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1.
Single cell Raman spectroscopy (SCRS) is a non-invasive and label-free technology, allowing in vivo and multiple parameter analysis of individual living cells. A single cell Raman spectrum usually contains more than 1000 Raman bands which provide rich and intrinsic information of the cell (e.g. nucleic acids, protein, carbohydrates and lipids), reflecting cellular genotypes, phenotypes and physiological states. A Raman spectrum serves as a molecular 'fingerprint' of a single cell, making it possible to differentiate various cells including bacterial, protistan and animal cells without prior knowledge of the cells. However, a key drawback of SCRS is the fact that spontaneous Raman signals are naturally weak; this review discusses recent research progress in significantly enhancing and improving the signal of spontaneous Raman spectroscopy, including resonance Raman spectroscopy (RRS), coherent anti-Stokes Raman spectroscopy (CARS), stimulated Raman spectroscopy (SRS) and surface enhanced Raman scattering (SERS). This review focuses on the biotechnological development and the associated applications of SCRS, including Raman activated cell sorting (RACS) and Raman imaging and mapping.  相似文献   

2.
We report the implementation of the transnasal image-guided high wavenumber (HW) Raman spectroscopy to differentiate tumor from normal laryngeal tissue at endoscopy. A rapid-acquisition Raman spectroscopy system coupled with a miniaturized fiber-optic Raman probe was utilized to realize real-time HW Raman (2800-3020 cm(-1)) measurements in the larynx. A total of 94 HW Raman spectra (22 normal sites, 72 tumor sites) were acquired from 39 patients who underwent laryngoscopic screening. Significant differences in Raman intensities of prominent Raman bands at 2845, 2880 and 2920 cm(-1) (CH(2) stretching of lipids), and 2940 cm(-1) (CH(3) stretching of proteins) were observed between normal and cancer laryngeal tissue. The diagnostic algorithms based on principal components analysis (PCA) and linear discriminant analysis (LDA) together with the leave-one subject-out, cross-validation method on HW Raman spectra yielded a diagnostic sensitivity of 90.3% (65/72) and specificity of 90.9% (20/22) for laryngeal cancer identification. This study demonstrates that HW Raman spectroscopy has the potential for the noninvasive, real-time diagnosis and detection of laryngeal cancer at the molecular level.  相似文献   

3.
Raman tensors for localized vibrations of base (A, U, G, and C), ribose and phosphate groups of double-stranded RNA have been determined from polarized Raman measurements on oriented fibers of the genomic RNA of bacteriophage phi6. Polarized Raman intensities for which electric vectors of both the incident and scattered light are polarized either perpendicular (I[bb]) or parallel (I[cc]) to the RNA fiber axis have been obtained by Raman microspectroscopy using 514.5-nm excitation. Similarly, the polarized Raman components, I(bc) and I(cb), for which incident and scattered vectors are mutually perpendicular, have been obtained. Spectra collected from fibers maintained at constant relative humidity in both H2O and D2O environments indicate the effects of hydrogen-isotopic shifts on the Raman polarizations and tensors. Novel findings are the following: 1) the intense Raman band at 813 cm(-1), which is assigned to phosphodiester (OPO) symmetrical stretching and represents the key marker of the A conformation of double-stranded RNA, is characterized by a moderately anisotropic Raman tensor; 2) the prominent RNA band at 1101 cm(-1), which is assigned to phosphodioxy (PO2-) symmetrical stretching, also exhibits a moderately anisotropic Raman tensor. Comparison with results obtained previously on A, B, and Z DNA suggests that tensors for localized vibrations of backbone phosphodiester and phosphodioxy groups are sensitive to helix secondary structure and local phosphate group environment; and 3) highly anisotropic Raman tensors have been found for prominent and well-resolved Raman markers of all four bases of the RNA duplex. These enable the use of polarized Raman spectroscopy for the determination of purine and pyrimidine base residue orientations in ribonucleoprotein assemblies. The present determination of Raman tensors for dsRNA is comprehensive and accurate. Unambiguous tensors have been deduced for virtually all local vibrational modes of the 300-1800 cm(-1) spectral interval. The results provide a reliable basis for future evaluations of the effects of base pairing, base stacking, and sequence context on the polarized Raman spectra of nucleic acids.  相似文献   

