Ioan Notingher

Raman microspectroscopy: a noninvasive tool for studies of individual living cells in vitro

There is an increasing need for noninvasive methods that are able to monitor individual live cells in vitro, including in vitro testing of chemicals and pharmaceuticals, monitoring the growth of engineered tissues and the development of cell-based biosensors. Raman spectroscopy is a pure optical technique based on inelastic scattering of laser photons by molecular vibrations of biopolymers, which provide a chemical fingerprint of cells or organelles without fixation, lysis or the use of labels and other contrast-enhancing chemicals. Changes in cells during the cell cycle, cell death, differentiation or during the interaction with various chemicals or materials involve biochemical changes that can be measured with high spatial (∼300 nm) and temporal (seconds to minutes) resolution. The latest technological developments, especially high-sensitivity charged coupled detectors and high-power near-infrared lasers, have spurred the growth of Raman microspectroscopy towards being a well established analytical tool. This review covers the recent applications of this technique, including studies of individual cells, both pro- and eukaryotes, and emphasizes the potential impact on modern scientific endeavors, such as tissue engineering and drug discovery.

In situ characterisation of living cells by Raman spectroscopy

We report the first Raman spectra of individual living and dead cells (MLE-12 line) cultured on bioinert standard poly-L-lysine coated fused silica and on bioactive 45S5 Bioglass R � measured at 785 nm laser excitation. At this excitation wavelength no damage was induced to the cells even after 40 minutes irradiation at 115 mW power, as indicated by cell morphology observation and trypan blue viability test. We show that shorter wavelength lasers, 488 nm and 514 nm, cannot be used because they induce damage to the cells at very low laser powers (5 mW) and short irradiation times (5-20 minutes). The most important differences between the spectra of living and dead cells are in the 1530-1700 cm −1 range, where the dead cells have strong peaks at 1578 cm −1 and 1607 cm −1 . Other differences occur around the DNA peak at 1094 cm −1 .O ur study establishes the feasibility of using the 785 nm laser for an in situ real-time non-invasive method to follow biological events (proliferation, differentiation, cell death, etc.) within individual cells cultured on bioactive scaffolds in their physiologic environment over long periods of time.

Discrimination between ricin and sulphur mustard toxicity in vitro using Raman spectroscopy

A Raman spectroscopy cell-based biosensor has been proposed for rapid detection of toxic agents, identification of the type of toxin and prediction of the concentration used. This technology allows the monitoring of the biochemical properties of living cells over long periods of time by measuring the Raman spectra of the cells non-invasively, rapidly and without use of labels (Notingher et al. 2004 doi:10.1016/j.bios.2004.04.008). Here we show that this technology can be used to distinguish between changes induced in A549 lung cells by the toxin ricin and the chemical warfare agent sulphur mustard. A multivariate model based on principal component analysis (PCA) and linear discriminant analysis (LDA) was used for the analysis of the Raman spectra of the cells. The leave-one-out cross-validation of the PCA-LDA model showed that the damaged cells can be detected with high sensitivity (98.9%) and high specificity (87.7%). High accuracy in identifying the toxic agent was also found: 88.6% for sulphur mustard and 71.4% for ricin. The prediction errors were observed mostly for the ricin treated cells and the cells exposed to the lower concentration of sulphur mustard, as they induced similar biochemical changes, as indicated by cytotoxicity assays. The concentrations of sulphur mustard used were also identified with high accuracy: 93% for 200 μM and 500 μM, and 100% for 1000 μM. Thus, biological Raman microspectroscopy and PCA-LDA analysis not only distinguishes between viable and damaged cells, but can also discriminate between toxic challenges based on the cellular biochemical and structural changes induced by these agents and the eventual mode of cell death.

Diagnosis of tumors during tissue-conserving surgery with integrated autofluorescence and Raman scattering microscopy.

Significance Histopathology is the standard method for diagnosis of cancer. However, this method requires time-consuming procedures for sectioning and staining of tissues, making histopathology impractical for use during surgery for most cancer types. We report a unique method based on two optical spectroscopy techniques—autofluorescence imaging and Raman scattering—that can accurately measure molecular differences between tumor cells and healthy tissue and allows diagnosis of tumors faster than histopathology, without requiring tissue sectioning or staining. Our study demonstrates the potential of this technique for diagnosis of tissues during cancer surgery, providing a quick and objective way to determine whether the tissue layers removed by the surgeon are clear of tumor. Tissue-conserving surgery is used increasingly in cancer treatment. However, one of the main challenges in this type of surgery is the detection of tumor margins. Histopathology based on tissue sectioning and staining has been the gold standard for cancer diagnosis for more than a century. However, its use during tissue-conserving surgery is limited by time-consuming tissue preparation steps (1–2 h) and the diagnostic variability inherent in subjective image interpretation. Here, we demonstrate an integrated optical technique based on tissue autofluorescence imaging (high sensitivity and high speed but low specificity) and Raman scattering (high sensitivity and high specificity but low speed) that can overcome these limitations. Automated segmentation of autofluorescence images was used to select and prioritize the sampling points for Raman spectroscopy, which then was used to establish the diagnosis based on a spectral classification model (100% sensitivity, 92% specificity per spectrum). This automated sampling strategy allowed objective diagnosis of basal cell carcinoma in skin tissue samples excised during Mohs micrographic surgery faster than frozen section histopathology, and one or two orders of magnitude faster than previous techniques based on infrared or Raman microscopy. We also show that this technique can diagnose the presence or absence of tumors in unsectioned tissue layers, thus eliminating the need for tissue sectioning. This study demonstrates the potential of this technique to provide a rapid and objective intraoperative method to spare healthy tissue and reduce unnecessary surgery by determining whether tumor cells have been removed.

