Relationship between raman spectroscopic lines and growth ofRhizobium japonicum

The Raman spectroscopic lines of liquid cultures ofRhizobium japonicum have been compared with electron microscopic examinations and growth measurements of these cells. The results showed that the significant Raman lines are related to the reproduction activities of the procaryotic cells.     

Raman Tweezers Spectroscopy of Live,Single Red and White Blood Cells

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.     

Raman spectroscopy and order in biological systems

The Raman spectra in the low 5-200 cm-1 frequency region of metabolically active E. coli cells have been analyzed to determine whether they are indicators of a possible in vivo underlying order by applying standard concepts derived from the Raman spectroscopy of crystalline systems with varying degrees of order. The analysis suggests that in-vivo space-time ordered structures involving amino acids associated with DNA exist since the low frequency lines of metabolically active cells can be assigned to lines seen in the spectra of crystals of given amino acids known to associate with DNA early in the lifetime of a cell.     

Raman spectroscopy and order in biological systems

The Raman spectra in the low 5–200 cm⁻¹ frequency region of metabolically activeE. coli cells have been analyzed to determine whether they are indicators of a possible in vivo underlying order by applying standard concepts derived from the Raman spectroscopy of crystalline systems with varying degrees of order. The analysis suggests that in-vivo space-time ordered structures involving amino acids associated with DNA exist since the low frequency lines of metabolically active cells can be assigned to lines seen in the spectra of crystals of given amino acids known to associated with DNA early in the lifetime of a cell.     

Near-infrared Raman Microspectroscopy Detects High-risk Human Papillomaviruses

BACKGROUND: Detecting human papillomaviruses (HPVs) infection in cervical cells is an exceedingly important part of the clinical management of cervical dysplasia. Current guidelines in women's health outline the need for both the Papanicolaou test as well as high-risk HPV testing. Testing for HPV is expensive, is time-consuming, and requires experienced technicians. METHODS: Two sets of near-infrared Raman microspectroscopy experiments were conducted using a Raman confocal microscope system. First, Raman spectra were acquired from four different cell culture lines, two positive for HPV (HeLa, SiHa), one negative for HPV, but malignant (C33A), and one normal, HPV-negative line (NHEK). The three malignant lines were all derived from cervical cells. Second, Raman spectra were acquired from deidentified patient samples that were previously tested for the presence of high-risk HPV. RESULTS: The spectra from the cell culture lines and the patient samples contained many statistically significant differences. Using sparse multinomial logistic regression to classify the data led to classification accuracies of 89% to 97% for the cell culture samples and 98.5% for the patient samples. CONCLUSIONS: Raman micro-spectroscopy can be used to detect HPV and differentiate among specific HPV strains. This technique may provide health providers with a new method for quickly testing cell samples for the presence of HPV.     

Assessment of Cell Line Models of Primary Human Cells by Raman Spectral Phenotyping

Researchers have previously questioned the suitability of cell lines as models for primary cells. In this study, we used Raman microspectroscopy to characterize live A549 cells from a unique molecular biochemical perspective to shed light on their suitability as a model for primary human pulmonary alveolar type II (ATII) cells. We also investigated a recently developed transduced type I (TT1) cell line as a model for alveolar type I (ATI) cells. Single-cell Raman spectra provide unique biomolecular fingerprints that can be used to characterize cellular phenotypes. A multivariate statistical analysis of Raman spectra indicated that the spectra of A549 and TT1 cells are characterized by significantly lower phospholipid content compared to ATII and ATI spectra because their cytoplasm contains fewer surfactant lamellar bodies. Furthermore, we found that A549 spectra are statistically more similar to ATI spectra than to ATII spectra. The spectral variation permitted phenotypic classification of cells based on Raman spectral signatures with >99% accuracy. These results suggest that A549 cells are not a good model for ATII cells, but TT1 cells do provide a reasonable model for ATI cells. The findings have far-reaching implications for the assessment of cell lines as suitable primary cellular models in live cultures.     

