Stanislav O. Konorov

Assessing differentiation status of human embryonic stem cells noninvasively using Raman microspectroscopy.

Raman microspectroscopy is an attractive approach for chemical imaging of biological specimens, including live cells, without the need for chemi-selective stains. Using a microspectrometer, near-infrared Raman spectra throughout the range 663 cm(-1) to 1220 cm(-1) were obtained from colonies of CA1 human embryonic stem cells (hESCs) and CA1 cells that had been stimulated to differentiate for 3 weeks by 10% fetal bovine serum on gelatin. Distributions and intensities of spectral bands attributed to proteins varied significantly between undifferentiated and differentiated cells. Importantly, compared to proteins and lipids, the band intensities of nucleic acids were dominant in undifferentiated cells with a dominance-reversal in differentiated cells. Thus, we could identify intensity ratios of particular protein-related bands (e.g., 757 cm(-1) tryptophan) to nucleic acid bands (784 cm(-1) DNA/RNA composite) that were effective in discriminating between spectra of undifferentiated and differentiated cells. We observed no discernible negative effects due to the laser exposure in terms of morphology, proliferation, or pluripotency of the stem cells. We conclude that Raman microscopy and complementary data processing procedures provide a rapid, noninvasive approach that can distinguish hESCs from differentiated cells. This is the first report to identify specific Raman markers for the differentiation status of hESCs.

Hollow-core photonic crystal fiber-optic probes for Raman spectroscopy

We have implemented a new Raman fiber-optic probe design based on a hollow-core photonic-crystal excitation fiber surrounded by silica-core collection fibers. The photonic-crystal fiber offers low attenuation at the pump radiation wavelength, mechanical flexibility, high radiation stability, and low background noise. Because the excitation beam is transmitted through air inside the hollow-core fiber, silica Raman scattering is much reduced, improving the quality of the spectra obtained using probes of this design. Preliminary results show that the new probe design decreases the Raman background from the silica by approximately an order of magnitude compared to solid-core silica Raman probes.

Absolute Quantification of Intracellular Glycogen Content in Human Embryonic Stem Cells with Raman Microspectroscopy

We present a method to perform absolute quantification of glycogen in human embryonic stem cells (hESCs) in situ based on the use of Raman microspectroscopy. The proposed quantification method was validated by comparison to a commonly used commercial glycogen assay kit. With Raman microspectroscopy, we could obtain the glycogen content of hESCs faster and apparently more accurately than with the kit. In addition, glycogen distributions across a colony could be obtained. Raman spectroscopy can provide reliable estimates of the in situ glycogen content in hESCs, and this approach should also be extensible to their other biochemical constituents as well as to other cell types.

Label-free determination of the cell cycle phase in human embryonic stem cells by Raman microspectroscopy.

The cell cycle is a series of integrated and coordinated physiological events that results in cell growth and replication. Besides observing the event of cell division it is not feasible to determine the cell cycle phase without fatal and/or perturbing invasive procedures such as cell staining, fixing, and/or dissociation. Raman microspectroscopy (RMS) is a chemical imaging technique that exploits molecular vibrations as a contrast mechanism; it can be applied to single living cells noninvasively to allow unperturbed analysis over time. We used RMS to determine the cell cycle phase based on integrating the composite 783 cm(-1) nucleic acid band intensities across individual cell nuclei. After correcting for RNA contributions using the RNA 811 cm(-1) band, the measured intensities essentially reflected DNA content. When quantifying Raman images from single cells in a population of methanol-fixed human embryonic stem cells, the histogram of corrected 783 cm(-1) band intensities exhibited a profile analogous to that obtained using flow-cytometry with nuclear stains. The two population peaks in the histogram occur at Raman intensities corresponding to a 1-fold and 2-fold diploid DNA complement per cell, consistent with a distribution of cells with a population peak due to cells at the end of G1 phase (1-fold) and a peak due to cells entering M phase (2-fold). When treated with EdU to label the replicating DNA and block cell division, cells with higher EdU-related fluorescence generally had higher integrated Raman intensities. This provides proof-of-principle of an analytical method for label-free RMS determination in situ of cell cycle phase in adherent monolayers or even single adherent cells.

Raman Microscopy-Based Cytochemical Investigations of Potential Niche-Forming Inhomogeneities Present in Human Embryonic Stem Cell Colonies

Measuring spatial and temporal patterns of cytochemical variation in human embryonic stem cell (hESC) colonies is necessary for understanding the role of cellular communication in spontaneous differentiation, the mechanisms of biological niche creation, and structure-generating developmental processes. Such insights will ultimately facilitate directed differentiation and therewith promote advances in tissue engineering and regenerative medicine. However, the patterns of cytochemical inhomogeneities of hESC colonies are not well studied and their causes are not fully understood. We used Raman spectroscopic mapping to contrast supra-cellular variations in cytochemical composition across pluripotent and partly differentiated hESC colonies to gain a better understanding of the early-stage (i.e., 5 days) effects of the differentiation process on the nature and evolution of these patterns. Higher protein-to-nucleic acid ratios, a differentiation status indicator observed previously using Raman spectroscopy, confirmed reported results that spontaneous differentiation is more pronounced on the edges of a colony than elsewhere. In addition, pluripotent and partly differentiated colonies also showed higher lipid concentrations relative to nucleic acids at colony edges in contrast to relative glycogen concentrations, which were up to 400% more pronounced in the colony centers compared to their edges. Pluripotent and partly differentiated colonies differed, with the latter having higher average protein-to-nucleic acid and lipid-to-nucleic acid ratios but a lower glycogen-to-nucleic acid ratio. In both cases, cell density, pluripotency, and high glycogen appeared to vary in tandem. Spatial variations in glycogen- and protein-to-nucleic acid ratios have features on the order of 100 μm and larger. These dimensions are consistent with those reported for stem cell niches and suggest that cytochemical inhomogeneities may provide colony-level information about niches and niche formation. These results demonstrate Raman mapping to be a potentially useful technique for revealing the complexities in the spatial organization of hESC cultures and thus for monitoring the evolution of engineered hESC niches.