4.
Raman spectra were obtained from aqueous solutions of the deoxyoligonucleotide d(CGCGAATTCGCG)2 (I), which has been suggested as a model for B-type DNA conformation. These spectra were compared with the Raman spectra of the aqueous solutions of several DNAs of natural origin taken under identical solution conditions. Since the model sequence has a high percent GC (66%), the Raman spectrum was compared with the Raman spectrum of the DNA from Micrococcus lysodeikticus (72% GC), and the spectra of the two different DNAs were found to be rather similar in both 50 mM salt and 6 M salt solutions. Computer-aided band-shape analysis of the backbone vibrational region of the Raman spectra shows the existence of several bands corresponding to different furanose ring puckers. This appears to indicate a heterogeneity of furanose ring pucker in both the model dodecamer and the native DNA. Significant differences were found in the intensity of the conformational marker band at 810 cm-1, which indicates corresponding differences in furanose ring pucker heterogeneities in these two high GC content DNAs. The Raman spectrum of the dodecamer (I) was used to analyze the Raman spectrum of the DNA inside the head of living intact salmon sperm. Sperm spectra were taken with both our conventional Raman spectrograph and a newly developed intracavity laser Raman microscope system. Although the DNA in the sperm head is required by packing considerations to be in a highly compact and condensed state, the Raman spectra of the intact sperm are almost identical with that of the model dodecamer (I) if the difference in base composition is taken into account.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

5.
An optical trap has been combined with a Raman spectrometer to make high-resolution measurements of Raman spectra of optically-immobilized, single, live red (RBC) and white blood cells (WBC) under physiological conditions. Tightly-focused, near infrared wavelength light (1064 nm) is utilized for trapping of single cells and 785 nm light is used for Raman excitation at low levels of incident power (few mW). Raman spectra of RBC recorded using this high-sensitivity, dual-wavelength apparatus has enabled identification of several additional lines; the hitherto-unreported lines originate purely from hemoglobin molecules. Raman spectra of single granulocytes and lymphocytes are interpreted on the basis of standard protein and nucleic acid vibrational spectroscopy data. The richness of the measured spectrum illustrates that Raman studies of live cells in suspension are more informative than conventional micro-Raman studies where the cells are chemically bound to a glass cover slip.  相似文献   

6.
《Biotechnology advances》2017,35(2):168-177
Optical labels are needed for probing specific target molecules in complex biological systems. As a newly emerging category of tags for molecular imaging in live cells, the Raman label attracts much attention because of the rich information obtained from targeted and untargeted molecules by detecting molecular vibrations. Here, we list three types of Raman probes based on different mechanisms: Surface Enhanced Raman Scattering (SERS) probes, bioorthogonal Raman probes, and Resonance Raman (RR) probes. We review how these Raman probes work for detecting and imaging proteins, nucleic acids, lipids, and other biomolecules in vitro, within cells, or in vivo. We also summarize recent noteworthy studies, expound on the construction of every type of Raman probe and operating principle, sum up in tables typically targeting molecules for specific binding, and provide merits, drawbacks, and future prospects for the three Raman probes.  相似文献   

7.
Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.  相似文献   

8.
This study aims to evaluate the diagnostic utility of the combined near-infrared (NIR) autofluorescence (AF) and Raman spectroscopy for improving in vivo detection of gastric cancer at clinical gastroscopy. A rapid Raman endoscopic technique was employed for in vivo spectroscopic measurements of normal (n=1098) and cancer (n=140) gastric tissues from 81 gastric patients. The composite NIR AF and Raman spectra in the range of 800-1800 cm(-1) were analyzed using principal component analysis (PCA) and linear discriminant (LDA) to extract diagnostic information associated with distinctive spectroscopic processes of gastric malignancies. High quality in vivo composite NIR AF and Raman spectra can routinely be acquired from the gastric within 0.5s. The integrated intensity over the range of 800-1800 cm(-1) established the diagnostic implications (p=1.6E-14) of the change of NIR AF intensity associated with neoplastic transformation. PCA-LDA diagnostic modeling on the in vivo tissue NIR AF and Raman spectra acquired yielded a diagnostic accuracy of 92.2% (sensitivity of 97.9% and specificity of 91.5%) for identifying gastric cancer from normal tissue. The integration area under the receiver operating characteristic (ROC) curve using the combined NIR AF and Raman spectroscopy was 0.985, which is superior to either the Raman spectroscopy or NIR AF spectroscopy alone. This work demonstrates that the complementary Raman and NIR AF spectroscopy techniques can be integrated together for improving the in vivo diagnosis and detection of gastric cancer at endoscopy.  相似文献   

