Transcript 100609_3
Single Molecule Spectroscopy (SMS) 2010/6/9 Miyasaka Lab. Iida Atsushi Contents • • • • Introduction -History of Single Molecule Spectroscopy (SMS) -Difference between ensemble and single-molecule measurement -Information obtained only by SMS Measurement -Principle -Single-molecule detection Confocal Microscope Wide-Field Mycroscope Representative results of SMS -Blinking -Spectral jump -Photon antibunching My work History of Single Molecule Spectroscopy • 1989 W. E. Moerner et al. First detection of single-molecule with FM spectroscopy • 1990 M. Orrit et al. Fluorescence excitation spectra of single molecules • 1992 T. Basche et al. Blinking, Spectral jump Difference between ensemble and single-molecule measurements The result of ensemble measurement Ensemble An emission spectrum of a single molecule “Single molecule” Information obtained only by SMS FRET : 励起エネルギー移動 SMS Ensemble measurement Intensity of red light Direct observation of dynamical state changes The signal is averaged. Information obtained only by SMS • Evaluation of nano-scale heterogeneity Fluorescent molecule Polymer Properties of a molecule depend on its microscopic environment . • Energy level • Life-time • Diffusion Principle Absorption I0 I I0 I Fluorescence Quite sensitive detector Single fluorescence molecules in dark space can be detected optically. Key : Reduction of the noise from the background Confocal Microscope Intensity trajectory Sample Life-time Objective lens Coincidence Pinhole Detector 3-dimentianal resolution Small background High temporal resolution Wide-field Microscope 2-dimentioanl resolution Objective lens ・translational diffusion coefficient High sensitivity camera ・rotational motion ・Many molecules can be observed at a time. Blinking Dye molecules in a polymer film (PMMA) non-luminescent Each molecule emits light frequently. Non-luminescent process, for example photo-ionization, relates with this phenomena. Spectral jump Host molecule p-terphenyl Guest molecule pentacene M. Oritt, J. Bernard, Phys. Rev. Lett., 65, 2716 (1990). Photon antibunching Beam splitter (50:50) Detector 1 A molecule emits one photon from its one excited state. Detector 2 Photons can not be divided. If you detect photons from a single molecule, there is no possibility to detect two photons by the detector 1 and 2 at the same time. Phenomenon that multiple photons do not exist at the same time. 12 Light pulse 125ns Interphoton arrival time Photon Photodetector 1 Photodetector 2 Events Photon antibunching -125 0 125 Delay /ns Ensemble Events Single molecule Coincidence -125 0 125 Delay /ns The coincidence event is not observed for single molecule system. 13 My Work water - octane Droplet is very small. specific surface area(比表面積) surface area volume Influence of surface is very big in ultrasmall droplet. - - - - - The surface is anionic. The interaction between surface and a solute depends on the size and the surface charge. My Work Transmission Fluorescence Dye molecule is moving in the droplet, and absorbs to the surface. Only one dye molecule exists in the droplet. My work surfactant ; triton x-100 (non-ionic) SDS (anionic) Triton x-100 The nano-scale environment around a solute is similar to that in the bulk octane. Triton x-100 A solute is diffusing center of the droplet. SDS SDS A solute exists in the different environment from the bulk octane. Life-time SDS<Triton x-100 (3.6ns) A solute is diffusing near the surface. (6.2ns) Life-time of the dye molecules in bulk octane is 6.4ns.