Evaluation of Microscopic Inhomogeneity in Solids Using Single

Molecules as Nanometer-sized Probes

Yoko MiyamotoMiyasaka Lab.

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ContentsI. Introduction

Single molecule detection: History and methods Single molecules as nano-probes in evaluating microscopic properties of materials

II. Single molecule imaging and its related techniquesWide-field microscopySingle molecule tracking

III. Evaluation of polymeric materials with SMIPrevious worksMy current research topic: Development of a multicolor detection system

IV. Summary

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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

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Each guest molecule is in different environment.

The result of ensemble measurement

Emission spectrum of a single molecule

Different electronic state → Position-dependent spectrum and lifetime

Single molecule detection

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The motions of individual guest molecules strongly depend on microenvironment.

Tracking the motion of guests can provide information on microscopic properties of materials:

・ Nanostructures・ Mobility of guests (Free volume in polymer, Host-Guest Interaction, etc. ) ・ Spatial heterogeneity ....

Single molecules as nano-probes in evaluating microscopic properties of materials

2D, but very high spatial resolutionMany molecules can be observed at a time.

Wide-field Microscopy

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Single molecule tracking

bgsY

y

sX

xIyxf })(

2

1)(

2

1exp{),( 2121

0

sin)(cos)( 001 yyxxx

cos)(sin)( 001 yyxxy

Actual precision of tracking ~ 5-10 nm in our experimental set up

X0 = 274.03 +/- 0.0339 pixel

Y0 = 148.17 +/- 0.0351 pixel

Previous works:Visualization of nanoscale heterogeneity in pol ｙ HEA films

Sample preparation using spin-casting method

Cover glass

Well cleaned cover glass

single molecule

Poly(2-hydroxyethylacrylate) [ polyHEA ]

N,N’-bis(2,6-dimethylphenyl) perylene-3,4,9,10-tetracarboxylic diimide

2.45 nm

1.22 nm

Trajectory (on glass substrate)

Time course of step size

Exp. Time: 500 ms

Nanoscale heterogeneity in the sample ?

Previous works:Visualization of nanoscale heterogeneity in pol ｙ HEA films

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Exposure time ~ 30.5 msExposure time ~ 500 ms

Previous works:Visualization of nanoscale heterogeneity in pol ｙ HEA films

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Exposure time: 30.5ms 　Silicon substrate

50 nm

100 nm

Previous works:Visualization of nanoscale heterogeneity in pol ｙ HEA films

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Motivation of my workSMS can reveal the inhomogeneity of polymers.

HOWEVER,we cannot obtain the direct information on the relation between

the motions of the guest molecule and the host polymer.

dual-color imaging

Dye labeled polymer chain

Guest dye

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Dual-color single molecule tracking system

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① 　励起側光学系

② 　検出側光学系

Evaluation of system

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PMMA 　 glass transition temperature (Tg) 82℃ 　→　 glassy state at room temperature 　

BPPBIN,N’-3,3-Dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4 ： 9,10-tetracarboxylic diimide

OON

O

OO

O

N

OO

6μm

BPTDIN,N’-3,3-Dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-terrylene-3,4:11,12-tetracarboxidiimide

O

N

O

O

N

O

N

O

O

O

N

O

800 750 700 650 600 550 500 Wave Length [nm]

吸収スペクトル 蛍光スペクトル

6μm

Evaluation of system

BPTDI BPPBI

15

-20

-10

0

10

20

x /

nm

806040200

number of frames6

5

4

3

2

1

0

Occ

uren

ce

-20 -10 0 10 20

Position[nm]

-20

-10

0

10

20

x /

nm

140120100806040200

number of frames8

6

4

2

0

Occ

uren

ce

20151050-5-10

Position[nm]

±20nm ±30nm

Poly (2-hydroxyethyl acrylate) (PolyHEA)

glass transition temperature (Tg) 17℃ 　→　 molecules show liquidity

Diffusion motions of guest molecules

BPPBIBPTDIDistribution of diffusion coefficient 16

14

12

10

8

6

4

2

0Num

ber

of O

ccur

renc

e

80x10-3604020Diffusion coefficient / m2s-1

14

12

10

8

6

4

2

0Num

ber

of O

ccur

renc

e

80x10-3604020

Diffusion coefficient / m2s-1

382

380

378

376

374

y / p

ixe

l

302826242220x / pixel

245

240

235

230

225

y / p

ixel

500495490485x / pixel

Summary• I have shown the significance of measuring single molecules

in evaluating microscopic properties of materials .

• Wide-field microscopy permitted the tracking of translational motions of individual molecules.

• To more clearly and precisely elucidate the relation between the motion of the guest molecule and that of the host polymer, we have developed a dual-color imaging system.

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Contents• Introduction -History of Single Molecule Spectroscopy -Difference between ensemble and single-molecule measurement -Information obtained only by SMS

• Measurement - Single-molecule 　 detection - Wide-field Microscope and Confocal Microscope - Single-molecule tracking

• My work

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Glass substrate

Polymer film

Spin-coat method

Cover glass

Well cleaned cover glass

single molecule

Wide-field microscopy

Difference between ensemble and single-molecule measurements

An emission spectrum of a single molecule

The result of ensemble measurement

“Single molecule”

Ensemble

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~ 10 nm

PDI in PMMA (Tg ~ 355K, Mw 15000 )

Apparent diffusion coefficient < 4.0 × 10-5 m2s-1

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Loose area

fast diffusion Packed area

slow diffusion

Free volume distribution, permittivity, density, viscosity, elasticity, etc.

Information obtained only by SMS

Inhomogeneity in nanometer scale

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N,N’ -bis(2,6-dimethylphenyl)- perylene-3,4,9,10-tetracarboxylic diimide (PDI)

PA08 (Nissan Chemical inc. )(Anti-reflection layer in lithography) 　　　　　　　　　

(n 1.5)≒

Ethyl lactate solution ofPA08 （ 0.36 wt ％）Irgacure 184 （ 0.022 wt ％）

Irgacure184 （ Ciba specialty chemicals ）　　　　　　　　　

O

OR

OR

CH2OR

n

Photo-curable polymeric material

2000 rpm

UV light (2.6 w/cm2)

Jpn. J. Appl. Phys. 2007, 46, p7279.

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Trajectories(UV irradiation (325 nm) duration ~ 1 sec)

70% of PDI molecules: D dropped to < 0.002 μm2s-1.

The rest 30% could diffuse as freely as in the non-reacted sample.

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Wide-field microscopy Confocal microscopy

3-dimentianal resolutionSmall backgroundHigh temporal resolution

2-dimentianal resolutionMany molecules can be Observed at a time.High spatial resolution

Wide-field Microscope and Confocal Microscope

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