Transcript Uncovering the Mechanisms of Strong Two

Observation of New Strong High-Frequency Feature in Two-Photon Absorption Spectrum of GFP and
its Description within Three-Level Model with Resonance Enhancement
Mikhail Drobizheva, Nikolay S. Makarova, Aleksander Rebanea, Thomas E. Hughesb
aDepartment
of Physics, Montana State University-Bozeman, USA
bDepartment of Cell Biology and Neuroscience, Montana State University-Bozeman, USA
Analysis
Two-Photon Absorption Spectra
Two-level system (lowest transitions: 1 and 2)
Wt-GFP (Clontech)
250
200
300
350
400
450
500
S1
550
2PA spectrum (absolute) of wt-GFP, pH 7.4,
measured in our laboratory
2PA spectrum (relative) of wt-GFP published
in[Xu, 1996]
Excitation spectrum of wt-GFP, pH 7.4, lreg = 510 nm
s2, GM
150
0
500
600
700
800
900
1000
1100
2PA laser wavelength, nm
Structure of GFP.
The chromophore
(yellow color) is
formed within the bbarrel structure.
His-tagged CFP
1PA wavelength, nm
250
300
350
450
h
500
2PA (set 1)
2PA (set 2)
Absorption
Excitation (lreg = 520nm)
Detection by 1-photon emission
mi0
mRFP and His-tagged
CFP samples.The
proteins are emitting
intense fluorescence
under UV light
illumination.
100
0
500
600
700
800
900
1000
10
28000
2PA
Absorption
Two-gaussian fit of 2PA
First Gaussian
peak
Second Gaussian
Excitation
30000
32000
peak # 6 (?)
#3
mi 0 m fi
2
 i 0 /  12
g (2 )
5
0
12000
13000
14000
Laser frequency, cm
15000
16000
-1
References
1. C. Xu, W. Zipfel, J.B. Shear, R.M. Williams, W.W. Webb, Proc. Nat. Acad. Sci., 93, 10763 (1996).
3. K. Winkler, J. Lindner, V. Subramaniam, T.M. Jovin, P. Vohringer, Phys. Chem. Chem. Phys., 4, 1072 (2002).
mRFP
4. Yu.P. Meshalkin, Opt. Spectr., 86, 53 (1999).
1PA wavelength, nm
400
26000
2. A.D. Xia, S. Wada, H. Tashiro, W.H. Huang, Arch. Biochem. Biophys., 372, 280 (1999).
2PA laser wavelength, nm
300
24000
-1
2
200
500
5. R.E. Campbell, O. Tour, A.E. Palmer, P.A. Steinbach, G.S. baird, D.A. Zacharias, R.Y. Tsien, PNAS, 99, 7877
(2002).
600
250
s2, GM
Example: peaks 3 and 6 of mRFP
2
6. R. Tsien, Ann. Rev. Biochem., 67, 509 (1998).
2PA (set 1)
2PA (set 2)
2PA (set 3)
Excitation
Absorption
7. M. Chattoraj, B.A. King, G.U. Bublitz, S.G. Boxer, PNAS, 93, 8362 (1996).
8. R. Nifosi, Y. Luo, J. Phys. Chem. B, 111, 505 (2007).
9. P.S. Tsai, B. Friedman, A.I. Ifarraguerri, B.D. Thompson, V. Lev-Ram, C.B. Schaffer, Q. Xiong, R.Y. Tsien,
J.A. Squier, D. Kleinfeld, Neuron, 39, 27 (2003).
Structure of mRFP.
The chromophore is
shown with red color.
(Picture is taken from
[5].)
100
50
10. W.R. Zipfel, R.M. Williams, W.W. Webb, Nat. Biotech., 21, 1369 (2003).
11. M. Drobizhev, F. Meng, A. Rebane, Y. Stepanenko, E. Nickel, C.W. Spangler, J. Phys. Chem. B, 110, 9802
(2006).
0
Specie
Peak,
form
l2PAm
nm
GFP
1 (B)
958
960
796
800
800
1 (A)
F2 = A C s2 I h lreg,
2
3(A?)
Histagged
CFP
800
1000
1200
F2,ref = A Cref s2, refI href (lreg),
[1]
[1]
[2]
15
3.8
120
7.6
160
[1]
[1]
[2]
Conclusions:
2
865
~850 (CFP)
840 (CFP)
637
<550
560 (2l1PA)
130
75 (CFP) [9]
733
760 calc
~720-760 (2l1PA)
6
<700
670 calc
685 (2l1PA)
|Dm|
D
1.0x104 [6]
8.2
6.8
15.8
<20
2.5x104 [6]
[3]
<80
3
em
M-1cm-1
>200
912
3
4
mRFP
<570
520-560 (2l1PA)
>515 (pump-probe)
s2m
GM
1
2
F2 Cref href (lreg )
s 2,ref
F2,ref Ch (lreg )
mfi
S0
s 2 ( )  C
Measured two-photon excited fluorescence signal:
s2 
Gf
300
Reference detector
h (lreg), href (lreg), C, and Cref are obtained from independent
one-photon measurements. As a result,
Sf
Transition frequency, cm
400
400
s 2m
n  10

.
