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.