Non-destructive analysis of the nuclei of transgenic living cells using laser tweezers and near-infrared raman spectroscopic technique

Transgenic cell lines of loblolly pine （Pinus taeda L.） were analyzed by a compact laser-tweezers-Raman-spectroscopy （LTRS） system in this investigation. A low power diode laser at 785 nm was used for both laser optical trapping of single transgenic cells and excitation for near-infrared Raman spectroscopy of the nuclei of synchronized cells, which were treated as single organic particles, at the S-phase of the cell cycle. Transgenic living cells with gfp and uidA genes were used as biological samples to test this LTRS technique. As expected, different Raman spectra were observed from the tested biological samples. This technique provides a high sensitivity and enables real-time spectroscopic measurements of transgenic cell lines. It could be a valuable tool for the study of the fundamental cell and molecular biological process by trapping single nucleus and by providing a wealth of molecular information about the nuclei of cells.     

The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy

Laser tweezers Raman spectroscopy (LTRS) was used to characterize the effect of different chemical fixation procedures on the Raman spectra of normal and leukemia cells. Individual unfixed, paraformaldehyde-fixed, and methanol-fixed normal and transformed lymphocytes from three different cell lines were analyzed with LTRS. When compared to the spectra of unfixed cells, the fixed cell spectra show clear, reproducible changes in the intensity of specific Raman markers commonly assigned to DNA, RNA, protein, and lipid vibrations (e.g. 785, 1230, 1305, 1660 cm(-1)) in mammalian cells, many of which are important markers that have been used to discriminate between normal and cancer lymphocytes. Statistical analyses of the Raman data and classification using principal component analysis and linear discriminant analysis indicate that methanol fixation induces a greater change in the Raman spectra than paraformaldehyde. In addition, we demonstrate that the spectral changes as a result of the fixation process have an adverse effect on the accurate Raman discrimination of the normal and cancer cells. The spectral artifacts created by the use of fixatives indicate that the method of cell preparation is an important parameter to consider when applying Raman spectroscopy to characterize, image, or differentiate between different fixed cell samples to avoid potential misinterpretation of the data.     

Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm

Raman microscope vibrational spectra have been recorded from single photoreceptor cells frozen at 77 K. Spectra of photostationary steady-state mixtures of visual pigments and their primary photoproducts were obtained from toad red rods (lambda max 502 nm), angelfish rods (lambda max 500 nm), gecko blue rods (lambda max 467 nm), and bullfrog green rods (lambda max 430 nm). All four photoproducts have enhanced low-wavenumber Raman lines at approximately 850, 875, and 915 cm-1 and show the anomalous decoupling of the 11- and 12-hydrogen out-of-plane (HOOP) wagging vibrations, as is observed in the bovine primary photoproduct. The low-wavenumber lines are enhanced in the resonance Raman spectrum by conformational distortion, and the uncoupling of the 11- and 12-hydrogen wags is caused by additional protein perturbations. The similarity of the HOOP modes in all four photoproducts indicates that the protein perturbations that uncouple the 11- and 12-hydrogen wags and that enhance the HOOP modes are very similar. Thus, these perturbations of the photoproduct Raman spectrum cannot be caused by the same protein-chromophore interactions that are responsible for wavelength regulation in these pigments.     

Multiplex targeting, tracking, and imaging of apoptosis by fluorescent surface enhanced Raman spectroscopic dots

We have developed multifunctional fluorescent surface enhanced Raman spectroscopic tagging material (F-SERS dots) composed of silver nanoparticle-embedded silica spheres with fluorescent organic dye and specific Raman labels for multiplex targeting, tracking, and imaging of cellular/molecular events in the living organism. In this study, F-SERS dots fabricated with specific target antibodies (BAX and BAD) were employed for the detection of apoptosis. The F-SERS dots did not show any particular toxicity in several cell lines. The F-SERS dots could monitor the apoptosis effectively and simultaneously through fluorescent images as well as Raman signals in both cells and tissues with high selectivity. Our results clearly demonstrate that F-SERS dots can be easily applicable to multiplex analysis of diverse cellular/molecular events important for maintaining cellular homeostasis.     

