Fluctuations and synchrony of RNA synthesis in nucleoli.

Ribosomal RNA (rRNA) sequences are synthesized at exceptionally high rates and, together with ribosomal proteins (r-proteins), are utilized as building blocks for the assembly of pre-ribosomal particles. Although it is widely acknowledged that tight regulation and coordination of rRNA and r-protein production are fundamentally important for the maintenance of cellular homeostasis, still little is known about the real-time kinetics of the ribosome component synthesis in individual cells. In this communication we introduce a label-free MicroRaman spectrometric approach for monitoring rRNA synthesis in live cultured cells. Remarkably high and rapid fluctuations of rRNA production rates were revealed by this technique. Strikingly, the changes in the rRNA output were synchronous for ribosomal genes located in separate nucleoli of the same cell. Our findings call for the development of new concepts to elucidate the coordination of ribosomal components production. In this regard, numerical modeling further demonstrated that the production of rRNA and r-proteins can be coordinated, regardless of the fluctuations in rRNA synthesis. Overall, our quantitative data reveal a spectacular interplay of inherently stochastic rates of RNA synthesis and the coordination of gene expression.

Organelle specific imaging in live cells and immuno-labeling using resonance Raman probe.

Raman microspectroscopy is one of the most powerful tools in molecular sensing, offering a non-invasive and comprehensive characterization of the intracellular environment. To analyze and monitor molecular content in specific cellular compartments, different parts of cellular architecture must be unambiguously identified to guide Raman image/spectra acquisition. In this regards, the development of Raman molecular probes, producing spectrally distinct and intense signal is of outmost practical importance. Here we report on a new generation of Raman molecular probes, designed for application in live cells and immuno-labeling, capable of providing unprecedentedly high detection sensitivity through Resonance Raman (RR) enhancement. In contrast to existing Raman markers, the proposed RR reporter is designed to produce RR enhancement under excitation in the visible spectral range, far away from absorption of cellular biomolecules. We show that this concept allows for facile identification of labeled cellular domains, simultaneously with mapping of the macromolecules using spontaneous Raman technique. We demonstrate the breakthrough potential of these RR probes for selective labeling and rapid Raman imaging of membranes as well as mitochondria in live cells. We also show that these resonant Raman probes open the way for Raman-based intracellular immuno-labeling.

Nucleolar molecular signature of pluripotent stem cells.

Induced pluripotent stem cells (iPSC) are generated by reprogramming somatic cells to the pluripotent state. Identification and quantitative characterization of changes in the molecular organization of the cell during the process of cellular reprogramming is valuable for stem cell research and advancement of its therapeutic applications. Here we employ quantitative Raman microspectroscopy and biomolecular component analysis (BCA) for a comparative analysis of the molecular composition of nucleoli in skin fibroblasts and iPSC derived from them. We report that the cultured fibroblasts obtained from different human subjects, share comparable concentrations of proteins, RNA, DNA, and lipids in the molecular composition of nucleoli. The nucleolar molecular environment is drastically changed in the corresponding iPSC. We measured that the transition from skin fibroblasts to iPSC is accompanied by a statistically significant increase in protein concentrations ~1.3-fold, RNA concentrations ~1.3-fold, and DNA concentrations ~1.4-fold, while no statistically significant difference was found for the lipid concentrations. The analysis of molecular vibrations associated with diverse aminoacids and protein conformations indicates that nucleoli of skin fibroblasts contain similar subsets of proteins, with prevalence of tyrosine. In iPSC, we observed a higher signal from tryptophan with an increase in the random coil and α helix protein conformations, indicating changes in the subset of nucleolar proteins during cell reprogramming. At the same time, the concentrations of major types of macromolecules and protein conformations in the nucleoli of iPSC and human embryonic stem cells (hESC) were found to be similar. We discuss these results in the context of nucleolar function and conclude that the nucleolar molecular content is correlated with the cellular differentiation status. The approach described here shows the potential for spectroscopically monitoring changes in macromolecular organization of the cell at different stages of reprogramming.

