Іnstitute of physics nasu the development of new organic probes for two-photon induced fluorescence...

Іnstitute of PhysicsNASU

The development of new organic probes for two-photon induced fluorescence microscopy application

Mykhailo V. Bondar and Olga V. Przhonska

Institute of Physics, Prospect Nauki, 46, Kiev-28, 03028, Ukraine

Іnstitute of PhysicsNASU

Outline

Stimulated and spontaneous transitions in organic molecules

Linear spectral characterization

Two-photon absorption spectra

Transient absorption spectroscopy and superfluorescence

Stimulated emission depletion properties

Conclusions

Two-photon absorbing organic molecules with efficient stimulated emission depletion (STED) for bioimaging

2

Іnstitute of PhysicsNASU

Stimulated and spontaneous transitions in organic molecule

Abs Fluor

S0

S1

SnESA

hv, 2hvStimulatedEmission

NR

NR

01() 3.8*10-21 () [M-1cm-1] ~ 10-16 cm2; 10() ~ 10-16 cm2; 2PA() ~ 10-48 cm4s

Simplified molecular electronic model

3

K.D. Belfield, et al., J. Phys. Chem. B, 2009, 113, 1701.

Іnstitute of PhysicsNASU

S1

S0

/1I

S2

S3

S4

1hv quenchpump

Ifl0 ~ Ip; (1-Ifl/Ifl

0) ~ Iq

pump quench

I(r,t) = I0exp[-(r2/r02 + t2/ 2)]

Delay 10 ps

dN1(r,t)/dt = N001I(r,t) - N1[1/ + 10I(r,t)]

N0(r,t) + N1(r,t) = NC

ò N1(r,t)dt = N1(r,p)

dN1(r,t)/dt = N1(r,t)2PEI2q(r,t)

(1 - Ifl/Ifl0) = 2PE{q(/8)1/2[rq

2/(rp2+rq

2)]}Iq2

Ifl0 ~ N1(r,p)rdrd

Ifl ~ N1q(r,p)rdrd

N1q(r,p) = N1(r,p)[1- 2PE(/2)1/2qI2

q(r)]

J.R. Lakowicz, et al., J. Photochem. Photobiol., 1994, 60, 546.

2hv quench

ESA

Fluorescence quenching method

4

Іnstitute of PhysicsNASU

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Fl

Norm

aliz

ed A

bso

rban

ce

Wavelength, nm

Abs#38 CHF

Norm

aliz

ed F

luore

scen

ce

pumpprobe

I(r,t) = I0exp[-(r2/r02 + t2/ 2)]

Delay 10 ps

0 1 2 3 40.00

0.05

0.10

0.15

0.20

0.25#38 CHF, cuv. 1mmexc. =400 nmquench. =1400 nm100 fs1 kHz

(IF

0-IF

)/IF

0

E2p, J

0.0 0.5 1.0 1.5

0.00

0.05

0.10

0.15

0.20

#38 CHF, cuv. 1mmexc. =400 nmquench. =750 nm100 fs1 kHz

(IF

0-IF

)/IF

0

Ep, J

y=A+B*xA=-0.02097B=0.13038R=0.9937

Fluorescence quenching method

5

K.D. Belfield, et al., J. Phys. Chem. B, 2009, 113, 1701.

Іnstitute of PhysicsNASU

MIRA 900-F

Ve

rdi-V

10

Legend EliteOPA

OperaSolo

Z

SM

F SF

DL

WP

800 nm, 1kHz

DC400 nm

P

SF

240–20000 nm

F

SampleOceanOptics

800 nm, 76 MHz, 200 fs

P ~ 100 fs

PD

PD

S

Experimental setup for pump-probe and single beam experiments

PD

600 800 10000.0

0.5

1.0#38 CHF1mm Cuv.1280 nm100 fs

Eq = 4 mkJ

Eq = 2.6 mkJ

Flu

ore

scence inte

nsity, a.u

.

Wavelength, nm

Eq=1.6 mkJ

6

K.D. Belfield, et al., ChemPhysChem, 2011, 12, 2755.

