Blue-Shifted Green Fluorescent Protein Homologues Are Brighter than Enhanced Green Fluorescent Protein under Two-Photon Excitation

Abstract

Fluorescent proteins (FPs) are indispensable markers for two-photon imaging of live tissue, especially in the brains of small model organisms. The quantity of physiologically relevant data collected, however, is limited by heat-induced damage of the tissue due to the high intensities of the excitation laser. We seek to minimize this damage by developing FPs with improved brightness. Among FPs with the same chromophore structure, the spectral properties can vary widely due to differences in the local protein environment. Using a physical model that describes the spectra of FPs containing the anionic green FP (GFP) chromophore, we predict that those that are blue-shifted in one-photon absorption will have stronger peak two-photon absorption cross sections. Following this prediction, we present 12 blue-shifted GFP homologues and demonstrate that they are up to 2.5 times brighter than the commonly used enhanced GFP (EGFP).

Figure 1

Correlation between σ_2,max and the peak 1PA position. The fitted curve is based on eq 6 in the text. Error bars are at ±13%.

Figure 2

eqFP486, Rosmarinus, amFP486/K68M, and dTFP0.2 spectra: 2PA, 1PA, and emission. The top axis shows the transition wavelengths for 1PA and emission spectra, and the bottom axis shows the laser wavelengths for the 2PA spectra. The left axis is the 2PA cross section, and the right axis is the 2P brightness, which is scaled equally to show differences between FPs. The 2PA fit is displayed as a guide to the eye. The 1PA and the fluorescence emission spectra (excitation 450 nm) are normalized to the intensity of the 2PA peak.

Figure 3

Absolute 1P (top) and 2P (bottom) brightness of Rosmarinus, EGFP, and mNeonGreen plotted versus the excitation wavelength. ε, extinction coefficient; φ, fluorescence quantum yield; σ₂, 2PA cross section.