Miyasaka Lab. Ikegami Takahiro 100nm Ke Xu, H. P. Babcock, X. Zhuang, Nature Methods, 2012, 9,...
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Transcript of Miyasaka Lab. Ikegami Takahiro 100nm Ke Xu, H. P. Babcock, X. Zhuang, Nature Methods, 2012, 9,...
Miyasaka Lab.Ikegami Takahiro
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
I. BackgroundMicroscopyFluorescence MicroscopySuper-resolution Microscopy ( STED, PALM & STORM )
II. My workPrincipleSimulationExperience
III. Summary
IV. Future work
I. BackgroundMicroscopyFluorescence MicroscopySuper-resolution Microscopy ( STED, PALM & STORM )
II. My workPrincipleSimulationExperience
III. Summary
IV. Future work
Have you ever used a microscope?
20 μm
Shigeru Amemiya, Jidong Guo, Hui Xiong, Darrick A. Gross, Anal Bioanal Chem, 2006, 386, 458–471.
500 nm
5 μm0.5 μm
Scanning Electron Microscopy ( SEM )
Atomic Force Microscopy ( AFM )
Fluorescence Microscopy
Various Microscopy
L. Schermelleh, R. Heintzmann, H. Leonhard, THE JOURNAL OF CELL BIOLOGY, 2010, 190, 165-175.
S. Sharma, R. W. Johnson, T. A. Desai, Biosensors and Bioelectronics, 2004, 20, 227-239.
Dye
Sample example
Imaging
Shtengel et al., PNAS. 2009, 10,1073.
Fluorescence microscopy
Observation target
・ Biological tissue
・ Polymer film
CCD camera
LaserScanning
Laser
GlassSiO2
Trajectory of dye in PolyHEAArai Yuhei, graduation thesis, 2014
3D trajectory of dye in PolyHEATaga Yuhei, thesis for master degree, 2014
・ Internal observation ・ Contactless
・ Time resolution
Advantage of fluorescence microscopy
Spatial resolution
Fluorescence Microscopyλ/2 ( 200 nm )≧
Scanning Electron Microscopy ( SEM )Atomic Force Microscopy ( AFM )( 0.1 nm )≧
<<
0.5 μm5 μm
L. Schermelleh, R. Heintzmann, H. Leonhard, THE JOURNAL OF CELL BIOLOGY, 2010, 190, 165-175.
Resolution of fluorescence microscopy
Low Resolution
High Resolution
Large LASER Spot
Small LASER Spot
Point Spread Function( PSF ) Fluorescence PSF
Objective
smaller thandiffraction limitSuper-ResolutionMicroscopy
STED ( Stimulated Emission depletion )Super-Resolution Microscopy
h(v)
v
Δν
FWHM
Dye : RhodamineB λSTED = 600 nm : STED beam wavelength λexc = 490 nm : Ecitation beam wavelength N.A.= 1.4 : Numerical aperture of objective
FWHM of effective PSF50 nm
S. W. Hell, J. Wichmann, OPICS LETTERS. 1994, 19, 11.
STED beam
Excitation beam
PALM ( PhotoActivated Localization Microscopy )& STORM ( Stochastic Optical Reconstruction Microscopy )
Super-Resolution Microscopy
CCD
camera
B. Huang, W. Wang, M. Bates, X. Zhuang, Science, 2008, 319, 810-813.
Low Resolution
Fluorescence PSF
Localization
(A) Normal
PALM & STORM
Normal (B) STORM
I. BackgroundMicroscopyFluorescence MicroscopySuper-resolution Microscopy ( STED, PALM & STORM )
II. My workPrincipleSimulationExperience
III. Summary
IV. Future work
diarylethene derivative (DE1)
1.6
1.2
0.8
0.4
0.0
Ab
s.
700600500400300wavelength / nm
0.6
0.4
0.2
0.0
Flu
o. In
ten
sity
Open-ring
Closed-ring
Fluo.
Fluorescent
UV(Φoc= 0.43)
Closed-formOpen-form
S S CH2OHHOH2C
FF
F F FF
Et
EtO O O O
Vis. (Φco= 1.6×10-4)
ΦF =0.88non-Fluorescent
S S CH2OHHOH2C
FF
F F FF
Et
EtO O O O
Super-resolution by using 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-formOpen-form
Fluorescent
1.0x1016
0.8
0.6
0.4
0.2
0.0
EF
S p
hoto
n nu
mbe
r
-400 -200 0 200 400position / nm
EFS
1.2x1014
1.0
0.8
0.6
0.4
0.2
0.0
EF
S p
hoto
n nu
mbe
r
-400 -200 0 200 400position / nm
EFS
250
200
150
100
50
FW
HM
/ n
m
6004002000Inter-spot dist. / nm
Relation between Inter-spot distance & FWHM
Vis. position = 0 nm
Vis. position= - 550 nm
FWHM = 230 nm
FWHM = 40 nm
※FWHM : 半値全幅
Simulation Laser & Fluorescence Intensity Distribution
parameterΦ : Ring reaction yieldI : IntensityC : Concentration
Laser
Dyes
PMMAcover glass
600
550
500
450
400
FW
HM
/ n
m
600400200Inter-spot dist. / nm
260
250
240
230 290280270260250240230220210200190180
250
200
150
100
50
Int.
coun
t
141210864Position / µm
Fluorescent intensity
EFS by Simulation1.2x10
14
1.0
0.8
0.6
0.4
0.2
0.0
EF
S p
ho
ton n
um
ber
-400 -200 0 200 400position / nm
EFS
Experimental resultGuestDE1
HostPMMA
※ Position of visible light was shifted to left.
1μm
ParameterSample preparationIntensity ( UV & Vis.)Irradiated position (Vis.)
Relation between Inter-spot distance & FWHM
Stage scan imaging with APD
3.0x1016
2.5
2.0
1.5
1.0
0.5
0.0-400 -200 0 200 400position
ph
oto
n n
um
ber
A
B
C
D
E
Measure photon number
※ Depended on the distribution of laser intensity
single molecule
PMMA cover glass
Condition
Principle
APD
Laser
A B C D E
Distribution of laser intensity
Objective
Stage
・ a few dye in several micrometers square
・ only a dye in laser light
・ 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
Lens
DM
Pinhole
Objective
Stage
3020
100
3020100
3020
100
3020100
1200
1000
800
600
400
200
Inte
nsity
12008004000position / nm
FWHM = 772 nm
UV & Vis. completely overlaped.
300nm
3000
2000
1000
Inte
nsity
12008004000position / nm
FWHM = 241 nm
UV
Vis.
Vis.
UV
300nm
Stage scan imaging with APD
UV & Vis. partly overlaped.
Laser spot model Stage scan imaging Distribution of photon number
Summary
・ I explained about super-resolution microscopes such as STED, STORM, and PALM.
・ We observed that the smaller UV & Visible light overlap was, the smaller a fluorescence spot size became.
UV
Vis.
UV beam Visible donuts beam EFS
Future work
・ Smaller spots than diffraction limit are made.
・ The visible donuts beam is used, and isotropic fluorescent spots is made.
・ Biological tissues or structures of polymer are modified by DE1, and they are observed.