Cellular processes are intrinsically complex and dynamic, in which a myriad of cellular components including nucleic acids, proteins, membranes, and organelles are involved and undergo spatiotemporal changes. Label-free Raman imaging has proven powerful for studying such dynamic behaviors in vivo and at the molecular level. To construct Raman images, univariate data analysis has been commonly employed, but it cannot be free from uncertainties due to severely overlapped spectral information. Here, we demonstrate multivariate curve resolution analysis for time-lapse Raman imaging of a single dividing yeast cell. A four-dimensional (spectral variable, spatial positions in the two-dimensional image plane, and time sequence) Raman data "hypercube" is unfolded to a two-way array and then analyzed globally using multivariate curve resolution. The multivariate Raman imaging thus accomplished successfully disentangles dynamic changes of both concentrations and distributions of major cellular components (lipids, proteins, and polysaccharides) during the cell cycle of the yeast cell. The results show a drastic decrease in the amount of lipids by ~50% after cell division and uncover a protein-associated component that has not been detected with previous univariate approaches.

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Anti-Stokes Raman scattering is used to monitor vibrational energy redistribution in the ambient temperature liquids nitromethane (NM-h3) and perdeuterated nitromethane (NM-d3) after ultrafast IR excitation of either the symmetric or asymmetric CH- or CD-stretch transitions. The instantaneous populations of most of the fifteen NM vibrations are determined with good accuracy, and a global fitting procedure with a master equation is used to fit all the data. The pump pulses excite not only CH- or CD-stretches but also certain combinations of bending and nitro stretching fundamentals. The coupled vibrations that comprise the initial state are revealed via the instantaneous rise of the anti-Stokes transients associated with each vibrational fundamental. In contrast to many other polyatomic liquids studied previously, there is little energy exchange among the CH-stretch (or CD-stretch) excitations, which is attributed to the nearly free rotation of the methyl group in NM. The vibrational cooling process, which is the multistep return to a thermalized state, occurs in three stages in both NM-h3 and NM-d3. In the first stage, the parent CH- or CD-stretch decays in a few picoseconds, exciting all lower-energy vibrations. In the second stage, the midrange vibrations decay in 10-15 ps, exciting the lower-energy vibrations. In the third stage, these lower-energy vibrations decay into the bath in tens of picoseconds. The initial excitations are thermalized in approximately 150 ps in NM-h3 and there is little dependence on which CH-stretch is excited. VC is somewhat faster in NM-d3 with more dependence on the initial CD-stretch, taking approximately 100 ps with symmetric CD-stretch excitation and approximately 120 ps with asymmetric CD-stretch excitation. Comparison is made with earlier nonequilibrium molecular dynamics simulations of VC [Kabadi, V. N.; Rice, B. M. Molecular dynamics simulations of normal mode vibrational energy transfer in liquid nitromethane. J. Phys. Chem. A 2004, 108, 532-540]. The simulations do a good job of reproducing the observed VC process and in addition they predicted the slow interconversion among CH-stretch excitations and the slower relaxation of the asymmetric CH-stretch now observed here.

In vivo time-lapse Raman imaging reveals highly dynamic and concerted changes in concentration and distribution of phospholipids and proteins during and after cell division of a single living Schizosaccharomyces pombe cell.

Ultrafast infrared-Raman spectroscopy is used to study vibrational energy dynamics of three molecules in aqueous solution (D(2)O) that serve as models for the building blocks of peptides. These are glycine-d(3) zwitterion (GLY), N-methylacetamide-d (NMA), and benzoate anion (BZ). GLY is the simplest amino acid, NMA a model compound with a peptide bond, and BZ a model for aromatic side chains. An ultrashort IR pulse pumps a parent CH-stretch on each solute. Anti-Stokes Raman monitors energy flow through the solutes' strongly Raman-active transitions. Stokes Raman of D(2)O stretching functions as a molecular thermometer to monitor energy dissipation from solute to solvent. A three-stage model is used to summarize the vibrational energy redistribution process and to provide a framework for discussing energy dynamics of different molecules. The initial CH-stretch excitation is found to be delocalized over some or all of the solute molecule in NMA and BZ but not in GLY. The overall time constants for energy dissipation are 7.2 ps for GLY, 4.9 ps for NMA, and 8.0 ps for BZ. CH-stretch energy in GLY is redistributed in a nearly statistical manner among observed GLY vibrations. In NMA the energy is distributed among about one-half of the observed vibrations, and in BZ much of the observed energy is channeled along a CH-stretch to the ring stretch pathway. The strongly Raman-active vibrations accurately represent the flow of vibrational energy through NMA but not through GLY or BZ.