4.83 1015 f 2 e10 m
Consequences:
1. Resonance enhancement of 2PA (see Table)
2. Possibility to extract real frequencies of 2PA
transitions
Si
1. Three fluorescent proteins, wt-GFP, His-tagged CFP and mRFP demonstrate
strong two-photon absorption (100 – 400 GM) in a wide spectral range, from
550 to 1000 nm.
SHG
For the reference molecule (with known s2):
2
Three-level system (higher transitions: 3,4,…)
h
Sample
where A constant, C-concentration, s2 – 2PA cross section, I laser intensity, h (lreg) – differential quantum efficiency of
fluorescence at registration wavelength,
Knowings 2 , ε10 , and ν10 , one can find :
Dμ10
2PA laser wavelength, nm
PC
m0
S0
600
PM, CCD or
IR detector
2
 3n 2 
f   2   Onsager's local field factor;
 2n  1 
n  refractiveindex
h
100
OPA
100 fs,
0.1 mJ,
1.1-2.2 nm
Spectrometer
Triax 550
Δμ10  μ1  μ0 , g (2ν )  lineshapefunction;
m10
150
n
2
h
Absorption spectrum of wt-GFP, pH 7.4
2PA cross section of tyrosine [Meshalkin, 1999]
2PA cross section of tryptophan [Meshalkin, 1999]
200
n
2
Dμ10 ε10 (2 )
2 (2 )4 f 4
2
2
15 f
σ2 
μ10 Δμ10 g (2ν )  4.83 10
(1),
2
5 (hnc)
n
ν10
50
Experimental Setup and
Method of 2PA Measurement
Regenerative Ti:sapphire
amplifier 150 fs, 1 kHz,
0.8 mJ, 770-800 nm
Perturbation theory gives [11]:
m1
1PA wavelength, nm
s2, GM
The knowledge of two-photon absorption (2PA)
properties of green fluorescent protein (GFP) and its
mutant variants is indispensable for smart design of
GFP-related probes for 2PA microscopy, including
genetically-encoded sensors of action potentials in
neurons. We study 2PA spectra of green fluorescent
protein in a wide spectral range from 550 to 1000 nm.
The 2PA spectrum of wt-GFP shows a moderatelystrong peak at 800 nm, which has been observed
previously by Webb and co-authors [1]. Our measured
peak cross section value, s2 = 120 ± 20 GM, correlates
rather well with the result of Xia, et al [2]. Most
intriguing is that we observe the onset of a new, rather
strong 2PA band at shorter excitation wavelengths,
llaser < 700 nm. This region was not explored before.
The maximum cross section, which we were able to
measure with our femtosecond fluorescence technique
is s2 ~ 200 GM at 580 nm. This rather strong 2PA can
be explained by the quantum-mechanical resonance
enhancement effect, occurring when the laser
frequency approaches one of the allowed one-photon
transitions. In our case this is the first one-photon
transition of the neutral A form (around 400 nm). The
new 2PA peak, observed here for GFP for the first
time, is inherently related to the higher electronic
transition(s) of the chromophore. This is corroborated
with a recent finding by Winkler et al. [3], that the A
form of wt-GFP possesses an excited Sn level, lying
somewhere lower than 38,800 cm-1, corresponding to
515 nm of laser wavelength in case of degenerate 2PA.
We also measure 2PA spectra of the red- and cyan FP
mutants and interpret them in terms of three-level
model.
s2(10/-1)
Abstract
[9]
[10]
[8]
[8]
11
3.2x104 [6]
150
>440
120
5.7x103 [5]
>210
7.3x103 [5]
50 calc [8]
~100
[8]
14
[7]
[7]
2. In most cases, 2PA and 1PA transition frequencies coincide. This is explained
by non-centrosymmetric structure of chromophores.
3. 2PA strength of the lowest S0 - S1 transition of GFP can be quantitatively
described with two-level model expression, which involves both the squares
of transition dipole moment and of permanent dipole moment change.
4. For higher-lying transitions, the 2PA cross section is resonantly enhanced
and can qualitatively be described within three-level model. These
transitions are, perhaps, due to higher (Sn) energy states of chromophores.
In the case of GFP these states can be tentatively assigned to the states,
mostly localized on tyrosine residue of the chromophore.
Acknowledgments: This work was supported by Center
for Bio-Inspired Nanomaterials (MSU, Bozeman).