Observation of New Strong HighFrequency Feature in TwoPhoton Absorption Spectrum of GFP and its Desc

1
Observation of New Strong High-Frequency Feature
in Two-Photon Absorption Spectrum of GFP and its
Description within Three-Level Model with
Resonance EnhancementMikhail Drobizheva,
Nikolay S. Makarova, Aleksander Rebanea, Thomas
E. HughesbaDepartment of Physics, Montana State
University-Bozeman, USAbDepartment of Cell
Biology and Neuroscience, Montana State
University-Bozeman, USA
Analysis
Two-Photon Absorption Spectra
Abstract 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 moderately-strong 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 lt 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.
Two-level system (lowest transitions 1 and 2)
Wt-GFP (Clontech)
Perturbation theory gives 11
Three-level system (higher transitions 3,4,)
Structure of GFP. The chromophore (yellow color)
is formed within the b-barrel structure.

• Consequences
• Resonance enhancement of 2PA (see Table)
• Possibility to extract real frequencies of 2PA
  transitions

His-tagged CFP
Example peaks 3 and 6 of mRFP
mRFP and His-tagged CFP samples.The proteins are
emitting intense fluorescence under UV light
illumination.
References 1. C. Xu, W. Zipfel, J.B. Shear, R.M.
Williams, W.W. Webb, Proc. Nat. Acad. Sci., 93,
10763 (1996). 2. A.D. Xia, S. Wada, H. Tashiro,
W.H. Huang, Arch. Biochem. Biophys., 372, 280
(1999). 3. K. Winkler, J. Lindner, V.
Subramaniam, T.M. Jovin, P. Vohringer, Phys.
Chem. Chem. Phys., 4, 1072 (2002). 4. Yu.P.
Meshalkin, Opt. Spectr., 86, 53 (1999). 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). 6. R. Tsien, Ann. Rev. Biochem., 67,
509 (1998). 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). 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).
mRFP
Experimental Setup and Method of 2PA Measurement
Regenerative Tisapphire amplifier 150 fs, 1 kHz,
0.8 mJ, 770-800 nm
Structure of mRFP. The chromophore is shown with
red color. (Picture is taken from 5.)
n
OPA 100 fs, 0.1 mJ, 1.1-2.2 nm
Detection by 1-photon emission

• Conclusions
• 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.
• 2. In most cases, 2PA and 1PA transition
  frequencies coincide. This is explained by
  non-centrosymmetric structure of chromophores.
• 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.
• 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.

n
Sample
Spectrometer Triax 550
SHG
PM, CCD or IR detector
Reference detector
PC
Measured two-photon excited fluorescence
signal F2 A C s2 I 2 h (lreg), where A-
constant, C-concentration, s2 2PA cross
section, I - laser intensity, h (lreg)
differential quantum efficiency of fluorescence
at registration wavelength, For the reference
molecule (with known s2) F2,ref A Cref s2,
refI 2 href (lreg), h (lreg), href (lreg), C,
and Cref are obtained from independent one-photon
measurements. As a result,
Acknowledgments This work was supported by
Center for Bio-Inspired Nanomaterials (MSU,
Bozeman).