Mikhail N. Slipchenko

Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis.

Spectroscopic imaging has been an increasingly critical approach for unveiling specific molecules in biological environments. Toward this goal, we demonstrate hyperspectral stimulated Raman loss (SRL) imaging by intrapulse spectral scanning through a femtosecond pulse shaper. The hyperspectral stack of SRL images is further analyzed by a multivariate curve resolution (MCR) method to reconstruct quantitative concentration images for each individual component and retrieve the corresponding vibrational Raman spectra. Using these methods, we demonstrate quantitative mapping of dimethyl sulfoxide concentration in aqueous solutions and in fat tissue. Moreover, MCR is performed on SRL images of breast cancer cells to generate maps of principal chemical components along with their respective vibrational spectra. These results show the great capability and potential of hyperspectral SRL microscopy for quantitative imaging of complicated biomolecule mixtures through resolving overlapped Raman bands.

High-Speed Vibrational Imaging and Spectral Analysis of Lipid Bodies by Compound Raman Microscopy

Cells store excess energy in the form of cytoplasmic lipid droplets. At present, it is unclear how different types of fatty acids contribute to the formation of lipid droplets. We describe a compound Raman microscope capable of both high-speed chemical imaging and quantitative spectral analysis on the same platform. We used a picosecond laser source to perform coherent Raman scattering imaging of a biological sample and confocal Raman spectral analysis at points of interest. The potential of the compound Raman microscope was evaluated on lipid bodies of cultured cells and live animals. Our data indicate that the in vivo fat contains much more unsaturated fatty acids (FAs) than the fat formed via de novo synthesis in 3T3-L1 cells. Furthermore, in vivo analysis of subcutaneous adipocytes and glands revealed a dramatic difference not only in the unsaturation level but also in the thermodynamic state of FAs inside their lipid bodies. Additionally, the compound Raman microscope allows tracking of the cellular uptake of a specific fatty acid and its abundance in nascent cytoplasmic lipid droplets. The high-speed vibrational imaging and spectral analysis capability renders compound Raman microscopy an indispensible analytical tool for the study of lipid-droplet biology.

Label-free quantitative analysis of lipid metabolism in living Caenorhabditis elegans

The ubiquity of lipids in biological structures and functions suggests that lipid metabolisms are highly regulated. However, current invasive techniques for lipid studies prevent characterization of the dynamic interactions between various lipid metabolism pathways. Here, we describe a noninvasive approach to study lipid metabolisms using a multifunctional coherent anti-Stokes Raman scattering (CARS) microscope. Using living Caenorhabditis elegans as a model organism, we report label-free visualization of coexisting neutral and autofluorescent lipid species. We find that the relative expression level of neutral and autofluorescent lipid species can be used to assay the genotype-phenotype relationship of mutant C. elegans with deletions in the genes encoding lipid synthesis transcription factors, LDL receptors, transforming growth factor β receptors, lipid desaturation enzymes, and antioxidant enzymes. Furthermore, by coupling CARS with fingerprint confocal Raman analysis, we analyze the unsaturation level of lipids in wild-type and mutant C. elegans. Our study shows that complex genotype-phenotype relationships between lipid storage, peroxidation, and desaturation can be rapidly and quantitatively analyzed in a single living C. elegans.

Use of helium nanodroplets for assembly, transport, and surface deposition of large molecular and atomic clusters

The utility of continuous beam of helium droplets for assembly, transport, and surface deposition of metal and molecular clusters is studied. Clusters of propyne having from about 10 to 10(4) molecules were obtained via sequential pickup of molecules by He droplets with average sizes in the range of 10(4)-10(7) atoms. The maximum attainable flux of the propyne molecules carried by He droplets was found to be in the range of (5-15)x10(15) molecules sr(-1) s(-1), being larger in larger droplets. The size of the clusters and the flux of the transported species are ultimately limited by the evaporative extinction of the entire helium droplet upon capture of particles. It is shown that the attenuation of the He droplet beam in the process of the cluster growth can be used in order to obtain the average size and the binding energy of the clusters. Furthermore, we used He droplets for assembling and surface deposition of gold and silver clusters having about 500 atoms. Typical deposition rate of metal atoms of about 3 x 10(15) atoms sr(-1) s(-1) is comparable to or larger than obtained with other beam deposition techniques. We propose that doping of He droplets by Au and Ag atoms in two separate pickup chambers leads to formation of the bimetal clusters having core-shell structure.