Іnstitute of PhysicsNASU

Linear photophysical and photochemical properties

C2H5

C2H5

N

O

NC CN

NC2H5

C2H5

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

3'-6'2'1'CHXTOLCHFTHFODCBDCM

Norm

alize

d A

bsorb

ance

Wavelength, nm

ACNCHXTHFTOLDCMCHFODCB

1-7

Norm

alize

d F

luore

scence

FPh 410-7

0 2 4 6 8 1010

100

1000CHX - 0.20 nsTOL - 0.95 nsCHF - 2.4 nsODCB - 1.9 nsTHF - 1.4 nsDCM - 0.63 ns (75%) 2.6 ns (25%)

Inte

nsi

ty, a

.u.

t, ns

t = tR QY; A = 1 / tR

IRF ~ 80 pslexc = 440 nm

300 400 500 600

0.0

0.1

0.2

0.3

Anis

otr

opy

Wavelength, nm

CHX

THF

pTHF

ODCB

CHF

TOL

DCM

Abs-ODCB

Abs-CHX

7

0.0 0.1 0.2 0.3

4000

5000

6000

7000

8000

Sto

kes S

hift, c

m-1

f

38 in:CHXTOLCHFODCBTHFDCMACN

constahc

fv

2

3

2

dEN

c

A

)()10ln()(1500

0101

2

01

Diode laser

(532 nm)

f = 5 cmsample

P0 = 85 mW/cm2

K.D. Belfield, et al., ChemPhysChem, 2011, 12, 2755.

Іnstitute of PhysicsNASU

Two-photon absorption spectra

400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

1.2800 1000 1200 1400 1600

10

100

1000

TOL DCM

Anis. pTHF

2PA

1PA

Norm

alize

d A

bsorb

ance

1PA Wavelength, nm

2P

A, G

M

2PA Wavelength, nm

N+NC6H13

C6H13C6H13

C6H13HO

HO

OH

OH

O

O- C6H13

C6H13

N

S

NC CN

N

C6H13

C6H13

400 500 600 7000.0

0.2

0.4

0.6

0.8

1.0

800 1000 1200 1400

200

400

600 TOL DCM

Anis. pTHF2PA

1PA

Norm

alize

d A

bsorb

ance

Anis

otr

opy

1PA Wavelength, nm

2P

A, G

M

2PA Wavelength, nm

SQ FD

8

Іnstitute of PhysicsNASU

Transient absorption spectroscopy

9

N+N

C6H13

C6H13C6H13

C6H13

HO

HO

OH

OH

O

O-

S1

S0

I

S2

S3

S4

Pump

650 nm

ESAProbe450 750 nm

I

/1TOL 3.0 ns 3.3 ns

–TOL

DCM

S1

S0

I

S2

S3

S4

Pump

650 nm

ESAProbe450 750 nm

I

/1TOL 3.0 ns 3.3 ns

–TOL

DCM

-0.5 0.0 0.5 1.0 1.5 2.0-0.3

-0.2

-0.1

0.0610 nm probe

630 nm probe

D

Delay, ps

640 nm probe

-0.5 0.0 0.5 1.0 1.5 2.0-0.3

-0.2

-0.1

0.0

660 nm probe

630 nm probe

D

Delay, ps

640 nm probe

-0.5 0.0 0.5 1.0 1.5 2.0 2.5

0.00

0.05

0.10

510 nm probe

490 nm probe

D

Delay, ps

470 nm probe

#44 TOL

#44 TOL #44 DCM

SQ

K.D. Belfield, et al., ChemPhysChem, 2013, 14, 1-14.

Іnstitute of PhysicsNASU

Transient absorption spectroscopy

10

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 520 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 540 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 680 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 720 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 740 nm

0 2 4 6 8 10

-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 840 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 880 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 920 nm

0 2 4 6 8 10-0,04

-0,02

0,00

0,02

0,04

D

Delay, ps

pr = 960 nm

FD in СН2Cl2

K.D. Belfield et al., J. Phys. Chem. C, 2013, 117, 11941.

