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