Optical-trapping confocal Raman microscopy enhances the capabilities of traditional Raman spectroscopy for the analysis of small particles by significantly reducing the sampling volume and minimizing background signal from the particle surroundings. Chemical composition and structural information can be obtained from optically trapped particles in aqueous solution without the need for labeling or extensive sample preparation. In this work, the challenges of measuring temperature dependent changes in suspended particles are addressed with the development of a small-volume, thermally conductive sample cell attached to a temperature-controlled microscope stage. To demonstrate its function, the gel to liquid-crystalline phase transitions of optically trapped lipid vesicles, composed of pure 1,2-ditridecanoyl-sn-glycero-3-phosphocholine (DTPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), were detected by changes in Raman spectra of the lipid bilayer. The Raman scattering data were found to correlate well with differential scanning calorimetry (DSC) results.

In recent years observations at the level of individual atoms and molecules became possible by micros-copy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholip-ids carrying one rhodamine dye molecule. For this methodology , fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bio-science-for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoi-chiometries of individual components. The ultimate goal of high-sensitivity detection schemes is observation on the single molecule level. This came into reach by the invention of scanning probe microscopy (1, 2), which has since brought a wealth of new insights (3). Optical methods allowed for detection of single atoms (4). The effective light conversion in fluorescent molecules made it possible to detect single fluorophores in liquids by confocal fluorescence mi-croscopy (5-8) and to perform high-resolution spectroscopy of single dye molecules at low temperature (9-12). The first true imaging of single dye molecules by optical means was achieved by scanning near-field optical microscopy (13). This method is unique in reaching a spatial resolution of 14 nm, much below the optical diffraction limit but restricted in its application to immobile objects. Very recently, single fluorescence labeled myosin molecules on immobilized actin filaments were imaged by conventional microscopy and illumination times of seconds (14). It would be of interest for many applications, especially in bioscience, to extend microscopy to visualization of single fluorophores in motion. To our knowledge, such imaging has not been reported to date. Here we show that the motion of single dye molecules can be visualized by conventional fluo-rescence microscopy by extending the time resolution into the millisecond range. For this, we used epifluorescence micros-copy with argon-ion laser excitation and imaging onto a highly-sensitive liquid-nitrogen-cooled CCD-camera. Optical parts were carefully selected to achieve an efficiency for the detection of emitted fluorescence as high as 3%, while scattered light was blocked effectively. For demonstration of the potentials of observing individual mobile molecules we have chosen a fluorescence-labelled lipid in a fluid lipid membrane as a most appropriate system. It uniquely permitted to use results obtained at high surface densities of labelled lipid for The publication costs of this article were defrayed in part by p...

The binding of glucagon-like peptide-1 (GLP-1) to a planar phospholipid bilayer is measured using single-molecule total internal reflection fluorescence microscopy. From several reports in the literature, GLP-1 has been shown to be a random coil in free solution, adopting a folded, α-helix conformation when intercalated into membrane environments. Single-molecule fluorescence measurements of GLP-1 binding to supported lipid bilayers show evidence of two populations of membrane-associated molecules having different residence times, suggesting weakly adsorbed peptides and strongly bound peptides in the lipid bilayer. The path to and from a strongly bound (folded, intercalated) state would likely include an adsorbed state as an intermediate, so that the resulting kinetics would correspond to a consecutive first-order reversible three-state model. In this work, the relationships between measured single-molecule residence times and the microscopic rates in a three-state kinetic model are derived and used to interpret the binding of GLP-1 to a supported lipid bilayer. The system of differential equations associated with the proposed consecutive-three state kinetics scheme is solved, and the solution is applied to interpret histograms of single-molecule, GLP-1 residence times in terms of the microscopic rates in the sequential two-step model. These microscopic rates are used to estimate the free energy barrier to adsorption, the fraction of peptides adsorbing to the membrane surface that successfully intercalate in the bilayer, the lifetime of inserted peptides in the membrane, and the free energy change of insertion into the lipid bilayer from the adsorbed state. The transition from a random coil in solution to a folded state in a membrane has been recognized as a common motif for insertion of membrane active peptides. Therefore, the relationships developed here could have wide application to the kinetic analysis of peptide-membrane interactions.

The microstructurally-induced heterogeneous stress fields arising in a series of Cr-doped polycrystalline alumina materials are mapped with sub-micrometer sub-grain size resolution using fluorescence microscopy. Analysis of the hyperspectral data sets generated during imaging enabled both the amplitude and position of the characteristic Cr R1 fluorescence peak to be determined at every pixel in an image. The peak amplitude information was used to segment the images into individual grains and grain boundary regions. The peak position information, in conjunction with measurements on single-crystal controls, was used to quantify overall stress distributions in the materials and provide stress scales for maps. The combined information enabled spatial variations in the stress fields in crystallographic axes to be mapped and compared directly with microstructural features such as grains and grain boundaries. The mean c-axis stresses in these materials were approximately 200 MPa with stress distribution widths of about 70 MPa, both increasing with average grain size. Greatest variations in stress were observed at grain junctions; no trend in the stress for individual grains with grain size was observed.