James P. R. Day

Quantitative coherent anti-Stokes Raman scattering (CARS) microscopy.

The ability to observe samples qualitatively at the microscopic scale has greatly enhanced our understanding of the physical and biological world throughout the 400 year history of microscopic imaging, but there are relatively few techniques that can truly claim the ability to quantify the local concentration and composition of a sample. We review coherent anti-Stokes Raman scattering (CARS) as a quantitative, chemically specific, and label-free microscopy. We discuss the complicating influence of the nonresonant response on the CARS signal and the various experimental and mathematical approaches that can be adopted to extract quantitative information from CARS. We also review the uses to which CARS has been employed as a quantitative microscopy to solve challenges in material and biological science.

Quantitative CARS Molecular Fingerprinting of Single Living Cells with the Use of the Maximum Entropy Method

This work was supported by the SENTAN project (Program-S) of the Japan Science and Technology Agency (JST). H. Kano gratefully acknowledges financial support from the Precursory Research for Embryonic Science and Technology (PRESTO) program of JST. The authors thank C. Onogi for providing the spontaneous Raman spectrum of yeast mitochondria, Leukos and Horus Laser companies for technical support, and Dr. F. Omura and H. Yomo (Suntory Co., Ltd.) for providing us with the yeast sample. We gratefully acknowledge J. Ukon (HORIBA, Ltd.) for assisting in the collaboration between the Japanese and French groups.

Label-Free Imaging of Lipophilic Bioactive Molecules during Lipid Digestion by Multiplex Coherent Anti-Stokes Raman Scattering Microspectroscopy

The digestion and absorption of lipophilic, bioactive molecules such as lipids, physiologically active nutrients (nutraceuticals), and drugs play a crucial role in human development and health. These molecules are often delivered in lipid droplets. Currently, the kinetics of digestion of these lipid droplets is followed by in vitro models that simulate gastrointestinal conditions, while phase changes within the lipid droplets are observed by light or electron microscopy. However real-time, spatially resolved information about the local chemical composition and phase behavior inside the oil droplet is not accessible from these approaches. This information is essential as the surface and phase behavior determine the local distribution of molecules in the oil droplets and thus may influence the rate of uptake, for example, by impairing the effective transfer of bioactive molecules to intestinal cells. We demonstrate the capability of multiplex coherent anti-Stokes Raman scattering (CARS) microspectroscopy to image the digestion process non-invasively, with submicrometer resolution, millimolar sensitivity, and without the need for labeling. The lipolysis of glyceryl trioleate emulsion droplets by porcine pancreatic lipase is imaged, and the undigested oil and the crystalline lipolytic products are distinguished by their different vibrational signatures. The digestion of droplets containing the phytosterol analogue ergosterol is also probed, and the crystals are observed to dissolve into the lipolytic products. The lipophilic drug progesterone and Vitamin D(3) are dissolved in glyceryl trioctanoate emulsion droplets, and the local concentration is mapped with millimolar sensitivity. The bioactive molecules are observed to concentrate within the droplets as the oil is hydrolyzed. This observation is ascribed to the low solubility of these molecules in the lipolytic products for this system. Neither the type of bioactive molecule nor the initial radius of the emulsion droplet had a large effect upon the rate of digestion under these conditions; lipolysis of the triglyceride by pancreatic lipase appears insensitive to the type of bioactive molecule in solution. These findings shed important new light on lipid digestion and open new possibilities for the chemical visualization of lipid digestion and phase changes in lipid droplets containing bioactive molecules, which in combination with other existing techniques will provide a full picture of this complex physicochemical process.

Label-Free Chemical Imaging of Catalytic Solids by Coherent Anti-Stokes Raman Scattering and Synchrotron-Based Infrared Microscopy†

Take a look inside: The combination of coherent anti-Stokes Raman scattering and synchrotron-based IR microscopy during the catalytic conversion of thiophene derivatives on zeolite crystals yields space- and time-resolved chemically specific information without the need for labeling (see picture). The thiophene reactant is mostly present in the center of the crystal, and the product is aligned within the straight pores of the zeolites.

Host–Guest Geometry in Pores of Zeolite ZSM‐5 Spatially Resolved with Multiplex CARS Spectromicroscopy

Pore relations: Reagent molecules form head-to-tail chains in the pores of ZSM-5 zeolite particles in the presence or absence of acidic sites, according to multiplex coherent anti-Stokes Raman scattering spectromicroscopy (mCARS). The molecular ordering in the pores makes it possible to characterize the crystallographic subunits of individual ZSM-5 particles with (sub)micrometer spatial resolution in three dimensions.

CARS microscopy for the visualization of micrometer-sized iron oxide MRI contrast agents in living cells

Micrometer-sized iron oxide particles (MPIOs) attract increasing interest as contrast agents for cellular tracking by clinical Magnetic Resonance Imaging (MRI). Despite the great potential of MPIOs for in vivo imaging of labeled cells, little is known on the intracellular localization of these particles following uptake due to the lack of techniques with the ability to monitor the particle uptake in vivo at single-cell level. Here, we show that coherent anti-Stokes Raman scattering (CARS) microscopy enables non-invasive, label-free imaging of MPIOs in living cells with sub-micron resolution in three dimensions. CARS allows simultaneous visualization of the cell framework and the MPIOs, where the particles can be readily distinguished from other cellular components of comparable dimensions, and localized inside the cell.

Coherent Anti-Stokes Raman Scattering Microspectroscopic Kinetic Study of Fast Hydrogen Bond Formation in Microfluidic Devices

The kinetics of a key noncovalent, hydrogen bonding interaction was studied in situ using coherent anti-stokes Raman scattering (CARS) microspectroscopy in a microfluidic device. The association of model compounds, pyridine and hexafluoroisopropanol, was quantitatively monitored with submicrometer resolution. Lower limits for the very high formation and dissociation rate constants of the model 1:1 pyridine-hexafluoroisopropanol hydrogen bonded complex in dichloromethane-d2 were determined to be k1 > 10(5) M(-1)s(-1) and k-1 > 333.3 s(-1), respectively.