100nm Ke Xu, H. P. Babcock, X. Zhuang, Nature Methods. 2012, 9, 185–188. Sub-diffraction limited point spread function achieved by using photo-switchable fluorescence of diarylethene derivatives Miyasaka Lab. Ikegami Takahiro

I. Introduction Optical microscopy Fluorescence microscopy Super-resolution microscopy ( e.g. STED, PALM & STORM) II. My work Principle Simulation Experience III. Summary IV. Future work Contents

Do you know what won the Nobel Prize in Physics 2014? Blue LED Isamu AkasakiHiroshi AmanoShuji Nakamura Photo: Nobelprize.org

Eric BetzigStefan W. HellWilliam E. Moerner Super resolution microscopy Then, do you know what won the Nobel Prize in Chemistry 2014? Photo: Nobelprize.org

Optical microscopy Observation target ・ Biological tissue ・ Polymer film Glass SiO 2 Trajectory of dye in PolyHEA Arai Yuhei, graduation thesis D trajectory of dye in PolyHEA Taga Yuhei, thesis for master degree One of the commonest methods to observe the microstructures Advantage ・ Internal structure observation ・ Non-destructive and non- invasive observation ・ High temporal resolution …etc Disadvantage ・ Low spatial resolution Optical microscopy λ/2 ( ≧ 200 nm) SEM ( ≧ 3 nm ) TEM ( ≧ 0.1 nm ) STM ( ≧ 0.1 nm )

Confocal microscopy Confocal fluorescence microscopy Dye Sample example Imaging Fluorescence microscopy Highest resolution in the optical microscopy CCD camera Laser or Stage Scanning Laser Resolution of optical microscopy Depended on laser spot size Laser intensity distribution Fluorescence spot ( Intensity distribution ) Objective Dye Counting photon number Wide field microscopy Diffraction limit 回折限界

Wide field fluorescence microscopy Dye Sample example Imaging with CCD Fluorescence microscopy Highest resolution in the optical microscopy CCD camera Laser Resolution of optical microscopy Point Spread Function 点広がり関数 Objective Dye Fluorescence Imaging Wide field microscopy Diffraction limit 回折限界

Excitation beam Fluorescence dye Objective Dumping beam STED ( Stimulated Emission depletion ) Super-Resolution microscopy S. W. Hell, J. Wichmann, OPICS LETTERS. 1994, 19, 11. Stefan W. Hell Stimulated emission

Eric Betzig PALM ( PhotoActivated Localization Microscopy ) Super-Resolution microscopy Normal image Blinking image ( 光の明滅 ) Localization ( 位置決定 ) Normal image PALM image STORM ( STochastic Optical Reconstruction Microscopy ) ⇒ Photoswitching E. Betzig, et al., Science, 313, (2006).

Excitation beam Fluorescence dye Objective Dumping beam Super-Resolution microscopy STED Microscopy Stimulated emission My work Photochemical reaction!! ⇒ Purpose Using weaker intensity beam to avoid breaking samples

I. Introduction Optical microscopy Fluorescence microscopy Super-resolution microscopy ( e.g. STED, PALM & STORM) II. My work Principle Simulation Experience III. Summary IV. Future work Contents

diarylethene derivative (DE1) Fluorescent UV (Φ oc = 0.43) Closed-form Open-form Vis. (Φ co = 1.6×10 -4 ) Φ F =0.88 non-Fluorescent Super-resolution by employing photo-switchable fluorescent molecule

PSF Objective Dye (DAE1) Principle Visible position is shifted. UV Vis. Effective fluorescent spot size is changed by modulating a overlap of UV and Visible light. ※ UV Vis. Closed-form Open-form Fluorescent

Relation between Inter-spot distance & FWHM Vis. position = 0 nm Vis. position= nm FWHM = 230 nm FWHM = 40 nm ※ FWHM : 半値全幅 Simulation Laser & Fluorescence Intensity Distribution parameter Φ : Reaction yield I : Intensity C : Concentration Laser DE1 PMMA cover glass

CCD camera Laser DE1 PMMA cover glass Imaging with CCD camera example Imaging Relation between Inter-spot distance & FWHM Vis. position = 0 nm Vis. position= nm FWHM = 230 nm FWHM = 40 nm ※ FWHM : 半値全幅

Imaging with CCD camera Guest DE1 Host PMMA ※ Position of visible light was shifted to left. 1μm Parameter Sample preparation Intensity ( UV & Vis.) Irradiated position (Vis.) Relation between Inter-spot distance & FWHM Fluorescent intensity FWHM 949 nm FWHM 334nm

DE1 example Imaging Stage Scanning LASER Stage scan imaging with APD single molecule cover glass Condition ･ a few dye in several micrometers square ･ only a dye in laser light PMMA

Stage scan imaging with APD A B C D E Measure photon number ※ Depended on the distribution of laser intensity Principle APD Laser A B C D E Distribution of laser intensity Objective Stage ・ Laser intensity is measured. ・ A fluorescence spot which is smaller than diffraction limit can be got. ・ The resolution is depended on the laser spot size and the step length of a stage. Optical setup Lens DM Pinhole Objective Stage

UV & Vis. completely overlaped. Vis. UV Stage scan imaging with APD UV & Vis. partly overlaped. Laser spot modelStage scan image Fluorescence Intensity Distribution FWHM 69.5 nm!! FWHM 222 nm With a confocal microscopy, smaller fluorescence spots were formed. Experimental condition Laser intensity : UV 3.01 nW, Vis 35.1 μW Sample : DE M, PMMA 1 w%

With doughnut beam Anisotropically reducing size Isotropically reducing size Excitation beam Objective Dumping beam Fluorescence spot Stage scan image Laser spot model

UVVisible Visible ( Doughtnut-shaped ) Visible ( Donut + Gaussian ) Effective Fluorescence Spot FWHM ～ 100 nm With doughnut beam Visible ( Gaussian-shaped )

FWHM 243 nm Experimental condition Laser intensity: UV nW, Vis 2.7 μW, Donut 8.28μW Sample: DE M, PMMA 1 w% Without Doughnut beam Vis. UV Laser spot model Stage scan image Fluorescence Intensity Distribution With doughnut-shaped beam, fluorescence spots isotropically became smaller. With Doughnut beam Doughnut With doughnut beam FWHM 183 nm!!

Summary ・ I explained about super-resolution microscopes such as STED, PALM, and STORM. ・ We formed fluorescence spots smaller than the diffraction limit size. ・ Fluorescence spots isotropically became smaller with the doughnut beam irradiation. UV Vis.

Future work ・ Fluorescence spot size are Isotropically reduced smaller than diffraction limit. ・ Nanoparticle of DE1 is made. ・ Biological tissues or structures of polymer are modified by DE1, and they are observed. T. Asahi, T.Sugiyama, H. Masuhara, Acco. of chem. res., 2008.