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
Colorectal cancer can be prevented if detected early (e.g., precancerous polyps‐adenoma). Endoscopic differential diagnosis of hyperplastic polyps (that have little or no risk of malignant transformation) and adenomas (that have prominent malignant latency) remains an unambiguous clinical challenge. Raman spectroscopy is an optical vibrational technique capable of probing biomolecular changes of tissue associated with neoplastic transformation. This work aims to apply a fiber‐optic simultaneous fingerprint (FP) and high wavenumber (HW) Raman spectroscopy technique for real‐time in vivo assessment of adenomatous polyps during clinical colonoscopy. We have developed a fiber‐optic Raman endoscopic technique capable of simultaneously acquiring both the FP (i.e., 800–1800 cm–1) and HW (i.e., 2800–3600 cm–1) Raman spectra from colorectal tissue subsurface (<200 µm) for real‐time assessment of colorectal carcinogenesis. In vivo FP/HW Raman spectra were acquired from 50 patients with 17 colorectal polyps during clinical colonoscopy. Prominent Raman spectral differences (p < 0.001) were found between hyperplastic (n = 118 spectra), adenoma (n = 184 spectra) that could be attributed to changes in inter‐ and intra‐cellular proteins, lipids, DNA and water structures and conformations. Simultaneous FP/HW Raman endoscopy provides a diagnostic sensitivity of 90.9% and specificity of 83.3% for differentiating adenoma from hyperplastic polyps, which is superior to either the FP or HW Raman technique alone. This study shows that simultaneous FP/HW Raman spectroscopy technique has the potential to be a clinically powerful tool for improving early diagnosis of adenomatous polyps in vivo during colonoscopic examination.
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. 相似文献
The biomolecular events resulting from the progression of hepatoblastoma remain to be elucidated. Fourier‐transform infrared (FTIR) and Raman spectroscopies are capable of noninvasively and accurately capturing the biochemical properties of biological tissue from its pathological status. Our aim was to probe critial biomolecular changes of liver accompanying the progression of pure foetal hepatoblastoma (PFH) by FTIR and Raman spectroscopies. Herein, biochemical alterations were both evident in the FTIR spectra (regions of 3100‐2800 cm?1 and 1800‐900 cm?1) and the Raman spectra (region of 1800‐400 cm?1) among normal, borderline and malignant liver tissues. Compared with normal tissues, the ratios of protein‐to‐lipid, α‐helix‐to‐β‐sheet, RNA‐to‐DNA, CH3 methyl‐to‐CH2 methylene, glucose‐to‐phospholipids, and unsaturated‐to‐saturated lipids intensities were significantly higher in malignant tissues, while the ratios of RNA‐to‐Amide II, DNA‐to‐Amide II, glycogen‐to‐cholesterol and Amide I‐to‐Amide II intensities were remarkably lower. These biochemical alterations in the transition from normal to malignant have profound implications not only for cyto‐pathological classification but also for molecular understanding of PFH progression. The successive changes of the spectral characteristics have been shown to be consistent with the development of PFH, indicating that FTIR and Raman spectroscopies are excellent tools to interrogate the biochemical features of different grades of PFH. 相似文献
The Fourier transform Raman and infrared (IR) spectra of the Ceramide 3 (CER3) have been recorded in the regions 200–3500 cm? 1 and 680–4000 cm? 1, respectively. We have calculated the equilibrium geometry, harmonic vibrational wavenumbers, electrostatic potential surfaces, absolute Raman scattering activities and IR absorption intensities by the density functional theory with B3LYP functionals having extended basis set 6-311G. This work is undertaken to study the vibrational spectra of CER3 completely and to identify the various normal modes with better wavenumber accuracy. Good consistency is found between the calculated results and experimental data for the IR and Raman spectra. 相似文献
