100nm Ke Xu, H. P. Babcock, X. Zhuang, Nature Methods. 2012, 9,
185188. Sub-diffraction limited point spread function achieved by
using photo-switchable fluorescence of diarylethene derivatives
Miyasaka Lab. Ikegami Takahiro
Slide 2
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
Slide 3
Do you know what won the Nobel Prize in Physics 2014? Blue LED
Isamu AkasakiHiroshi AmanoShuji Nakamura Photo: Nobelprize.org
http://www.nobelprize.org/nobel_prizes/physics/laureates/2014/
Slide 4
Eric BetzigStefan W. HellWilliam E. Moerner Super resolution
microscopy Then, do you know what won the Nobel Prize in Chemistry
2014? Photo: Nobelprize.org
http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2014/
Slide 5
Optical microscopy Observation target Biological tissue Polymer
film Glass SiO 2 Trajectory of dye in PolyHEA Arai Yuhei,
graduation thesis. 2014 3D trajectory of dye in PolyHEA Taga Yuhei,
thesis for master degree. 2014 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 /2 ( 200 nm) SEM ( 3 nm ) TEM ( 0.1 nm ) STM ( 0.1 nm
)
Slide 6
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
Slide 7
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 Objective Dye Fluorescence Imaging Wide field
microscopy Diffraction limit
Slide 8
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
Slide 9
Eric Betzig PALM ( PhotoActivated Localization Microscopy )
Super-Resolution microscopy Normal image Blinking image ( )
Localization ( ) Normal image PALM image STORM ( STochastic Optical
Reconstruction Microscopy ) Photoswitching E. Betzig, et al.,
Science, 313, 1642-1645 (2006).
Slide 10
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
Slide 11
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
Slide 12
diarylethene derivative (DE1) Fluorescent UV ( oc = 0.43)
Closed-form Open-form Vis. ( co = 1.610 -4 ) F =0.88
non-Fluorescent Super-resolution by employing photo-switchable
fluorescent molecule
Slide 13
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
Slide 14
Relation between Inter-spot distance & FWHM Vis. position =
0 nm Vis. position= - 550 nm FWHM = 230 nm FWHM = 40 nm FWHM :
Simulation Laser & Fluorescence Intensity Distribution
parameter : Reaction yield I : Intensity C : Concentration Laser
DE1 PMMA cover glass
Slide 15
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= - 550 nm FWHM = 230 nm FWHM =
40 nm FWHM :
Slide 16
Imaging with CCD camera Guest DE1 Host PMMA Position of visible
light was shifted to left. 1m Parameter Sample preparation
Intensity ( UV & Vis.) Irradiated position (Vis.) Relation
between Inter-spot distance & FWHM Fluorescent intensity FWHM
949 nm FWHM 334nm
Slide 17
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
Slide 18
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
Slide 19
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 W Sample : DE1 10 -11 M, PMMA 1 w%
FWHM 243 nm Experimental condition Laser intensity: UV 0.378
nW, Vis 2.7 W, Donut 8.28W Sample: DE1 10 -9 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!!
Slide 23
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.
Slide 24
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.