Estimation of poly A secondary structure from Raman scattering in acid aqueous solution.

In the frequency region 600–1600 cm^?1 the Raman spectra of acidic aqueous solutions of poly (rA) consist of several well-resolved lines. Four of these lines at 725, 1303, 1336 and 1508 cm^?1 demonstrate the Raman hypochromic effect of poly (rA) at pH-values of 5.73 and 5.35 as a function of the temperature.The results suggest that Raman intensity measurements are sensitive to order-disorder transitions of aqueous polynucleotides.     

The resonance Raman frequencies of the Fe-CO stretching and bending modes in the CO complex of cytochrome P-450cam

Resonance Raman spectra of the ferrous CO complex of cytochrome P-450cam have been observed both in its camphor-bound and free states. Upon excitation at 457.9 nm, near the absorption maximum of the Soret band, the ferrous CO complex of the camphor-bound enzyme showed an anomalously intense Raman line at 481 cm-1 besides the strong Raman lines at 1366 and 674 cm-1 for the porphyrin vibrations. The Raman line at 481 cm-1 (of the 12C16O complex) shifted to 478 cm-1 upon the substitution by 13C16O and to 473 cm-1 by 12C18O without any detectable shift in porphyrin Raman lines. This shows that the line at 481 cm-1 is assignable to Fe-CO stretching vibration. By the excitation at 457.9 nm, a weak Raman line was also observed at 558 cm-1, which was assigned to the Fe-C-O bending vibration, because it was found to shift by -14 cm-1 on 13C16O substitution while only -3 cm-1 on 12C18O substitution. These stretching and bending vibrations of the Fe-CO bond were not detected with the excitation at 413.1 nm, though the porphyrin Raman lines at 1366 and 674 cm-1 were clearly observed. When the substrate, camphor, was removed from the enzyme, the Fe-CO stretching vibration was found to shift to 464 cm-1 from 481 cm-1, while no detectable changes were found in porphyrin Raman lines. This means that the bound substrate interacts predominantly with the Fe-CO portion of the enzyme molecule.     

The pH dependence of the resonance raman spectra and structural alterations at heme moieties of various c-type cytochromes.

The pH dependence of resonance Raman spectra were studied for ferrous and ferric cytochromes c, c2, c3, c-551, and c-555. The frequencies of the 1565 cm-1 (ferric) and 1539 cm-1 lines (ferrous) were sensitive to the replacement of the sixth ligand. The titration curve for the 1565 cm-1 line of cytochrome c was parallel with that for the 695 nm band. The pH dependence of the 1539 cm-1 line of ferrous cytochrome c3 suggested the stepwise replacement of the sixth ligand of its four hemes, although such pH dependence was not recognized for the Raman spectra of other ferrous cytochromes investigated. The relative intensities of three Raman lines at 1639, 1587, and 1561 cm-1 of ferric protoporphyrin bis-imidazole complex were changed clearly by the presence of detergents. The relative intensities of the corresponding three Raman lines of cytochromes b5 and c were close to those of the ferric porphyrin complex in the presence and absence of detergents, respectively, suggesting an appreciable difference in their heme environments. Reduced hemin in detergent solution, unexpectedly, gave the Raman spectrum of ferric low spin type.     

Studies of viral structure by Raman spectroscopy. I. R17 virus and R17 RNA

The laser-excited Raman spectrum of the RNA virus, R17, is shown to contain a large number of Raman lines assignable to scattering by vibrations of the nucleotide residues of RNA and the amino-acid residues of protein capsomers. The Raman lines from specific nucleotide vibrations in the phage are compared with their counterparts in the spectrum of protein-free RNA to suggest many similarities of RNA structure in the phage and protein-free states. However, the average configuration of guanine residues in the phage is apparently very different from that of protein-free RNA, suggesting that guanine plays an important role in RNA-protein interactions.     