Optically generated reconfigurable photonic structures of elastic quasiparticles in frustrated cholesteric liquid crystals.

We describe laser-induced two-dimensional periodic photonic structures formed by localized particle-like excitations in an untwisted confined cholesteric liquid crystal. The individual particle-like excitations (dubbed "Torons") contain three-dimensional twist of the liquid crystal director matched to the uniform background director field by topological point defects. Using both single-beam-steering and holographic pattern generation approaches, the periodic crystal lattices are tailored by tuning their periodicity, reorienting their crystallographic axes, and introducing defects. Moreover, these lattices can be dynamically reconfigurable: generated, modified, erased and then recreated, depending on the needs of a particular photonic application. This robust control is performed by tightly focused laser beams of power 10-100 mW and by low-frequency electric fields at voltages ~10 V applied to the transparent electrodes.

Nonlinear optical imaging and Raman microspectrometry of the cell nucleus throughout the cell cycle.

Fundamental understanding of cellular processes at molecular level is of considerable importance in cell biology as well as in biomedical disciplines for early diagnosis of infection and cancer diseases, and for developing new molecular medicine-based therapies. Modern biophotonics offers exclusive capabilities to obtain information on molecular composition, organization, and dynamics in a cell by utilizing a combination of optical spectroscopy and optical imaging. We introduce here a combination of Raman microspectrometry, together with coherent anti-Stokes Raman scattering (CARS) and two-photon excited fluorescence (TPEF) nonlinear optical microscopy, to study macromolecular organization of the nucleus throughout the cell cycle. Site-specific concentrations of proteins, DNA, RNA, and lipids were determined in nucleoli, nucleoplasmic transcription sites, nuclear speckles, constitutive heterochromatin domains, mitotic chromosomes, and extrachromosomal regions of mitotic cells by quantitative confocal Raman microspectrometry. A surprising finding, obtained in our study, is that the local concentration of proteins does not increase during DNA compaction. We also demonstrate that postmitotic DNA decondensation is a gradual process, continuing for several hours. The quantitative Raman spectroscopic analysis was corroborated with CARS/TPEF multimodal imaging to visualize the distribution of protein, DNA, RNA, and lipid macromolecules throughout the cell cycle.

Biophotonic probing of macromolecular transformations during apoptosis.

We introduce here multiplex nonlinear optical imaging as a powerful tool for studying the molecular organization and its transformation in cellular processes, with the specific example of apoptosis. Apoptosis is a process of self-initiated cell death, critically important for physiological regulation and elimination of genetic disorders. Nonlinear optical microscopy, combining the coherent anti-Stokes Raman scattering (CARS) microscopy and two-photon excited fluorescence (TPEF), has been used for analysis of spatial distribution of major types of biomolecules: proteins, lipids, and nucleic acids in the cells while monitoring their changes during apoptosis. CARS imaging revealed that in the nuclei of proliferating cells, the proteins are distributed nearly uniformly, with local accumulations in several nuclear structures. We have found that this distribution is abruptly disrupted at the onset of apoptosis and is transformed to a progressively irregular pattern. Fluorescence recovery after photobleaching (FRAP) studies indicate that pronounced aggregation of proteins in the nucleoplasm of apoptotic cells coincides with a gradual reduction in their mobility.

Realignment-enhanced coherent anti-Stokes Raman scattering and three-dimensional imaging in anisotropic fluids.

We apply coherent anti-Stokes Raman Scattering (CARS) microscopy to characterize director structures in liquid crystals. We demonstrate that the polarized CARS signal in these anisotropic fluids strongly depends on alignment of chemical bonds/molecules with respect to the collinear polarizations of Stokes and pump/probe excitation beams. This dependence allows for the visualization of the bond/molecular orientations via polarized detection of the CARS signal and thus for CARS polarization microscopy of liquid crystal director fields, as we demonstrate using structures in nematic, cholesteric, and smectic liquid crystals. On the other hand, laser-induced director realignment at powers above a well-defined threshold provides the capability for all-optical CARS signal enhancement in liquid crystals. Moreover, since the liquid crystalline alignment can be controlled by electric and magnetic fields, this demonstrates the feasibility of CARS signal modulation by applying external fields to these materials.

