Search

Identifying cell response to a chemotherapy drug treatment, in particular at the single cell level, is an important issue in patient management. This study aims at evaluating the effect of gemcitabine on single living cells using micro-Raman imaging. We used as a model the non-small lung cancer cell line, Calu-1, exposed to cytostatic doses (1 nM to 1 μM for 24 h and 48 h) of gemcitabine, an antitumor drug currently used in the treatment of lung cancer. Following drug treatment as a function of doses and incubation times, the Raman maps of single living cells were acquired. Cell biomolecules (DNA, RNA, and proteins) were chemically extracted and their spectral signatures used to evaluate their respective distribution in the cellular spectral information of control and treated cells. The quantification of these distributions reveals a significant effect of 100 nM gemcitabine at 48 h incubation (concomitant decrease of nucleic acids and increase of proteins). PCA analyses performed both on nuclear and extracted biomolecules spectra show a time-dependent effect of the drug. These promising results reveal that effects of subtoxic doses can be monitored at the single cell level highlighting the importance of such studies for clinical applications. [ABSTRACT FROM AUTHOR]

Over the last few years, there has been an increased interest in the study of stem cells in biomedicine for therapeutic use and as a source for healing diseased or injured organs/tissues. More recently, vibrational spectroscopy has been applied to study stem cell differentiation. In this study, we have used both synchrotron based FTIR and Raman microspectroscopies to assess possible differences between human pluripotent (embryonic) and multipotent (adult mesenchymal) stem cells, and how O2concentration in cell culture could affect the spectral signatures of these cells. Our work shows that infrared spectroscopy of embryonic (pluripotent) and adult mesenchymal (multipotent) stem cells have different spectral signatures based on the amount of lipids in their cytoplasm (confirmed with cytological staining). Furthermore, O2concentration in cell culture causes changes in both the FTIR and Raman spectra of embryonic stem cells. These results show that embryonic stem cells might be more sensitive to O2concentration when compared to mesenchymal stem cells. While vibrational spectroscopy could therefore be of potential use in identifying different populations of stem cells further work is required to better understand these differences. [ABSTRACT FROM AUTHOR]

Single-cell studies have important implications in biomedicine. An accurate investigation of biochemical behaviour and status requires a biomolecular probe such as vibrational microscopy. Amongst other approaches, synchrotron infrared microspectroscopy is an appropriate analytical tool for single-cell investigation. However, it is important to understand the precise origin of spectral differences as they are directly related to the cell biochemistry. Beside biomolecular changes, physical properties can interfere in the resulting information, and the two effects need separating. Both cells and nuclei induce Mie scattering effects due to their equivalent size with the probe wavelength. This results in a large modification of the spectra, and its precise contribution has to be determined in order to extract the true spectral information. On this basis, we carried out this study in order to evaluate the exact contribution of cell nuclei to Mie scattering. To this purpose, we isolated whole cancer cell nuclei and obtained, for the first time, their FTIR spectra with good signal to noise ratio. The synchrotron-based FTIR (S-FTIR) spectra of nuclei showed changes in lipids, proteins, and DNA absorptions when compared to spectra of whole lung cancer cells. Importantly, we estimated the Mie scattering properties of single cells and single nuclei spectra and were consequently able to separate optical and chemical properties of single cells and nuclei. This is the first study which sheds new light on the identification of the precise spectral biomarkers of a whole cell and those of the cell nucleus. [ABSTRACT FROM AUTHOR]

Non-melanoma skin cancer includes basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and Bowen's disease. The differential diagnosis of these lesions is sometimes difficult and relies on the histopathological examination of surgical specimens. However, a precise differential diagnosis is crucial for an accurate therapy and thus better patient care. FTIR spectral micro-imaging was applied directly on formalin-fixed paraffin-embedded samples of non-melanoma skin cancers. Chemometric and multivariate statistical analyses were developed to generate an automated IR-based histology without any chemical dewaxing. Different prediction models were developed using linear discriminant analysis combined with data reduction by Principal Component Analysis (PCA) or by wavenumber selection using statistical tests or genetic algorithms. Pseudo-colour maps were reconstructed and compared to conventional histology procedures. High correlation was obtained between the prediction maps and the histology which proves the great potential of FTIR spectroscopy for the differential diagnosis of skin carcinomas. [ABSTRACT FROM AUTHOR]

Raman microspectroscopy allows probing subcellular compartments and provides a unique spectral fingerprint indicative of endogenous molecular composition. Although several spectroscopic cell studies have been reported on fixed samples, only few attempts concern single growing cells. Here, we have tested different optical substrates that would best preserve cell integrity and allow direct measurement of Raman spectra at the single living cell level. Calu-1 lung cancer cells were used as a model and their morphology and growth were assessed on Raman substrates like quartz, calcium fluoride, and zinc selenide. Data show that quartz was the most appropriate taking into consideration both cell morphology and proliferation rate (47% on quartz vs.55% of BrdU-positive cells on conventional plastic). Using quartz, 40 cells were analysed and Raman spectra were collected from nuclei and cytoplasms using a 785 nm laser excitation of 30 mW at the sample, in the spectral range of 580–1750 cm−1, and an acquisition time of 2 × 10 sec/spectrum. Discriminant spectral information related to nucleus and cytoplasm were extracted by multivariate statistical methods and attributed to nucleic acids, lipids, and proteins. Finally, Raman spectral imaging was performed to show the distribution of these components within the cell. [ABSTRACT FROM AUTHOR]