9.
We have performed resonance enhanced Raman measurements of retinal isomers in solution (all-trans, 11-cis, 9-cis, and 13-cis) and cetyltrimethylammonium bromide (CTAB) detergent extracts of bovine rhodopsin near physiological temperatures (17 degrees C). In order to measure these photolabile systems, we have developed a general technique which allows Raman measurements of any photosensitive material. This technique involves imposing a molecular velocity transverse to the Raman exciting laser beam sufficient to ensure that any given molecule moves through the beam so that it has little probability of absorbing a photon. We have also measured the resonance Raman spectra of crystals of the same retinal isomers. The data show that each isomer has a distinct and characteristic Raman spectra and that the spectrum of 11-cis-retinal is quite similar but not identical with that of rhodopsin and similarly for 9-cis-retinal compared with isorhodopsin. In agreement with previous work, the Raman data demonstrate that retinal and opsin are joined by a protonated Schiff base. Due to the fact that the Raman spectra of 11-cis-retinal (solution) and rhodopsin show bands near 998 and 1018 cm(-1), a spectral region previously assigned to C-Me stretching motions, it is suggested that 11-cis-retinal in solution is compased of a mixture of 12-s-trans and 12-s-cis, and that the conformation of rhodopsin is (perhaps distorted) 12-s-trans.  相似文献   

10.
The current sensitivity of standard fluorescence-based protein detection limits the use of protein arrays in research and clinical diagnosis. Here, we use functionalized, macromolecular single-walled carbon nanotubes (SWNTs) as multicolor Raman labels for highly sensitive, multiplexed protein detection in an arrayed format. Unlike fluorescence methods, Raman detection benefits from the sharp scattering peaks of SWNTs with minimal background interference, affording a high signal-to-noise ratio needed for ultra-sensitive detection. When combined with surface-enhanced Raman scattering substrates, the strong Raman intensity of SWNT tags affords protein detection sensitivity in sandwich assays down to 1 fM--a three-order-of-magnitude improvement over most reports of fluorescence-based detection. We use SWNT Raman tags to detect human autoantibodies against proteinase 3, a biomarker for the autoimmune disease Wegener's granulomatosis, diluted up to 10(7)-fold in 1% human serum. SWNT Raman tags are not subject to photobleaching or quenching. By conjugating different antibodies to pure (12)C and (13)C SWNT isotopes, we demonstrate multiplexed two-color SWNT Raman-based protein detection.  相似文献   

11.
The resonant Raman enhancement of hemoglobin (Hb) in the Q band region allows simultaneous identification of oxy- and deoxy-Hb. The heme vibrational bands are well known at 532 nm, but the technique has never been used to determine microvascular Hb oxygen saturation (So(2)) in vivo. We implemented a system for in vivo noninvasive measurements of So(2). A laser light was focused onto areas of 15-30 microm in diameter. Using a microscope coupled to a spectrometer and a cooled detector, Raman spectra were obtained in backscattering geometry. Calibration was performed in vitro using blood at several Hb concentrations, equilibrated at various oxygen tensions. So(2) was estimated by measuring the intensity of Raman signals (peaks) in the 1,355- to 1,380-cm(-1) range (oxidation state marker band nu(4)), as well as from the nu(19) and nu(10) bands (1,500- to 1,650-cm(-1) range). In vivo observations were made in microvessels of anesthetized rats. Glass capillary path length and Hb concentration did not affect So(2) estimations from Raman spectra. The Hb Raman peaks observed in blood were consistent with earlier Raman studies using Hb solutions and isolated cells. The correlation between Raman-based So(2) estimations and So(2) measured by CO-oximetry was highly significant for nu(4), nu(10), and nu(19) bands. The method allowed So(2) determinations in all microvessel types, while diameter and erythrocyte velocity could be measured in the same vessels. Raman microspectroscopy has advantages over other techniques by providing noninvasive and reliable in vivo So(2) determinations in thin tissues, as well as in solid organs and tissues in which transillumination is not possible.  相似文献   

