress in Flow Cytometry‐sorted Buffalo Sperm

Previous reports of the ability of melatonin to scavenge a variety of toxic oxygen and nitrogen-based reactants suggest that melatonin could be an effective antioxidant for protecting sperm. In this study, flow cytometry and laser tweezers Raman spectroscopy were used to evaluate the effect of melatonin on buffalo sperm quality to optimize sperm sex-sorting procedures. In fresh sperm incubated in the presence or absence of melatonin (10(-4) m) for 1, 24, 48 h or 72 h at 27°C, the mitochondrial activity was significantly higher than in a non-melatonin control (p < 0.05). Also, during the flow-sorting process, sperm in melatonin-supplemented groups had higher (p < 0.05) mitochondrial activity than the control. The intensity of Raman spectra from sperm frozen in media supplemented with melatonin was significantly weaker than that for non-melatonin-treated groups, except for a band at 1302 per cm. Thus, melatonin helps to protect buffalo sperm from reactive oxygen species induced by staining, sorting and freezing and increases semen quality after the freezing-thawing processes. Furthermore, the results indicate the high potential of the laser tweezers Raman spectroscopy technique for rapid, effective and non-invasive assessment of the quality of sperm cells.

Power dependent oxygenation state transition of red blood cells in a single beam optical trap

Laser tweezers Raman spectroscopy (LTRS) was used to demonstrate that a red blood cell (RBC) in a single beam optical trap transitions from an oxygenated to a partially deoxygenated state with increasing trapping power. Continuous switching between the two states is possible by repeatedly cycling between low and high trapping powers. Alterations in the hemoglobin conformation and interactions due to cell folding in the trap are proposed to be responsible for the transition. This study demonstrates that mechanically induced biochemical changes by optical forces need to be considered when applying single beam optical tweezers for cell analysis. LTRS holds promise as a functional assay to characterize normal and diseased RBCs based on their biochemical response to the forces of a single beam optical trap.

Raman spectroscopy of individual monocytes reveals that single-beam optical trapping ofmononuclear cells occurs by their nucleus

We show that laser tweezers Raman spectroscopy of eukaryotic cells with a significantlylarger diameter than the tight focus of a single-beam laser trap leads to optical trapping ofthe cell by its optically densest part, i.e. typically the cell’s nucleus. Raman spectraof individual optically trapped monocytes are compared with location-specificRaman spectra of monocytes adhered to a substrate. When the cell’s nucleus isstained with a fluorescent live cell stain, the Raman spectrum of the DNA-specificstain is observed only in the nucleus of individual monocytes. Optically trappedmonocytes display the same behavior. We also show that the Raman spectra ofindividual monocytes exhibit the characteristic Raman signature of cells thathave not yet fully differentiated and that individual primary monocytes can bedistinguished from transformed monocytes based on their Raman spectra. This workprovides further evidence that laser tweezers Raman spectroscopy of individualcells provides meaningful biochemical information in an entirely non-destructivefashion that permits discerning differences between cell types and cellular activity.

Monitoring and rapid quantification of total carotenoids in Rhodotorula glutinis cells using laser tweezers Raman spectroscopy

Rhodotorula glutinis is known to accumulate large amounts of carotenoids under certain culture conditions, which have very important industrial applications. So far, the molecular mechanism of regulating carotenogenesis is still not well understood. To better understand the carotenogenesis process, it requires methods that can detect carotenogenesis rapidly and reliably in single live cells. In this paper, a method based on laser tweezers Raman spectroscopy (LTRS) was developed to directly detect carotenoids, as well as other important biological molecules in single live R. glutinis cells. The data showed that the accumulation of carotenoids and lipids occurred mainly in the late exponential and stationary phases when the cell growth was inhibited by nutrient limitation. Meanwhile, the carotenoid concentration changed together with the concentration of nucleic acids, which increased in the first phase and decreased in the last phase of the culture. These data demonstrate that LTRS is a rapid, convenient, and reliable method to study the carotenogenesis process in vivo.

Parallel Analysis of Individual Biological Cells Using Multifocal Laser Tweezers Raman Spectroscopy

We report on the development and characterization of a multifocal laser tweezers Raman spectroscopy (M-LTRS) technique for parallel Raman spectral acquisition of individual biological cells. Using a 785-nm diode laser and a time-sharing laser trapping scheme, multiple laser foci are generated to optically trap single polystyrene beads and suspension cells in a linear pattern. Raman signals from the trapped objects are simultaneously projected through the slit of a spectrometer and spatially resolved on a charge-coupled device (CCD) detector with minimal signal crosstalk between neighboring cells. By improving the rate of single-cell analysis, M-LTRS is expected to be a valuable method for studying single-cell dynamics of cell populations and for the development of high-throughput Raman based cytometers.

Intracellular ethanol accumulation in yeast cells during aerobic fermentation: a Raman spectroscopic exploration

To investigate the intracellular ethanol accumulation in yeast cells by using laser tweezers Raman spectroscopy (LTRS). Ethanol accumulation in individual yeast cells during aerobic fermentation triggered by excess glucose was studied using LTRS. Its amount was obtained by comparing intracellular and extracellular ethanol concentrations during initial process of ethanol production. We found that (i) yeasts start to produce ethanol within 3 min after triggering aerobic fermentation, (ii) average ratio of intracellular to extracellular ethanol is 1.54 ± 0.17 during the initial 3 h after addition of 10% (w/v) excess glucose and (iii) the accumulated intracellular ethanol is released when aerobic fermentation is stimulated with decreasing glucose concentration. Intracellular ethanol accumulation occurs in initial stage of a rapid aerobic fermentation and high glucose concentration may attribute to this accumulation process. This work demonstrates LTRS is a real-time, reagent-free, in situ technique and a powerful tool to study kinetic process of ethanol fermentation. This work also provides further information on the intracellular ethanol accumulation in yeast cells.