Polarized Raman microspectroscopy can provide precious information regarding the orientation and ordering of the molecules in a sample without staining or particular preparation. This technique is used for the first time on a human skin section to probe the molecular modifications of the surrounding dermis in superficial basal cell carcinoma. Spectra using polarized and conventional Raman microspectroscopies were recorded on dermis bordering either the tumor or healthy epidermis. Band areas and spectral decomposition on selected vibrations were computed. Significant differences in dermal collagen vibration bands are detected using both polarized and conventional micro-spectroscopies, but the spectral changes between tumor and healthy tissues are enhanced using polarized Raman microspectroscopy. The analysis of these spectral differences highlights structural modifications of the triple helix of collagen. We see polarized Raman microspectroscopy as a potential tool that could be implemented for clinical analyses to guide clinicians and surgeons in the treatment of aggressive skin cancers. The information obtainable could also help better elucidate the molecular mechanisms induced in basal cell carcinoma development. [ABSTRACT FROM AUTHOR]

The aim of this tutorial review is to give an overview of the state of the art of intracellular applications of analytical SERS spectroscopy. We pay particular attention to nanoparticle-based SERS spectroscopy since this currently dominates the published literature on non-disturbing analysis of live cells. We describe recent advances in this domain due to the development of multispectral imaging and to the combined use of SERRS (surface-enhanced resonance Raman scattering) and fluorescence spectroscopy. Finally, a perspective view is given on the tip-based approaches like tip-enhanced Raman spectroscopy (TERS) which allow micrometric and nanometric resolution. [ABSTRACT FROM AUTHOR]

FTIR spectral imaging was applied on formalin-fixed paraffin-embedded biopsies from colon and skin cancerous lesions. These samples were deposited onto different substrates (zinc selenide and calcium fluoride respectively) and embedded using two types of paraffin. Formalin fixation followed by paraffin embedding is the gold standard in tissue storage. It can preserve molecular structures and it is compatible with immunohistochemistry. However, paraffin absorption bands are significant in the mid-infrared region and can mask some molecular vibrations of the tissue. Direct data processing was applied on spectral images without any chemical dewaxing of the tissues. Extended Multiplicative Signal Correction was used to correct the spectral contribution from paraffin. For this purpose, the signal of paraffin was modelled using Principal Component Analysis and paraffin spectra were removed from the raw images based on an outlier detection. Then, pseudo-colour images were computed by K-means clustering in order to highlight histological structures of interest. This robust chemometrics methodology was applied on the two samples. Tumour areas were successfully demarcated from the rest of the tissue in both colon and skin independently of the embedding material and of the substrate. [ABSTRACT FROM AUTHOR]

The aim of this study was to investigate the correlation patterns between Fourier transform infrared (FT-IR) and Raman microspectroscopic data obtained from pork muscle tissue, which helped to improve the interpretation and band assignment of the observed spectral features. The pork muscle tissue was subjected to different processing factors, including aging, salting, and heat treatment, in order to induce the necessary degree of variation of the spectra. For comparing the information gained from the two spectroscopic techniques with respect to the experimental design, multiblock principal component analysis (MPCA) was utilized for data analysis. The results showed that both FT-IR and Raman spectra were mostly affected by heat treatment, followed by the variation in salt content. Furthermore, it could be observed that IR amide I, II, and III band components appear to be effected to a different degree by brine-salting and heating. FT-IR bands assigned to specific protein secondary structures could be related to different Raman C–C stretching bands. The Raman C–C skeletal stretching bands at 1031, 1061, and 1081 cm−1 are related to the IR bands indicative of aggregated β-structures, while the Raman bands at 901 cm−1 and 934 cm−1 showed a strong correlation with IR bands assigned to a α-helical structures. At the same time, the IR band at 1610 cm−1, which formerly was assigned to tyrosine in spectra originating from pork muscle, did not show a correlation to the strong tyrosine doublet at 827 and 852 cm−1 found in Raman spectra, leading to the conclusion that the IR band at 1610 cm−1 found in pork muscle tissue is not originating from tyrosine. [ABSTRACT FROM AUTHOR]

Diffusion-time distribution analysis (DDA) has been used to explore the plasma membrane fluidity of multidrug-resistant cancer cells (LR73 carcinoma cells) and also to characterize the influence of various membrane agents present in the extracellular medium. DDA is a recent single-molecule technique, based on fluorescence correlation spectroscopy (FCS), well suited to retrieve local organization of cell membrane. The method was conducted on a large number of living cells, which enabled us to get a detailed overview of plasma membrane microviscosity, and plasma membrane micro-organization, between the cells of the same line. Thus, we clearly reveal the higher heterogeneity of plasma membrane in multidrug-resistant cancer cells in comparison with the nonresistant ones (denoted sensitive cells). We also display distinct modifications related to a membrane fluidity modulator, benzyl alcohol, and two revertants of multidrug resistance, verapamil and cyclosporin-A. A relation between the distribution of the diffusion-time values and the modification of membrane lateral heterogeneities is proposed. [ABSTRACT FROM AUTHOR]