Far-field imaging of non-fluorescent species with subdiffraction resolution

Super-resolution optical microscopy is opening a new window to unveil the unseen details on the nanoscopic scale. Current far-field super-resolution techniques rely on fluorescence as the read-out1–5. Here, we demonstrate a scheme for breaking the diffraction limit in far-field imaging of non-fluorescent species by using spatially controlled saturation of electronic absorption. Our method is based on a pump-probe process where a modulated pump field perturbs the charge-carrier density in a sample, thus modulating the transmission of a probe field. A doughnut shape laser beam is then added to transiently saturate the electronic transition in the periphery of the focal volume, thus the induced modulation in the sequential probe pulse only occurs at the focal center. By raster scanning the three collinearly aligned beams, high-speed sub-diffraction-limited imaging of graphite nano-platelets was performed. This technique potentially enables super-resolution imaging of nano-materials and non-fluorescent chromophores, which may remain out of reach for fluorescence-based methods.

Hybrid femtosecond/picosecond coherent anti-Stokes Raman scattering for high-speed gas-phase thermometry

We demonstrate hybrid femtosecond/picosecond (fs/ps) coherent anti-Stokes Raman scattering for high-speed thermometry in unsteady high-temperature flames, including successful comparisons with a time- and frequency-resolved theoretical model. After excitation of the N(2) vibrational manifold with 100 fs broadband pump and Stokes beams, the Raman coherence is probed using a frequency-narrowed 2.5 ps probe beam that is time delayed to suppress the nonresonant background by 2 orders of magnitude. Experimental spectra were obtained at 500 Hz in steady and pulsed H(2)-air flames and exhibit a temperature precision of 2.2% and an accuracy of 3.3% up to 2400 K. Strategies for real-time gas-phase thermometry in high-temperature flames are also discussed, along with implications for kilohertz-rate measurements in practical combustion systems.

Single-shot gas-phase thermometry using pure-rotational hybrid femtosecond/picosecond coherent anti-Stokes Raman scattering

High-repetition-rate, single-laser-shot measurements are important for the investigation of unsteady flows where temperature and species concentrations can vary significantly. Here, we demonstrate single-shot, pure-rotational, hybrid femtosecond/picosecond coherent anti-Stokes Raman scattering (fs/ps RCARS) thermometry based on a kHz-rate fs laser source. Interferences that can affect nanosecond (ns) and ps CARS, such as nonresonant background and collisional dephasing, are eliminated by selecting an appropriate time delay between the 100-fs pump/Stokes pulses and the pulse-shaped 8.4-ps probe. A time- and frequency-domain theoretical model is introduced to account for rotational-level dependent collisional dephasing and indicates that the optimal probe-pulse time delay is 13.5 ps to 30 ps. This time delay allows for uncorrected best-fit N2-RCARS temperature measurements with ~1% accuracy. Hence, the hybrid fs/ps RCARS approach can be performed with kHz-rate laser sources while avoiding corrections that can be difficult to predict in unsteady flows.

Ultrahigh-frame-rate OH fluorescence imaging in turbulent flames using a burst-mode optical parametric oscillator

Burst-mode planar laser-induced fluorescence (PLIF) imaging of the OH radical is demonstrated in laminar and turbulent hydrogen-air diffusion flames with pulse repetition rates up to 50 kHz. Nearly 1 mJ/pulse at 313.526 nm is used to probe the OH P(2)(10) rotational transition in the (0,0) band of the A-X system. The UV radiation is generated by a high-speed-tunable, injection-seeded optical parametric oscillator pumped by a frequency-doubled megahertz-rate burst-mode Nd:YAG laser. Preliminary kilohertz-rate wavelength scanning of the temperature-broadened OH transition during PLIF imaging is also presented for the first time (to our knowledge), and possible strategies for spatiotemporally resolved planar OH spectroscopy are discussed.

Infrared intensity in small ammonia and water clusters

Helium droplet technique has been used in order to measure the strength of the infrared absorption in small ammonia and water clusters as a function of size. Hydrogen bonding in ammonia and water dimers causes an enhancement of the intensity of the hydrogen stretching bands by a factor of four and three, respectively. Two types of the hydrogen bonded clusters show different size dependence of the infrared intensity per hydrogen bond. In ammonia (NH3)2 and (NH3)3 it is close to the crystal value. In water clusters, it increases monotonically with cluster size being in tetramers, a factor of two smaller than in the ice. The measured infrared intensity in water clusters is found to be a factor of two to three smaller as compared to the results of numerical calculations.

Multimodal Nonlinear Optical Microscopy

Because each nonlinear optical (NLO) imaging modality is sensitive to specific molecules or structures, multimodal NLO imaging capitalizes the potential of NLO microscopy for studies of complex biological tissues. The coupling of multiphoton fluorescence, second harmonic generation, and coherent anti-Stokes Raman scattering (CARS) has allowed investigation of a broad range of biological questions concerning lipid metabolism, cancer development, cardiovascular disease, and skin biology. Moreover, recent research shows the great potential of using CARS microscope as a platform to develop more advanced NLO modalities such as electronic-resonance-enhanced four-wave mixing, stimulated Raman scattering, and pump-probe microscopy. This article reviews the various approaches developed for realization of multimodal NLO imaging as well as developments of new NLO modalities on a CARS microscope. Applications to various aspects of biological and biomedical research are discussed.