New all-optical method for measuring molecular permanent dipole moment difference using two-photon absorption spectroscopy

Stark effect, in combination with spectral hole burning and single-molecule spectroscopy, has been a fruitful technique to study permanent electric dipole moment of molecules in condensed phase. However, because measuring Stark shifts relies on external fields and narrow line- or hole-widths, the applicability of this method at ambient conditions required by most biological systems has remained limited. Here we demonstrate a new all-optical method for measuring the molecular dipole moment difference between ground and excited states using two-photon absorption (2PA) spectroscopy. We show that the value and orientation of the static dipole moment difference can be determined from the corresponding absolute 2PA cross-section. We use this new method to determine for the first time the strength of local electric field Eloc=0.1–1.0×108 V/cm inside beta-barrel of Fruit series of red fluorescent proteins. Because our method does not rely on external field and is applicable in liquid solutions, it is well suited for the study of biological systems.