Bioresorbable and bioactive composite materials based on polylactide foams filled with and coated by Bioglass® particles for tissue engineering applications

Poly(DL-lactide) (PDLLA) foams and bioactive glass (Bioglass®) particles were used to form bioresorbable and bioactive composite scaffolds for applications in bone tissue engineering. A thermally induced phase separation process was applied to prepare highly porous PDLLA foams filled with 10 wt % Bioglass® particles. Stable and homogeneous layers of Bioglass® particles on the surface of the PDLLA/Bioglass® composite foams as well as infiltration of Bioglass® particles throughout the porous network were achieved using a slurry-dipping technique. The quality of the bioactive glass coatings was reproducible in terms of thickness and microstructure. In vitro studies in simulated body fluid (SBF) were performed to study the formation of hydroxyapatite (HA) on the surface of the PDLLA/Bioglass® composites, as an indication of the bioactivity of the materials. Formation of the HA layer after immersion in SBF was confirmed by X-ray diffraction and Raman spectroscopy measurements. The rate of HA formation in Bioglass®-coated samples was higher than that observed in non-coated samples. SEM analysis showed that the HA layer thickness rapidly increased with increasing time in SBF in the Bioglass®-coated samples. The high bioactivity of the developed composites suggests that the materials are attractive for use as bioactive, resorbable scaffolds in bone tissue engineering.

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 µM 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. J. Cell. Biochem.

In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering.

Raman micro‐spectroscopy was used to discriminate between different types of bone cells commonly used in tissue engineering of bone, with the aim of developing a method of phenotypic identification and classification. Three types of bone cells were analysed: human primary osteoblasts (HOB), retroviral transfected human alveolar bone cells with SV40 large T antigen (SV40 AB), and osteoblast‐like human osteosarcoma derived MG63 cell line. Unsupervised principal component analysis (PCA) and linear discriminant analysis (LDA) of the Raman spectra succeeded in discriminating the osteosarcoma derived MG63 cells from the non‐tumour cells (HOB and SV40 AB). No significant differences were observed between the Raman spectra of the HOB and SV40 AB cells, confirming the biochemical similarities between the two cell types. Difference spectra between tumour and non‐tumour cells suggested that the spectral discrimination is based on the fact that MG63 osteosarcoma derived cells are characterised by lower concentrations of nucleic acids and higher relative concentrations of proteins compared to the non‐tumour bone cells. A supervised classification model (LDA) was built and showed high cross‐validation sensitivity (100%) and specificity (95%) for discriminating the MG63 cells and the non‐tumour cells, with 96% of the cells being correctly classified either as tumour or non‐tumour derived cells. This study proves the feasibility of using Raman spectroscopy to identify in situ phenotypic differences in living cells.

Cytoplasmic RNA in Undifferentiated Neural Stem Cells: A Potential Label-Free Raman Spectral Marker for Assessing the Undifferentiated Status

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ~700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (~1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA.

Toward label-free Raman-activated cell sorting of cardiomyocytes derived from human embryonic stem cells

Raman micro-spectroscopy (RMS) has been recently proposed for label-free phenotypic identification of human embryonic stem cells (hESC)-derived cardiomyocytes. However, the methods used for measuring the Raman spectra led to acquisition times of minutes per cell, which is prohibitive for rapid cell sorting applications. In this study we evaluated two measurement strategies that could reduce the measurement time by a factor of more than 100. We show that sampling individual cells with a laser beam focused to a line could eliminate the need of cell raster scanning and achieve high prediction accuracies (>95% specificity and >96% sensitivity) with acquisition times ∼ 5 seconds per cell. However, the use of commercially-available higher power lasers could potentially lead to sorting speeds of ∼ 10 cells per s. This would start to progress RMS to the field of cell sorting for applications such as enrichment and purification of hESC-derived cardiomyocytes.

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

BACKGROUND Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation. METHODS EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24h intervals. RESULTS The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340cm(-1), 1083cm(-1), 937cm(-1), 858cm(-1), 577cm(-1) and 482cm(-1). The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs. CONCLUSIONS The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation. GENERAL SIGNIFICANCE As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.