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.

Monitoring the mineralisation of bone nodules in vitro by space- and time-resolved Raman micro-spectroscopy.

Raman microscopy was used as a label-free method to study the mineralisation of bone nodules formed by mesenchymal stem cells cultured in osteogenic medium in vitro. Monitoring individual bone nodules over 28 days revealed temporal and spatial changes in the crystalline phase of the hydroxyapatite components of the nodules.

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.

Noninvasive detection and imaging of molecular markers in live cardiomyocytes derived from human embryonic stem cells.

Raman microspectroscopy (RMS) was used to detect and image molecular markers specific to cardiomyocytes (CMs) derived from human embryonic stem cells (hESCs). This technique is noninvasive and thus can be used to discriminate individual live CMs within highly heterogeneous cell populations. Principal component analysis (PCA) of the Raman spectra was used to build a classification model for identification of individual CMs. Retrospective immunostaining imaging was used as the gold standard for phenotypic identification of each cell. We were able to discriminate CMs from other phenotypes with >97% specificity and >96% sensitivity, as calculated with the use of cross-validation algorithms (target 100% specificity). A comparison between Raman spectral images corresponding to selected Raman bands identified by the PCA model and immunostaining of the same cells allowed assignment of the Raman spectral markers. We conclude that glycogen is responsible for the discrimination of CMs, whereas myofibril proteins have a lesser contribution. This study demonstrates the potential of RMS for allowing the noninvasive phenotypic identification of hESC progeny. With further development, such label-free optical techniques may enable the separation of high-purity cell populations with mature phenotypes, and provide repeated measurements to monitor time-dependent molecular changes in live hESCs during differentiation in vitro.

Non-invasive hydrodynamic imaging in plant roots at cellular resolution.

A key impediment to studying water-related mechanisms in plants is the inability to non-invasively image water fluxes in cells at high temporal and spatial resolution. Here, we report that Raman microspectroscopy, complemented by hydrodynamic modelling, can achieve this goal - monitoring hydrodynamics within living root tissues at cell- and sub-second-scale resolutions. Raman imaging of water-transporting xylem vessels in Arabidopsis thaliana mutant roots reveals faster xylem water transport in endodermal diffusion barrier mutants. Furthermore, transverse line scans across the root suggest water transported via the root xylem does not re-enter outer root tissues nor the surrounding soil when en-route to shoot tissues if endodermal diffusion barriers are intact, thereby separating 'two water worlds'.

TEM Tomography of Pores with Application to Computational Nanoscale Flows in Nanoporous Silicon Nitride (NPN).

Silicon nanomembrane technologies (NPN, pnc-Si, and others) have been used commercially as electron microscopy (EM) substrates, and as filters with nanometer-resolution size cut-offs. Combined with EM, these materials provide a platform for catching or suspending nanoscale-size structures for analysis. Usefully, the nanomembrane itself can be manufactured to achieve a variety of nanopore topographies. The size, shapes, and surfaces of nanopores will influence transport, fouling, sieving, and electrical behavior. Electron tomography (ET) techniques used to recreate nanoscale-sized structures would provide an excellent way to capture this variation. Therefore, we modified a sample holder to accept our standardized 5.4 mm × 5.4 mm silicon nanomembrane chips and imaged NPN nanomembranes (50⁻100 nm thick, 10⁻100 nm nanopore diameters) using transmission electron microscopy (TEM). After imaging and ET reconstruction using a series of freely available tools (ImageJ, TomoJ, SEG3D2, Meshlab), we used COMSOL Multiphysics™ to simulate fluid flow inside a reconstructed nanopore. The results show flow profiles with significantly more complexity than a simple cylindrical model would predict, with regions of stagnation inside the nanopores. We expect that such tomographic reconstructions of ultrathin nanopores will be valuable in elucidating the physics that underlie the many applications of silicon nanomembranes.

Applications of Raman micro-spectroscopy to stem cell technology: label-free molecular discrimination and monitoring cell differentiation.

Stem cell therapy is widely acknowledged as a key medical technology of the 21st century which may provide treatments for many currently incurable diseases. These cells have an enormous potential for cell replacement therapies to cure diseases such as Parkinson's disease, diabetes and cardiovascular disorders, as well as in tissue engineering as a reliable cell source for providing grafts to replace and repair diseased tissues. Nevertheless, the progress in this field has been difficult in part because of lack of techniques that can measure non-invasively the molecular properties of cells. Such repeated measurements can be used to evaluate the culture conditions during differentiation, cell quality and phenotype heterogeneity of stem cell progeny. Raman spectroscopy is an optical technique based on inelastic scattering of laser photons by molecular vibrations of cellular molecules and can be used to provide chemical fingerprints of cells or organelles without fixation, lysis or use of labels and other contrast enhancing chemicals. Because differentiated cells are specialized to perform specific functions, these cells produce specific biochemicals that can be detected by Raman micro-spectroscopy. This mini-review paper describes applications of Raman micro-scpectroscopy to measure moleculare properties of stem cells during differentiation in-vitro. The paper focuses on time- and spatially-resolved Raman spectral measurements that allow repeated investigation of live stem cells in-vitro.

Spectral depth profiling of arbitrary surfaces by thermal emission decay-Fourier transform infrared spectroscopy.

We report a new spectroscopic technique that combines step-scanning Fourier transform infrared spectroscopy with opto-thermal transient emission radiometry (OTTER) in order to provide near-surface depth-resolved spectra in the range 700-1800 cm(-1). It works nondestructively, without contact, with samples of arbitrary shape and size, without requiring prior preparation. The depth of surface probed depends on the thermal diffusivity of the sample; for organic materials it is approximately 10 microm. With homogeneous samples, absolute absorption coefficients can be measured. With two-layered samples, the technique proved able to distinguish between the spectral properties of the top layer and the substrate and to estimate the thickness of the top layer. We present a theoretical analysis with the main design features of the instrumentation and software, together with studies of homogeneous and layered samples, to validate the methods and illustrate the potential of the technique for practical applications.

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.