12.
Propagating surface plasmon (PSP) excitation, based on the total internal reflection configuration, was introduced into the nanoparticle (NP)-plane junction Raman spectroscopy. Experimental results demonstrated that silver nanospheres within the propagation region of PSP are effectively activated and detected by CCD camera due to their impressive Raman enhancement, which presents around 20 times improvement compared with the conventional NP-induced PSP/LSP co-enhanced Raman spectroscopy. This impressive Raman enhancement along with its high reproducibility of NP-plane junctions makes our configuration an attractive candidature for the PSP-assisted gap-mode surface-enhancement Raman spectroscopy and tip-enhanced Raman spectroscopy.  相似文献   

13.
A method using confocal Raman microspectroscopy for the detection of cellular proteins in single intact cells was developed. Two approaches were used to improve the detection of these cellular components. First, compounds with high Raman scattering were investigated for potential use as Raman labels. Raman labels were conjugated to either biomolecules or biotin and used as markers in the detection of cellular enzymes and receptors. Second, silver colloids were used to increase the surface-enhanced Raman scatter (SERS) of these Raman labels. Cresyl violet and dimethylaminoazobenzene are Raman labels that provide very sensitive SERS detection by a confocal Raman microscope with a HeNe laser at wavelength of 632.8 nm. The detection of 12-lipoxygenase and cyclooxygenase-1 in single bovine coronary artery endothelial cells and the binding of angiotensin II to its receptors in zona glomerulosa cells was demonstrated.  相似文献   

14.
When zeaxanthin ([3R,3R']-beta, beta-carotene-3,3'diol) is inserted into phospholipid dispersions and the latter heated through their gel-liquid crystal phase transitions, large changes are noted in the resonance Raman and absorption spectra of the carotenoid molecule. By analogy with the data of Carey and co-workers (J. Raman Spectrosc. 6:282) who studied the aggregation of zeaxanthin in acetone-water solutions, it is suggested that the carotenoid aggregates in the phospholipid gel state while forming a monomer in liquid crystal phases. The alterations in both the visible absorption and resonance Raman data have been used to monitor phospholipid phase behavior in dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine, (DSPC) one-component systems and binary mixtures. The phase diagram obtained for the binary system, as constructed from visible absorption and resonance Raman data, is compared with that of Shimshick and McConnell (Biochemistry. 12:2351) obtained from electron spin resonance (ESR) studies. Although the agreement between absorption and ESR data is generally satisfactory, onset temperatures for phase separation at low DSPC mole fractions deduced from resonance Raman measurements are several degrees lower than those from the other methods. Nevertheless, the use of zeaxanthin as a resonance Raman and visible absorption probe behavior will be useful in some situations where ordinary Raman spectroscopic data cannot be obtained easily. The advantage of the resonance Raman approach is illustrated in a study of the phase behavior of a phospholipid extract of a cel- mutant of Neurospora crassa. A phase separation region is observed with onset and completion temperatures of -19 and -6 degrees C, respectively.  相似文献   

15.
The self-complementary dodecamer d(CGCAAATTTGCG) crystallizes as a double helix of the B form and manifests a Raman spectrum with features not observed in Raman spectra of either DNA solutions or wet DNA fibers. A number of Raman bands are assigned to specific nucleoside sugar and phosphodiester conformations associated with this model B-DNA crystal structure. The Raman bands proposed as markers of the crystalline B-DNA structure are compared and contrasted with previously proposed markers of Z-DNA and A-DNA crystals. The results indicate that the three canonical forms of DNA can be readily distinguished by Raman spectroscopy. However, unlike Z-DNA and A-DNA, which retain their characteristic Raman fingerprints in aqueous solution, the B-DNA Raman spectrum is not completely conserved between crystal and solution states. The Raman spectra reveal greater heterogeneity of nucleoside conformations (sugar puckers) in the DNA molecules of the crystal structure than in those of the solution structure. The results are consistent with conversion of one-third of the dG residues from the C2'-endo/anti conformation in the solution structure to another conformation, deduced to be C1'-exo/anti, in the crystal. The dodecamer crystal also exhibits unusually broad Raman bands at 790 and 820 cm-1, associated with the geometry of the phosphodiester backbone and indicating a wider range of (alpha, zeta) backbone torsion angles in the crystal than in the solution structure. The results suggest that backbone torsion angles in the CGC and GCG sequences, which flank the central AAATTT sequence, are significantly different for crystal and solution structures, the former containing the greater diversity.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