Zinc Oxide Nanocrystals for Non-resonant Nonlinear Optical Microscopy in Biology and Medicine.

In this paper we show that biocompatible zinc oxide (ZnO) nanocrystals (NCs) having non-centrosymmetric structure can be used as non-resonant nonlinear optical probes for targeting in bioimaging applications in vitro by use of the second order processes of second harmonic and sum frequency generation, as well as the third order process of four wave mixing. These non-resonant processes provide advantages above and beyond traditional two-photon bioimaging: (i) the probes do not photo-bleach; (ii) the input wavelength can be judiciously selected; and (iii) no heat is dissipated into the cells, ensuring longer cell viability and ultimately longer imaging times. ZnO NCs have been synthesized in organic media by using a non-hydrolytic sol-gel process, and subsequently dispersed in aqueous media using phospholipid micelles, and incorporated with the biotargeting molecule folic acid (FA). Sum Frequency, Second Harmonic and non-resonant four wave mixing non-linear signals from this stable dispersion of ZnO NCs, targeted to the live tumor (KB) cells were used for imaging. Robust intracellular accumulation of the targeted (FA incorporated) ZnO nanocrystals could be observed, without any indication of cytotoxicity.

Optical trapping of director structures and defects in liquid crystals using laser tweezers.

We demonstrate optical manipulation of structures and defects in liquid crystals (LCs). The effective refractive index depends on the LC molecular orientations and the laser beam's polarization. We use the orientation-mediated refractive index contrast for the laser trapping in LCs with a homogeneous composition, but with spatially-varying patterns of molecular orientations. Tightly-focused polarized beams allow for optical trapping of disclinations and their clusters, dislocations and oily streaks, cholesteric fingers and focal conic domains, etc. We calculate the optical gradient forces for typical structures and explain the trapping properties at low laser powers. We also show that when a high-power beam causes local molecular realignment, the laser trapping properties change for two reasons: (1) the refractive index pattern and optical gradient forces are modified; (2) additional elastic structural forces arise to minimize the elastic free energy.

Laser trapping in anisotropic fluids and polarization-controlled particle dynamics.

Anisotropic fluids are widespread, ranging from liquid crystals used in displays to ordered states of a biological cell interior. Optical trapping is potentially a powerful technique in the fundamental studies and applications of anisotropic fluids. We demonstrate that laser beams in these fluids can generate anisotropic optical trapping forces, even for particles larger than the trapping beam wavelength. Immersed colloidal particles modify the fluid's ordered molecular structures and locally distort its optic axis. This distortion produces a refractive index "corona" around the particles that depends on their surface characteristics. The laser beam can trap such particles not only at their center but also at the high-index corona. Trapping forces in the beam's lateral plane mimic the corona and are polarization-controlled. This control allows the optical forces to be reversed and cause the particle to follow a prescribed trajectory. Anisotropic particle dynamics in the trap varies with laser power because of the anisotropy of both viscous drag and trapping forces. Using thermotropic liquid crystals and biological materials, we show that these phenomena are quite general for all anisotropic fluids and impinge broadly on their quantitative studies using laser tweezers. Potential applications include modeling thermodynamic systems with anisotropic polarization-controlled potential wells, producing optically tunable photonic crystals, and fabricating light-controlled nano- and micropumps.

Dynamics of multiple trapping by a single-beam laser tweezer.

A multiple-trap single-beam scanning laser tweezer system was developed and characterized. Different stationary and mobile multiple-trap modes were generated for polystyrene beads in a water environment. Trapping efficiency and stability were investigated for several dynamic parameters such as transition time between the sites, waiting time on a single site, number of trapping sites, and IR laser power. Optimal parameters for efficient generation of complex arrays and matrices were determined. We demonstrate an example of a single laser beam multiple-trap application by measuring the trap's stiffness in water for our laser tweezer setup.