In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.     

Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells

The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.     

In vitro toxicology evaluation of pharmaceuticals using Raman micro-spectroscopy

Raman micro-spectroscopy combined with multivariate analysis was employed to monitor real-time biochemical changes induced in living cells in vitro following exposure to a pharmaceutical. The cancer drug etoposide (topoisomerase II inhibitor) was used to induce double-strand DNA breaks in human type II pneumocyte-like cells (A549 cell-line). Raman spectra of A549 cells exposed to 100 microM etoposide were collected and classical least squares (CLS) analysis used to determine the relative concentrations of the main cellular components. It was found that the concentrations of DNA and RNA significantly (P < 0.05) decreased, whilst the concentration of lipids significantly (P < 0.05) increased with increasing etoposide exposure time as compared to control untreated A549 cells. The concentration of DNA decreased by 27.5 and 87.0% after 24 and 48 h exposure to etoposide respectively. Principal components analysis (PCA) successfully discriminated between treated and untreated cells, with the main variance between treatment groups attributed to changes in DNA and lipid. DNA fragmentation was confirmed by Western blot analysis of apoptosis regulator protein p53 and cell metabolic activity determined by MTT assay. The over-expression of p53 protein in the etoposide treated cells indicated a significant level of DNA fragmentation and apoptosis. MTT tests confirmed that cellular metabolic activity decreased following exposure to etoposide by 29.4 and 61.2% after 24 and 48 h, respectively. Raman micro-spectroscopy may find applications in the toxicology screening of other drugs, chemicals and new biomaterials, with a range of cell types.     

Cytotoxicity of novel derivatives of the spin trap EMPO

Free radicals are involved in different regulatory and pathological processes. The formation of superoxide in living cells or whole organisms is of major interest. ESR spin trapping allows identification of the radicals if proper spin traps are available. Our study investigates the toxicity of novel derivatives of the spin trap EMPO to cultured human lung carcinoma cells (A549), breast carcinoma cells (SKBR3), colon carcinoma cells (SW480) as well as to human fibroblasts (F2000). A dose-dependent decrease of the cell number was observed for all spin traps. At 100mM BuMPO, t-BuMPO and s-BuMPO caused pronounced cell loss (>90%) and increased LDH-release, while DEPMPO, EMPO, PrMPO and i-PrMPO caused only moderate cell loss (<60%) without any effect on the LDH-release after 24h. At 10mM and 50mM the latter agents even decreased LDH-release. 10mM and 50mM of i-PrMPO as well as 10mM PrMPO increased intracellular GSH content acting like antioxidants, whereas 50mM s-BuMPO and PrMPO decreased GSH content by 67% and 38%, respectively. Staining for apoptotic nuclei did not reveal any differences between controls and treated cultures indicating necrotic cell death possibly due to membrane toxicity. The following toxicity ranking was obtained: t-BuMPO>BuMPO>s-BuMPO>PrMPO>i-PrMPO approximately DEPMPO approximately EMPO. The least toxic compounds were DEPMPO (LD(50)=143 mM for SW480, 117 mM for A549 or 277 mM for F2000) and i-PrMPO (LD(50)=114 mM for SKBR3), the most toxic one was t-BuMPO (LD(50)=5-6mM for all cell types). In conclusion, up to 50mM i-PrMPO (t(1/2)=18.8 min) and up to 10 mM s-BuMPO (t(1/2)=26.3 min) can be recommended for further investigation of superoxide in biological systems.     

Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy

The structure, transformation, and bioactivity of single living Schizosaccharomyces pombe cells at the molecular level have been studied in vivo by time- and space-resolved Raman spectroscopy. A time resolution of 100 s and a space resolution of 250 nm have been achieved with the use of a confocal Raman microspectrometer. The space-resolved Raman spectra of living S. pombe cells at different cell cycle stages were recorded in an effort to elucidate the molecular compositions of organelles, including nuclei, cytoplasm, mitochondria, and septa. The time- and space-resolved measurement of the central part of a dividing yeast cell showed continuous spectral evolution from that of the nucleus to those of the cytoplasm and mitochondria and finally to that of the septum, in accordance with the transformation during the cell cycle. A strong Raman band was observed at 1602 cm(-)(1) only when cells were under good nutrient conditions. The effect of a respiration inhibitor, KCN, on a living yeast cell was studied by measuring the Raman spectra of its mitochondria. A sudden disappearance of the 1602 cm(-)(1) band followed by the change in the shape and intensity of the phospholipid bands was observed, indicating a strong relationship between the cell activity and the intensity of this band. We therefore call this band "the Raman spectroscopic signature of life". The Raman mapping of a living yeast cell was also carried out. Not only the distributions of molecular species but also those of active mitochondria in the cell were successfully visualized in vivo.     

