1. Single Molecule Spectroscopy (SMS) 2010/6/9 Miyasaka Lab. Iida Atsushi

2. 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

3. 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

4. Difference between ensemble and single-molecule measurements “Single molecule” An emission spectrum of a single molecule Ensemble The result of ensemble measurement

5. Information obtained only by SMS Direct observation of dynamical state changes Ensemble measurement The signal is averaged. SMS FRET : 励起エネルギー移動 Intensity of red light

6. Information obtained only by SMS Evaluation of nano-scale heterogeneity Energy level Life-time Diffusion Fluorescent molecule Polymer Properties of a molecule depend on its microscopic environment.

7. Principle I0I0 I0I0 II Absorption Quite sensitive detector Fluorescence Single fluorescence molecules in dark space can be detected optically. Key : Reduction of the noise from the background

8. Confocal Microscope Objective lens Sample Pinhole Detector Intensity trajectory Life-time Coincidence 3-dimentianal resolution Small background High temporal resolution

9. Wide-field Microscope Objective lens High sensitivity camera 2-dimentioanl resolution ・ Many molecules can be observed at a time. ・ translational diffusion coefficient ・ rotational motion

10. Blinking Dye molecules in a polymer film (PMMA) Each molecule emits light frequently. non-luminescent Non-luminescent process, for example photo-ionization, relates with this phenomena.

11. Spectral jump M. Oritt, J. Bernard, Phys. Rev. Lett., 65, 2716 (1990). p-terphenyl pentacene Host molecule Guest molecule

12. 12 Photon antibunching Beam splitter (50:50) Detector 1 Detector 2 A molecule emits one photon from its one excited state. 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. Photons can not be divided. Phenomenon that multiple photons do not exist at the same time.

13. 13 125ns Coincidence Delay /ns Events 0 Ensemble 125-125 Single molecule Delay /ns Events 0125-125 Photon Photodetector 2 The coincidence event is not observed for single molecule system. Photodetector 1 Interphoton arrival time Light pulse Photon antibunching

14. My Work The interaction between surface and a solute depends on the size and the surface charge. - - - - - - octane water specific surface area ( 比表面積 ) Influence of surface is very big in ultrasmall droplet. The surface is anionic. Droplet is very small. surface area volume

15. My Work Dye molecule is moving in the droplet, and absorbs to the surface. Transmission Fluorescence Only one dye molecule exists in the droplet.

16. My work SDS Triton x-100 Life-time SDS<Triton x-100 (6.2ns) (3.6ns) surfactant ; triton x-100 (non-ionic) SDS (anionic) Life-time of the dye molecules in bulk octane is 6.4ns. Triton x-100 SDS The nano-scale environment around a solute is similar to that in the bulk octane. A solute is diffusing center of the droplet. A solute exists in the different environment from the bulk octane. A solute is diffusing near the surface.