16.
A polarized laser (488.0 nm) Raman spectroscopic measurement has been made of a single crystal of adenosine triphosphoric acid trihydrate (ATP.3H2O), whose space group is P2(1)2(1)2(1), and whose dimensions are about 10 microns x 100 microns x 100 microns along the crystallographic a, b and c axes, respectively. For each Raman band in the 300-1800 cm-1 range, relative scattering intensities, Ibb and Icc, which correspond to the bb and cc components of the Raman tensor of the crystal, have been determined. In an aqueous solution of ATP, the depolarization ratio of each Raman band has also been determined. On the basis of these experimental results, some discussions are given of the shapes and orientations of the Raman scattering tensors localized in the base residue, ribose portion, and phosphodiester portions. A similar type of study has been made with a few DNA fibers.  相似文献   

17.
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.”  相似文献   

18.
Raman spectroscopy in chemical bioanalysis   总被引:4,自引:0,他引:4  
Advances in instrumentation are making Raman spectroscopy the tool of choice for an increasing number of (bio)chemical applications. Raman is an interesting option for several reasons, including the sensitivity to small structural changes, non-invasive sampling capability, minimal sample preparation, and high spatial resolution in the case of Raman micro-spectroscopy. Herein we discuss the most recent technical approaches employed, from the well-known surface enhanced resonance Raman spectroscopy to non-linear Raman techniques such as coherent anti-stokes Raman spectroscopy (CARS) and related techniques. Relevant applications of Raman spectroscopy in the fields of clinical pathology, in vivo and ex vivo imaging, classification and detection of microorganisms and chemical analysis in the past three years are also included.  相似文献   

19.
With (resonance) Raman microscospectroscopy, it is possible to investigate the chemical constitution of a very small volume (0.5 fl) in a living cell. We have measured resonance Raman spectra in the cytoplasm of living normal, myeloperoxidase (MPO)-deficient, and cytochrome b558-deficient neutrophils and in isolated specific and azurophilic granule fractions, using an excitation wavelength of 413.1 nm. Similar experiments were performed after reduction of the redox centers by the addition of sodium dithionite. The specific and azurophilic granules in both redox states appeared to have clearly distinguishable Raman spectra when exciting at a wavelength of 413.1 nm. The azurophilic granules and the cytochrome b558-deficient neutrophils showed Raman spectra similar to that of the isolated MPO. The spectra of the specific granules and the MPO-deficient neutrophils corresponded very well to published cytochrome b558 spectra. The resonance Raman spectrum of the cytoplasmic region of normal neutrophilic granulocytes could be fitted with a combination of the spectra of the specific and azurophilic granules, which shows that the Raman signal of neutrophilic granulocytes mainly originates from MPO and cytochrome b558, at an excitation wavelength of 413.1 nm.  相似文献   

20.
Measuring Raman spectra through an optical fibre is usually complicated by the high intrinsic Raman scatter of the fibre material. Common solutions such as the use of multiple fibres and distal optics are complex and bulky. We demonstrate the use of single novel hollow‐core negative‐curvature fibres (NCFs) for Raman and surface‐enhanced Raman spectroscopy (SERS) sensing using no distal optics. The background Raman emission from the silica in the NCF was at least 1000× smaller than in a conventional solid fibre, while maintaining the same collection efficiency. We transmitted pump light from a 785‐nm laser through the NCF, and we collected back the weak Raman spectra of different distal samples, demonstrating the fibre probe can be used for measurements of weak Raman and SERS signals that would otherwise overlap spectrally with the silica background. The lack of distal optics and consequent small probe diameter (<0.25 mm) enable applications that were not previously possible.   相似文献   

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