Dynamics of beta2-adrenergic receptor-ligand complexes on living cells

The agonist-induced dynamic regulation of the beta(2)-adrenergic receptor (beta(2)-AR) on living cells was examined by means of fluorescence correlation spectroscopy (FCS) using a fluorescence-labeled arterenol derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62 nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and 19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1) nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x 10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2) (hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8) cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x 10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand complex was detected immediately after addition of the ligand, whereas hindered mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38% of total binding) and of 15-20 min in A549 cells (up to 40% of total binding). Thus, the receptor-ligand complexes with low mobility were formed during receptor regulation. Consistently, stimulation of receptor internalization using the adenylate cyclase activator forskolin shifted the ratio of receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and immunocytochemical studies confirmed the appearance of endocytosed receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane after stimulation with the agonist terbutaline (1 microM). This regulatory receptor internalization was blocked after preincubation with propranolol and with a cholesterol-complexing saponin alpha-hederin.     

Gamma-glutamylcysteine synthetase activity in human lung epithelial (A549) cells: factors influencing its measurement

Despite the central role of gamma-glutamylcysteine synthetase (gammaGCS) in lung antioxidant defenses, the limited studies of the activity of this enzyme in respiratory cells have produced variable results. This study has examined the factors, which may influence the measurement of gammaGCS activity in cultured human lung epithelial cells (A549). Although a source of potential error, gammaGCS activity in A549 cell extracts did not vary significantly when appropriately assayed by three different methods or after removal of the endogenous inhibitor, glutathione (GSH). However, gammaGCS activity did increase significantly during the early stages of cell proliferation (3.50 +/- 0.31 vs. 2.35 +/- 0.16 nmol/min/10(6) cells for baseline, p < .001) and thereafter returned to baseline levels during the later stages of cell growth. Variations in initial plating density also significantly altered gammaGCS activity (3.11 +/- 0.14 vs. 4.04 +/- 0.50 nmol/min/10(6) cells, at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) and GSH content (45.43 +/- 4.43 vs. 63.64 +/- 3.28 nmol/10(6) cells at 0.25 x 10(5) and 0.58 x 10(5) cells/cm2, respectively, p < .001) during the early stages of cell proliferation. In addition, gammaGCS activity and GSH content were highest in A549 cells grown in medium containing cystine as the predominant sulfur-containing amino acid. These results suggest that gammaGCS activity of A549 cells is strongly dependent on initial plating density, stage of cell growth and sulfur amino acid content of the medium and may account for some of the variation in values reported by different investigators. Whether gammaGCS has an important role in the early phase of cell proliferation needs further investigation.     

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.     

Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy

Raman micro-spectroscopy is a laser-based technique which enables rapid and non-invasive biochemical analysis of cells and tissues without the need for labels, markers or stains. Previous characterization of the mammalian cell cycle using Raman micro-spectroscopy involved the analysis of suspensions of viable cells and individual fixed and/or dried cells. Cell suspensions do not provide cell-specific information, and fixing/drying can introduce artefacts which distort Raman spectra, potentially obscuring both qualitative and quantitative analytical results. In this article, we present Raman spectral characterization of biochemical changes related to cell cycle dynamics within single living cells in vitro. Raman spectra of human osteosarcoma cells synchronized in G(0)/G(1), S, and G(2)/M phases of the cell cycle were obtained and multivariate statistics applied to analyze the changes in cell spectra as a function of cell cycle phase. Principal components analysis identified spectral differences between cells in different phases, indicating a decrease in relative cellular lipid contribution to Raman spectral signatures from G(0)/G(1) to G(2)/M, with a concurrent relative increase in signal from nucleic acids and proteins. Supervised linear discriminant analysis of spectra was used to classify cells according to cell cycle phase, and exhibited 97% discrimination between G(0)/G(1)-phase cells and G(2)/M-phase cells. The non-invasive analysis of live cell cycle dynamics with Raman micro-spectroscopy demonstrates the potential of this approach to monitoring biochemical cellular reactions and processes in live cells in the absence of fixatives or labels.     