Nucleic acid derivatives studied by preresonance and resonance Raman spectroscopy in the ultraviolet region

Raman spectra of AMP, UTP, GMP, and CMP, and of their bromo-derivatives (8 Br-ATP, 8-bromo-adenosine, 8-bromo-guanosine, 5-bromo-deoxyuridine, 5-bromo-cytidine), are reported. They are obtained using excitation wavelengths of 457.9 nm (ionized continuous argon laser) and of 300 nm (tunable pulsed dye laser). Comparison of spectra leads to the following observations: (1) preresonances Raman effects on nucleotides spectra at 300 nm; (2) resonance Raman effects on bromoderivatives spectra at 300 nm; (3) in dilute solution (10^?4M), shifts and enhancements of Raman lines of the bromo-derivatives with respect to the corresponding lines of nucleoctides. On the basis of these comparisons, the assignments of the Raman lines are discussed, This provide the necessary background for the understanding of the properties of selected groups in DNA in dilute solution. The new experimental set-up for measurements of Raman spectra using excitation in the uv regions is described. It is specially designed to incorporate the pulsed feature of the excitation laser and for correcting of the instabilities of the sourse.     

Raman spectroscopy of mercury (II) binding to two filamentous viruses: Ff (fd, M13, f1) and Pf1

Ff and Pf1 are filamentous bacteriophages. Each contains, in a central core region surrounded by protein, a circular single-stranded DNA molecule, and it is known that the DNA bases are sites of Hg(II) binding. In the present study, Raman spectra were obtained for the two viruses in the presence of increasing amounts of Hg(II), with ratios (m) of Hg(II) added per nucleotide residue in the range 0 less than m less than 2.0. Hg(II) binding to the viruses induces Raman intensity changes in previously assigned Raman lines of viral DNA, demonstrating metal binding to the DNA bases, but also in many lines assigned to protein. The overall structures of the viruses do not change with Hg(II) binding, and the Raman spectra indicate little, if any, change in protein secondary structure. Changes in certain protein Raman lines induced by Hg(II) binding to the DNA for low values of m are attributed to altered interactions between solvent and protein side chains, aliphatic groups being the most affected. The nature of such changes for both viruses suggests DNA-protein linkage. In Pf1, lines assigned to ring vibrations of all four bases are perturbed upon initial addition of Hg(II) to m = 0.25. In Ff, however, lines assigned to base ring vibrations are not perturbed until m greater than or equal to 0.5. The results provide additional evidence for fundamentally different DNA structures in Ff and Pf1.     

Evidence of dithionite contribution to the low-frequency resonance Raman spectrum of reduced and mixed-valence cytochrome c oxidase.

The resonance Raman spectra of deoxygenated solutions of mixed-valence cyanide-bound and fully reduced cytochrome oxidase derivatives that have been reduced in the presence of aqueous or solid sodium dithionite exhibit two new low-frequency lines centered at 474 and 590 cm-1. These lines were not observed when the reductant system was changed to a solution containing ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). Under enzyme turnover conditions, the addition of dithionite to the reoxidized protein (the 428-nm or "oxygenated" form) increases the intensity of these lines, while reoxidation and rereduction of the enzyme in the presence of ascorbate/TMPD resulted in the absence of both lines. Our data suggest that both lines must have contributions from species formed from aqueous dithionite, presumably the SO2 species, since these two lines are also observed in the Raman spectrum of a solution of aqueous dithionite, but not in the spectrum of an ascorbate/TMPD solution. Since heme metal-ligand stretch vibrations are expected to appear in the low-frequency region from 215 to 670 cm-1, our results indicate that special care should be exercised during the interpretation of the cytochrome a3 resonance Raman spectrum.     