In situ analysis by microspectroscopy reveals triterpenoid compositional patterns within leaf cuticles of Prunus laurocerasus

The cuticular waxes on the leaves of Prunus laurocerasus are arranged in distinct layers differing in triterpenoid concentrations (Jetter et al., Plant Cell Environ 23:619–628, 2000). In addition to this transversal gradient, the lateral distribution of cuticular triterpenoids must be investigated to fully describe the spatial distribution of wax components on the leaf surfaces. In the present investigation, near infrared (NIR) Raman microspectroscopy, coherent anti-Stokes Raman scattering (CARS) microscopy, and third harmonic generation (THG) spectroscopy were employed to map the triterpenoid distribution in isolated cuticles from adaxial and abaxial sides of P. laurocerasus leaves. The relative concentrations of ursolic acid and oleanolic acid were calculated by treating the cuticle spectra as linear combinations of reference spectra from the major compounds found in the wax. Raman maps of the adaxial cuticle showed that the triterpenoids accumulate to relatively high concentrations over the periclinal regions of the pavement cells, while the very long chain aliphatic wax constituents are distributed fairly evenly across the entire adaxial cuticle. In the analysis of the abaxial cuticles, the triterpenoids were found to accumulate in greater amounts over the guard cells relative to the pavement cells. The very long chain aliphatic compounds accumulated in the cuticle above the anticlinal cell walls of the pavement cells, and were found at low concentrations above the periclinals and the guard cells.

Complete characterization of molecular vibration using frequency resolved gating

The authors propose a new approach to vibration spectroscopy based on the coherent anti-Stokes Raman scattering of broadband ultrashort laser pulses. The proposed method reveals both the amplitude and the phase of molecular vibrations by utilizing the cross-correlation frequency resolved optical gating (XFROG) technique. The spectrum of the anti-Stokes pulse is measured as a function of the time delay between the laser-induced molecular vibrations and a well characterized broadband femtosecond probe pulse. The iterative XFROG algorithm provides a simultaneous complete characterization of molecular vibrations both in frequency and time domains with high resolution. They demonstrate experimentally the feasibility of the proposed method and show one of its potential applications in disentangling the time behavior of a mixture of vibrationally excited molecules. The technique of femtosecond pulse shaping is used for further improvement of accuracy and stability against noise.

Lorentzian Amplitude and Phase Pulse Shaping for Nonresonant Background Suppression and Enhanced Spectral Resolution in Coherent Anti-Stokes Raman Scattering Spectroscopy and Microscopy

Femtosecond coherent anti-Stokes Raman scattering (CARS) spectroscopy offers several advantages over spontaneous Raman spectroscopy due to the inherently high sensitivity and low average power deposition in the sample. Femtosecond CARS can be implemented in a collinear pump/probe beam configuration for microspectroscopy applications and has emerged as a powerful technique for chemical imaging of biological specimens. However, one serious limitation of this approach is the presence of a high nonresonant background component that often obscures the resonant signals of interest. We report here an innovative pulse-shaping method based on Lorentzian amplitude and phase spectral modulation of a broadband femtosecond probe pulse that yields spectra with both high spectral resolution and no nonresonant background. No further mathematical analysis is needed to extract Raman spectra. The utility of the proposed method for CARS microscopy is demonstrated using a mixture of polystyrene and latex beads, as well as dry-fixed embryonic stem cells.

Narrowband spectroscopy by an all-optical correlation of broadband laser pulses

High-peak-power ultrafast lasers are widely used in nonlinear spectroscopy, but often limit its spectral resolution because of the broad frequency bandwidth of ultrashort laser pulses. Improving the resolution by achieving spectrally narrow excitation of, or emission from, the resonant medium by means of multiphoton interferences has been the focus of many recent developments in ultrafast spectroscopy. We demonstrate an alternative approach in which high resolution is exercised by detecting narrow spectral correlations between broadband excitation and emission optical fields. All-optical correlation analysis, easily incorporated into a traditional spectroscopic setup, enables direct, robust, and simultaneous detection of multiple narrow resonances in a single measurement.

Non-resonant background suppression by destructive interference in coherent anti-Stokes Raman scattering spectroscopy

Coherent anti-Stokes Raman scattering (CARS) with femtosecond interaction pulses has become a popular and powerful spectroscopic method. Non-resonant background is one of the most limiting factors for implementing this method more widely. We propose a new approach that suppresses the non-resonant background contribution to the measured signal in CARS spectroscopy while simultaneously yielding high spectral resolution. The method is based on femtosecond pulse shaping of probe, Stokes and pump beams. Destructive interference suppresses the non-resonant background, resulting only in the resonant contribution being detected.