Electrosprayed Janus Particles for Combined Photo-Chemotherapy

This work is a proof of concept study establishing the potential of electrosprayed Janus particles for combined photodynamic therapy-chemotherapy. Sub-micron-sized particles of polyvinylpyrrolidone containing either an anti-cancer drug (carmofur) or a photosensitiser (rose bengal; RB), and Janus particles containing both in separate compartments were prepared. The functional components were present in the amorphous form in all the particles, and infrared spectroscopy indicated that intermolecular interactions formed between the different species. In vitro drug release studies showed that both carmofur and RB were released at approximately the same rate, with dissolution complete after around 250 min. Cytotoxicity studies were undertaken on model human dermal fibroblasts (HDF) and lung cancer (A549) cells, and the influence of light on cell death explored. Formulations containing carmofur as the sole active ingredient were highly toxic to both cell lines, with or without a light treatment. The RB formulations were non-toxic to HDF when no light was applied, and with photo-treatment caused large amounts of cell death for both A549 and HDF cells. The Janus formulation containing both RB and carmofur was non-toxic to HDF without light, and only slightly toxic with the photo-treatment. In contrast, it was hugely toxic to A549 cells when light was applied. The Janus particles are thus highly selective for cancer cells, and it is hence proposed that such electrosprayed particles containing both a chemotherapeutic agent and photosensitiser have great potential in combined chemotherapy/photodynamic therapy.     

RNA content and chromatin structure of CHO cells arrested in metaphase by colcemid

To evaluate the stability of cells arrested in metaphase, cell viability, RNA content, and chromatin structure (the latter probed by the DNA in situ sensitivity to acid-induced denaturation) were studied in uniform-age mitotic CHO cell populations maintained either at 37 degrees C (in the presence of Colcemid) or at 0-4 degrees C for up to 6 h. No significant changes in cell viability and RNA content were seen throughout the experiment for both groups of cells. The sensitivity of DNA in situ to denaturation was significantly increased during the initial 40 min of cell arrest in mitosis. However, no further chromatin changes for up to 6 h were evident regardless of whether cells were kept at 37 degrees C with Colcemid or at 0-4 degrees C in its absence. The data indicate that neither significant deterioration of metaphase cells nor progressive chromatin changes are expected during stathmokinesis experiments in vitro or during the metaphase cell arrest in cytogenetic studies lasting up to 6 h. Also, no RNA turnover can be detected in mitotic cells during this time interval.     

Analysis of intracellular state based on controlled 3D nanostructures mediated surface enhanced Raman scattering

Near-infrared surface-enhanced Raman spectroscopy (SERS) is a powerful technique for analyzing the chemical composition within a single living cell at unprecedented resolution. However, current SERS methods employing uncontrollable colloidal metal particles or non-uniformly distributed metal particles on a substrate as SERS-active sites show relatively low reliability and reproducibility. Here, we report a highly-ordered SERS-active surface that is provided by a gold nano-dots array based on thermal evaporation of gold onto an ITO surface through a nanoporous alumina mask. This new combined technique showed a broader distribution of hot spots and a higher signal-to-noise ratio than current SERS techniques due to the highly reproducible and uniform geometrical structures over a large area. This SERS-active surface was applied as cell culture system to study living cells in situ within their culture environment without any external preparation processes. We applied this newly developed method to cell-based research to differentiate cell lines, cells at different cell cycle stages, and live/dead cells. The enhanced Raman signals achieved from each cell, which represent the changes in biochemical compositions, enabled differentiation of each state and the conditions of the cells. This SERS technique employing a tightly controlled nanostructure array can potentially be applied to single cell analysis, early cancer diagnosis and cell physiology research.     