C6H13

C6H13

N

S

NC CN

N

C6H13

C6H13

FD

400 600 800 1000 12000.0

0.2

0.4

0.6

0.8

1.0

1.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

CHXTOLTHFDCM

FluorescenceAbsACNCHXTHFTOLDCM

Norm

aliz

ed A

bso

rban

ce

Wavelength, nm

Norm

aliz

ed F

luore

scen

ce

Іnstitute of PhysicsNASU

Superfluorescence properties

11

650 700 7500,0

3,0x103

6,0x103

9,0x103

650 700 750

0,0

0,2

0,4

0,6

0,8

1,0

1,2

EP

1- 1.5 J2- 2.8 J3- 4.0 J

Inte

nsi

ty, c

ou

nts

/sec

Wavelength, nm

1

2

3a

No

rma

lize

d f

luo

res

ce

nc

e

4

650 700 7500,0

3,0x105

6,0x105

9,0x105

1,2x106 650 700 750

0,0

0,2

0,4

0,6

0,8

1,0

1,2

3

2

1

EP

1- 0.125 J2- 0.4 J3- 0.6 J

4

c

Inte

nsi

ty, c

ou

nts

/sec

Wavelength, nm

x103

No

rma

lize

d f

luo

res

ce

nc

e

0 15 30 45 600

1x106

2x106

3x106

4x106

1 2 30,0

5,0x102

1,0x103

1,5x103

2,0x103

Inte

nsi

ty, c

ou

nts

/sec

Pulse Energy, J

b

Inte

nsi

ty, c

ou

nts

/sec

Pulse Energy, J

0,0 1,5 3,0 4,50

1x106

2x106

3x106

4x106

0,0 0,2 0,4 0,60,0

5,0x105

1,0x106

1,5x106

Inte

nsi

ty, c

ount

s/se

c

Pulse Energy, J

dIn

ten

sity

, co

un

ts/s

ec

Pulse Energy, J

С 7.5·10-5 М (a, b)

С 1.8·10-3М (c, d)

Pump 650 nm, 100 fs, 1 кHz

K.D. Belfield, et al., ChemPhysChem, 2013, 14, 1-14.

N+N

C6H13

C6H13C6H13

C6H13

HO

HO

OH

OH

O

O-

Іnstitute of PhysicsNASU

Two-photon absorption and STED spectra

400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

1.2800 1000 1200 1400 1600

0

500

1000

1500

2000

2500

No

rma

lize

d A

bso

rba

nc

e, F

luo

res

cen

ce

1PA wavelength, nm

2

31'

1

2PA wavelength, nm

5

2P

E G

M,

10 ,

a.u

.

41hvSTED

2PA2hvSTED

(A. Penzkofer, et al., Chem. Phys., 1990, 142, 123.)

)(8

)(0

2

4

Eqcn RF

em

12

K.D. Belfield, et al., ChemPhysChem, 2011, 12, 2755.

0.5 1.0 1.50.00

0.05

0.10

0.15

0.20

830 nm

750 nm

710 nm

1 - I F

/ I

F0

Eq / J

a

20 40 60 800.00

0.01

0.02

0.03

0.04

b

1400 nm

1360 nm

1 - I F

/ I

F0

Eq2 / J

1280 nm

Іnstitute of PhysicsNASU

0 2 4 6 80.0

0.1

0.2

0.3

1 - F / F

0

Eq, J

580 nm

520 nm

640 nm

600 nm

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

1.2

0

2

4

6

8

Norm

aliz

ed A

bso

rbance

and F

luore

scence

Wavelength, nm

10*1

017

, cm

2

C2H5C2H5

N

S

H3C

H3C

O

# 40

#40 in Toluene

1hv quench Abs Em Em

CHFTOL

Pq

qp

qqFF E

rrchII

)(

)(2/1

20

20

100

K.D. Belfield et al., ChemPhysChem, 2012, 13, 3481.

0 10 20 300.00

0.02

0.04

0.06

0.08

1400 nm

1360 nm1240 nm

1 - F / F

0

E2, J

1320 nm

300 400 500 600 700 8000.0

0.2

0.4

0.6

0.8

1.0

1.2600 800 1000 1200 1400 1600

0

150

300

450

600

750

Norm

aliz

ed A

bso

rban

cean

d F

luore

scen

ce

1PA Wavelength, nm 2P

E, G

M

2PA and 2PE Wavength, nm

x)/8(/1 2/150 FF II

2hv quench

Abs Em

2PA 2PE

22

02

02

022

22

)2(

)()(x P

qqpq

q

qPEq Errrch

Two-photon absorption and STED spectra

13

Іnstitute of PhysicsNASU

Bioimaging application of new fluorene derivative

Images of HCT 116 cells incubated with 1. (A) DIC; (B) One photon fluorescence image; (C) 3D reconstruction from overlaid two-photon fluorescence images (Ex: 940 nm; Power: 120 mW; Em. short-pass filter 800 nm).

14K.D. Belfield, et al., ChemPhysChem, 2011, 12, 2755. X. Wang, et al., Biomed. Opt. Express, 2010, 1, 453.

Images of Hela cells incubated with SNP-DBF-NHFA (20 μM, 2 h). (b) 3D reconstruction from overlaid two-photon fluorescence images (Ex: 740 nm; Power: 30 mW; Em. short-pass filter 690 nm) 10 μm grid, (c) 2P-FLIM image (Ex: 740 nm; Power: 30 mW).

Іnstitute of PhysicsNASU

Fluorescence quenching methodology is a promising

technique for STED investigations

Two-photon stimulated emission spectra were obtained in

a broad spectral range and high STED cross sections were

observed for fluorene molecule

New fluorene derivatives with large two-photon

absorption,

efficient STED and high photostability has a good

potential

for application in fluorescence bioimaging

Conclusions

15