A metalorganic gaseous doping approach for constructing nitrogen‐doped carbon polyhedron catalysts embedded with single Fe atoms is reported. The resulting catalysts are characterized using scanning transmission electron microscopy, X‐ray photoelectron spectroscopy, and X‐ray absorption spectroscopy; for the optimal sample, calculated densities of Fe–Nx sites and active N sites reach 1.75812 × 1013 and 1.93693 × 1014 sites cm‐2, respectively. Its oxygen reduction reaction half‐wave potential (0.864 V) is 50 mV higher than that of 20 wt% Pt/C catalyst in an alkaline medium and comparable to the latter (0.78 V vs 0.84 V) in an acidic medium, along with outstanding durability. More importantly, when used as a hydrogen–oxygen polymer electrolyte membrane fuel cell (PEMFC) cathode catalyst with a catalyst loading as low as 1 mg cm‐2 (compared with a conventional loading of 4 mg cm‐2), it exhibits a current density of 1100 mA cm‐2 at 0.6 V and 637 mA cm‐2 at 0.7 V, with a power density of 775 mW cm‐2, or 0.775 kW g–1 of catalyst. In a hydrogen–air PEMFC, current density reaches 650 mA cm‐2 at 0.6 V and 350 mA cm‐2 at 0.7 V, and the maximum power density is 463 mW cm‐2, which makes it a promising candidate for cathode catalyst toward high‐performance PEMFCs. 相似文献
Strong light (800μmol photons/m^2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSII RC as determined by HPLC after light treatment were as follows: with Increasing illumination time chlorophyll (Chl) a and β-carotene (β-car) content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illuminatlon, the initial bleaching occurred maximally at 680 nm but that with Increasing Illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed Initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 mln light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination. After Illumination, the fluorescence emission Intensity changed and the fluorescence maximum blue shifted, showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm^-1 (υ101), 1 159 cm^-1 (υ2), 1 006 cm^-1 (υ3), 966 cm^-1 (υ4) for 488.0 nm excitation and 1 525 cm^-1 (υ1), 1 159 cm^-1 (υ2), 1 007 cm^-1 (υ3), 968 cm^-1 (υ4) for 514.5 nm excitation. It was confirmed that two spectroscopically different β-car molecules exist In the PSII RC. After light treatment for 20 mln, band positions and bandwidths were unchanged. This indicates that carotenoid configuration Is not the parameter that regulates photoprotectlon in the PSII RC. 相似文献
Resonance Raman spectra have been recorded from ferri-cytochromec bound to phospholipid vesicles composed of dimyristoyl phosphatidylglycerol (DMPG), dioleoyl phosphatidylglycerol (DOPG)
or dioleoyl phosphatidylglycerol-dioleoyl phasphatidylcholine (DOPG-OPC) (70 : 30 mole/mole). Lipid binding induces very significant
conformational changes in the protein molecule. The resonance Raman spectra differ in their content of bands originating from
two different conformational species, I and II, of the protein, and from two different spin and coordination states of the
heme in conformation II. Data of sufficiently high precision were obtained that the spectra of the individual species could
be quantitated by a constraint interative fitting routine using single Lorentzian profiles. In the high frequency, or marker
band region (1200 to 1700 cm−1), the frequencies, half widths and relative intensities of the individual bands could be estimated from previous surface
enhanced resonance Raman measurements on cytochromec adsorbed on a silver electrode. These were then further optimized to yield both the spectral parameters and relative contents
of the different species. In the low frequency, or finger-print, region (200 to 800 cm−1), the spectral parameters of the individual species were obtained from difference spectra derived by sequential subtraction
between the spectra of ferri-cytochromec in the three different lipid systems, using the relative proportions of the species derived from the marker band region.
These parameters were then subsequently refined by iterative optimization. The optimized spectral parameters in both frequency
regions for the six-coordinated low spin states I and II, and for the five-coordinated high spin state II are presented. The
proportion of state II, in which hence the heme crevice assumes an open structure, and of the five-coordinated high spin configuration,
is found to increase on binding ferri cytochromec to negatively charged lipid vesicles. The extent of this conformational change increases in the order: DOPG-DOPC<DOPG<DMPG,
with a parallel decrease of the proportion of the conformational state I, whose structure is similar to that of the uncomplexed
ferri-cytochrome c in solution. Similar conformational changes are found for ferro-cytochromec compared to those obtained with the oxidized species on binding to lipids. The present work is essential for studies which
seek to analyze, in any detailed fashion, the conformational transitions in the heme protein which take place in response
to changes in the lipid environment. 相似文献