Apoptosis in sulfur mustard treated A549 cell cultures

The chemical warfare agent sulfur mustard (SM) is a strong alkylating agent that leads to erythema and ulceration of the human skin several hours after exposure. Although SM has been intensively investigated, the cellular mechanisms leading to cell damage remain unclear. Apoptosis, necrosis and direct cell damage are discussed. In this study we investigated apoptotic cell death in pulmonary A549 cells exposed to SM (30-1000 microM, 30 min). 24 h after SM exposure DNA breaks were stained with the TUNEL method. Additionally, A549 cells were lysed and cellular protein was transferred to SDS page and blotted. Whole PARP as well as PARP cleavage into the p89 fragment, an indicator of apoptosis, were detected by specific antibodies. SM concentration dependent increase in TUNEL positive cells and PARP cleavage showed that SM is an inducer of apoptosis. It has been previously suggested that AChE is activated during apoptotic processes and may be involved in apoptosis regulation. Therefore, we examined AChE activity in A549 cells upon induction of apoptosis by SM (100-500 microM). Increased AChE activity was found in SM treated A549 cell cultures examined as determined by the Ellman's assay and by western blot. AChE activity showed a strong correlation with TUNEL positive cells. However, the broad caspase inhibitor zVAD and the PARP-inhibitor 3-aminobenzamide had no protective effect on A459 cells measured with AChE activity and frequency of TUNEL positive cells. In summary, our studies demonstrate that AChE activity may be a potential marker of apoptosis in A549 cells after SM injury. To what extent AChE is involved in apoptosis regulation during SM poisoning has to be further investigated.     

Escin reduces cell proliferation and induces apoptosis on glioma and lung adenocarcinoma cell lines

Aesculus hippocastanum (the horse chestnut) seed extract has a wide variety of biochemical and pharmacological effects including anti-inflammatory, antianalgesic, and antipyretic activities. The main active compound of this plant is escin. It is known that several medicinal herbs with anti-inflammatory properties have been found to have a role in the prevention and treatment of cancer. In the present study, the cytotoxic effects of escin in the C6 glioma and A549 cell lines were analyzed by MTT. Apoptotic effects of escin on both cell lines were evaluated by Annexin V binding capacity with flow cytometric analysis. Structural and ultrastructural changes were also evaluated using transmission electron microscopy. The results indicated that escin has potent antiproliferative effects against C6 glioma and A549 cells. These effects are both dose and time dependent. Taken together, escin possesses cell cycle arrest on G0/G1 phase and selective apoptotic activity on A549 cells as indicated by increased Annexin V-binding capacity, bax protein expression, caspase-3 activity and morphological changes obtained from micrographs by transmission electron microscopy.     

周期性机械牵张对肺泡Ⅱ型上皮细胞Cyr61表达的影响

目的研究周期性牵张肺泡Ⅱ型上皮细胞株A549细胞对Cyr61表达的影响。方法对肺泡Ⅱ型上皮细胞株A549细胞施加周期性机械牵张应力。加载频率0.5 Hz,加载时间2 h,加载应力分别为5%,15%,30%。加载应力为15%,加载频率0.5 Hz,加载时间分别0,15 min,30 min,60 min,120 min。每个实验均设立空白对照即不给予机械应力。用PCR法测定Cyr61 mRNA的表达,用western法测定Cyr61蛋白含量。结果随着加载应力的增加和加载时间的延长,Cyr61蛋白含量和mRNA表达均增加(P<0.05);施加不同加载幅度后,Cyr61蛋白含量和mRNA表达均增加(P<0.05)。结论肺泡Ⅱ型上皮细胞IL-8的产生和释放与周期性的机械牵张应力呈强度和时间依赖性。     

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.     

An NQO1-Initiated and p53-Independent Apoptotic Pathway Determines the Anti-Tumor Effect of Tanshinone IIA against Non-Small Cell Lung Cancer