Raman microspectroscopic study of biomolecular structure inside living adhesive cells

Cells adhesion is very important for many physiological processes. Using advanced Raman microspectroscopic technique, we selected T Leukemia cells (Jurkat) as the materials and obtained simultaneously conformation information of various biomolecules inside the whole living cells. By comparing the Raman microspectroscopic spectra of single and adhesive cancer cells, we found for the first time that when cells adhered, the conformation of the biomolecules (DNA, protein, carbohydrates and lipids) inside the cells had different changes: (i) the backbone of double-stranded DNA maintained orderly B-form or modified B-form conformation, whereas the groups of its deoxyribose and bases were modified; (ii) the conformational changes of the main chain and the side chain in the protein were obviously variant. The lines intensity belonging to a-helix and p-sheet decreased, while that of p-turn increased. Tyrosine and tryptophane residues of the protein changed from "buried state" to "exposed state"; the lines intensi     

Cell classification with low‐resolution Raman spectroscopy (LRRS)

The identification of individual eukaryotic and prokaryotic cells is the backbone of clinical pathology and provides crucial information about the genesis and progression of a disease. While most commonly fluorescent‐label based methods are applied, label‐free methods, such as Raman spectroscopy, are elegant alternatives. A major disadvantage of Raman spectroscopy is the low signal yield resulting in long acquisition times, making it impractical for high‐throughput clinical analysis. As a rule, Raman‐based cell identification relies on high‐resolution Raman spectra. This comes at a cost of detected Raman photons. In this letter we show that while the proper biochemical characterization of cells requires high‐resolution Raman spectra, the proper classification of cells does not. By varying the slit‐width between 50 µm and 500 µm it is possible to show that detected Raman signal from eukaryotic cells increased up to seven‐fold. Raman‐based cell classification was performed on three cancer cell lines: Jurkat, MiaPaca2, and Capan1, at three different resolutions 8 cm^–1, 24 cm^–1, and 48 cm^–1. Moreover, we have simulated the resolution decrease due to low‐diffraction gratings by binning neighboring pixels together. In both cases the cells were well classifiable using support vectors machine (SVM).

For anyone working in the field of Raman spectroscopy this picture of Sir C.V. Raman is recognizable, even with reduced spatial resolution. Raman spectra of eukaryotic cells can also be recognized even with six fold reduced spectral resolution.     

Four- and five-coordinate species in nickel-reconstituted hemoglobin and myoglobin: Raman identification of the nickel-histidine stretching mode

Nickel(II)-reconstituted hemoglobin (NiHb) and myoglobin (NiMb) and model Ni porphyrins have been investigated by Soret-resonance Raman difference spectroscopy. Two sets of frequencies for the oxidation-state and core-size marker lines in the region from 1300 to 1700 cm-1 indicate two distinct sites in NiHb. Only one of these sites is evident in the Raman spectra of NiMb. This result is consistent with the UV-visible absorption spectrum of NiHb, which shows two Soret bands at 397 and 420 nm and one Soret at 424 nm for NiMb. Excitation at the blue Soret component of NiHb with 406.7-nm laser radiation preferentially enhances the set of Raman marker lines typical of Ni-protoporphyrin IX [Ni(ProtoP )] in noncoordinating solvents. The wavelength of the blue Soret component and the Raman spectrum indicate four-coordination for this site in NiHb. Laser excitation in the red Soret band enhances a set of lines whose frequencies are compatible with neither four- nor six-coordinate frequencies but are intermediate between the two. The red Soret band of the proteins is also considerably less red shifted than six-coordinate Ni-porphyrin models. These results suggest that Ni in the second site possesses a single axial ligand. Raman spectra of 64Ni-reconstituted and natural abundance Ni-reconstituted hemoglobins, obtained simultaneously in a Raman difference spectrometer, have identified the Ni-ligand stretch at 236 cm-1. The line shifts to 229 cm-1 for the 64Ni-reconstituted Hb. For a pure Ni-ligand stretch a 10-cm-1 shift would be predicted.(ABSTRACT TRUNCATED AT 250 WORDS)