1. Energy transitions: the mitigators of absorption and fluorescence in FPs are the π electrons present in the chromophore. Upon excitation these electrons move into an excited, or higher energy state. The movement from the initial or ground state (S0) to the excited state (S1 or Sn) is called an electronic transition whose frequency is proportional to the energy difference between the two states. Higher-energy transitions (S0→Sn) occur with absorption at shorter (more energetic) wavelengths and lower-energy transitions (S0→S1) occur with absorption at longer (less energetic) wavelengths.
2. Cross section (σ2), GM, and two-photon brightness: The cross section (σ2) for two-photon absorption measured in GM (1 GM = 10-50 cm4 s) is the probability in which the fluorophore can simultaneously absorb a pair of photons per second at the unit average intensity of the incident light (1 photon/s * cm2) . To obtain two-photon brightness (σ2×ϕ), the cross section (σ2) is multiplied by the fluorophore's quantum yield of fluorescence (ϕ).
3. Stokes shift: difference between excitation maximum and fluorescence emission maximum.
4. Kasha's rule states that fluorescence is independent of the mode or wavelength of excitation and thus exciting different transitions of a molecule will yield the same fluorescence emission spectrum. When exciting a higher-energy (shorter wavelength) transition, fluorescence does not occur from that state. Instead some of the energy is lost in the form of heat and emission occurs from the same lower-energy state as is the case when the lowest-energy state is excited directly .
5. Vibronic transitions: In addition to a change of electronic states upon optical excitation, a vibrational state of a molecule can also change. This implies that while in the ground (electronic and vibrational) state atoms are mostly occupying their near-equilibrium positions. In a vibrational excited state these atoms start to oscillate (acquire more kinetic energy) along certain normal coordinates of a molecule. If both electronic and vibrational states change upon excitation, this transition corresponds to the molecule acquiring a quantum of vibrational energy in the electronically excited state that sum to form a vibronic transition. This transition always occurs higher in energy (at shorter wavelength) than the pure electronic transition in the absorption spectrum .
Protein expression and purification
The DNA encoding mKalama1, mAmetrine, the EBFP series, G1, G3, and the mTFP series were positioned in the pBAD plasmid (Invitrogen) such that a fusion protein is produced with an N-terminal 6xHis tag. Transformed E.coli (Top10, Invitrogen) were grown overnight in 4 mL LB + ampicillin. 200 mL of 0.2% arabinose, LB + ampicillin media was then inoculated with 1 mL of the overnight culture and allowed to grow at 34°C for 20 hours. The variants ECFP and mCerulean were positioned in the pRSET plasmid. For expression and purification protocol see . Cells were lysed (Bugbuster, Novagen) and affinity purified with Ni-NTA His Bind Resin (Novagen). The proteins were eluted in imidazol buffer pH 8.
Fluorescence lifetime, fluorescence quantum yield, extinction coefficient and concentration measurements
These methods have been previously described in detail in . Briefly, fluorescence quantum yields for ECFP, mCerulean, G1, G3, mWasabi, and the mTFP series were measured relative to fluorescein. The fluorescence quantum yields for mKalama1, mAmetrine, and the EBFP series were measured relative to 9,10-Diphenylanthracene in cyclohexane. Extinction coefficients per chromophore and concentrations of mature chromophore were measured using the Strickler-Berg relation as described in . See Table 2 for the data relevant to determining extinction coefficient using Strickler-Berg method.
Two-photon absorption spectra and cross section measurements
Absolute 2PA cross sections were measured using relative fluorescence technique with coumarin 485 in methanol used as a standard for ECFP, mCerulean, mAmetrine, mKalama, and the EBFP series and fluorescein as a standard for G1, G3, mWasabi, and the mTFP series. For a description of the methods see .
Finding the optimal fluorescent proteins for TPLSM depends upon knowing the detailed structure of the 2PA spectra over a wide range of excitation wavelengths. Here we are continuing our work to use a common set of standards, and an all-optical approach of determining chromophore concentration, to quantitatively compare the cross sections of a broad series of FPs . This approach is important because previous data obtained in different laboratories have varied [1, 3, 4, 17, 18].
To test the two-photon dual labeling approach with an imaging application HEK 293 cells were transiently transfected using lipofectamine 2000 (Invitrogen), with either mKalama1 or tagRFP in a CMV expression plasmid. The cells were subsequently mixed and re-plated so that they could be imaged simultaneously with one excitation wavelength (780 nm) and detected with two different PMTs (band pass filtered at 425-500 nm for mKalama1 and 550-575 nm for tagRFP). Excitation at 780 nm is most likely suboptimal, but this was as close to 760 nm that the Ti:sapphire laser used in the confocal microscope could be tuned to. To analyze the cross talk between the blue and red channels, the intensities for both the red and blue signals were plotted pixel by pixel.