NQO1 is an emerging and promising therapeutic target in cancer therapy. This study was to determine whether the anti-tumor effect of tanshinone IIA (TSA) is NQO1 dependent and to elucidate the underlying apoptotic cell death pathways. NQO1(+) A549 cells and isogenically matched NQO1 transfected and negative H596 cells were used to test the properties and mechanisms of TSA induced cell death. The in vivo anti-tumor efficacy and the tissue distribution properties of TSA were tested in tumor xenografted nude mice. We observed that TSA induced an excessive generation of ROS, DNA damage, and dramatic apoptotic cell death in NQO1(+) A549 cells and H596-NQO1 cells, but not in NQO1(-) H596 cells. Inhibition or silence of NQO1 as well as the antioxidant NAC markedly reversed TSA induced apoptotic effects. TSA treatment significantly retarded the tumor growth of A549 tumor xenografts, which was significantly antagonized by dicoumarol co-treatment in spite of the increased and prolonged TSA accumulations in tumor tissues. TSA activated a ROS triggered, p53 independent and caspase dependent mitochondria apoptotic cell death pathway that is characterized with increased ratio of Bax to Bcl-xl, mitochondrial membrane potential disruption, cytochrome c release, and subsequent caspase activation and PARP-1 cleavage. The results of these findings suggest that TSA is a highly specific NQO1 target agent and is promising in developing as an effective drug in the therapy of NQO1 positive NSCLC.     

Real‐time detection of single‐living pancreatic β‐cell by laser tweezers Raman spectroscopy: High glucose stimulation

Glucose acts as a β‐cell stimulus factor and leads to cellular responses that involve a large amount of biomolecule formation, relocation, and transformation. We hypothesize that information about these changes can be obtained in real‐time by laser tweezers Raman spectroscopy. To test this hypothesis, repeated measurements designs in accordance with the application of Raman spectroscopy detection were used in the current experiment. Single rat β‐cells were measured by Raman spectroscopy in 2.8 mmol/l glucose culture medium as a basal condition. After stimulation with high glucose (20 mmol/l), the same cells were measured continuously. Each cell was monitored over a total time span of 25 min, in 5 min intervals. During this period of time, cells were maintained at an appropriate temperature controlled by an automatic heater, to provide near‐physiological conditions. It was found that some significant spectral changes induced by glucose were taking place during the stimulation time course. The most noticeable changes were the increase of spectral intensity at the 1002, 1085, 1445, and 1655 cm^?1 peaks, mainly corresponding to protein and lipid. We speculate that these changes might have to do with β‐cell protein and lipid synthesis. Using laser tweezers Raman spectroscopy in combination with glucose stimulation, optical spectral information from rat β‐cells was received and analyzed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 587–594, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Lignin radicals in the plant cell wall probed by Kerr-gated resonance Raman spectroscopy

Lignin radicals are crucial intermediates for lignin biosynthesis in the cell wall of vascular plants. In this work they were for the first time, to our knowledge, selectively observed in wood cell walls by laser-based Kerr-gated resonance Raman spectroscopy, and the observations were supported by density functional theory prediction of their vibrational properties. For dry wood cells a lignin radical Raman band is observed at 1,570 cm(-1) irrespective of species. For wet beech cells they were generated in situ and observed at 1,606 cm(-1). DFT/B3LYP/6-31+G(d) modeling results support that in beech they are formed from syringyl (S) phenolic moieties and in spruce from guaiacyl (G) phenolic moieties. The observed lignin radical band is predicted as G is approximately 1,597 cm(-1) and S is approximately 1,599 cm(-1), respectively, and is assigned the (Wilson notation) nu(8a) phenyl ring mode. The RR band probes lignin radical properties, e.g., spin density distribution, and these respond to charge polarization or hydrogen bonding to proximate water molecules. These observations can be crucial for an understanding of the factors that control cell wall structure during biosynthesis of vascular plants and demonstrate the unique potential of RR spectroscopy of lignin radicals.     

Histological mapping of biochemical changes in solid tumors by FT-IR spectral imaging

Fourier-transform infrared (FT-IR) spectral imaging was used for analyzing biochemical changes in tumor cells. Metabolic parameters of human lung A549/8 adenocarcinoma and U87 glioma cells were compared under stress conditions in culture along with tumor progression after cell implantation onto the chick embryo chorio-allantoic membrane. In cell culture, glucose consumption and lactic acid release were higher in U87 cells. A549/8 cells were less sensitive to oxidative stress as observed through changes in fatty acyl chains. In vivo biochemical mapping of highly (U87) vs. poorly (A549/8) angiogenic tumors provided results comparable to culture models. Therefore, FT-IR imaging allows detecting subtle chemical changes in tumors, which might be useful for diagnosis.