Absolute Optical Cross Section of Individual Fluorescent Molecules

Abstract

Besides having revealed transitions masked in the bulk, optical spectroscopy of single molecules has enabled the observation of elementary molecular quantum events, such as the absorption and emission of a photon. Single photon transitions are quantified by a cross section that can be regarded as the photon capture area of a molecule. Therefore, absolute optical cross sections can readily be measured in the bulk from the decrease (by absorption) or the gain (by stimulated emission) of the number of photons in a beam. Unfortunately, this method is not applicable to individual molecules, because the resulting change in intensity is negligible. If the fluorescence quantum efficiency Qem is known, the molecular absorption cross section can be calculated from the fluorescence emission. However, this method re-introduces the hallmark of ensemble averaging, because Qem is known only from the bulk. As a result, the absolute photon capture area of a specific molecule has remained elusive. Here we report on the measurement of absolute cross sections of stimulated emission at room temperature. No a priori information about the individual molecule is needed. In addition, we demonstrate the instant control of a molecule.s excited state by light and thus, as a matter of fact, also the viability of pump–probe experiments with individual molecules at room temperature. Molecular optical absorption usually takes place from the vibrationally relaxed ground state S0 to a hot Franck–Condon state S1* of the first excited singlet state (Figure 1a). After vibrational relaxation, which occurs on a (sub)picosecond time scale, organic fluorophores emit within a few nanoseconds with a probability given by the fluorescence quantum yield. Alternatively, the S1!S0 transition can be enforced by light, that is by stimulated emission. Stimulated emission has been employed to probe the temporal evolution of the excited state in a pump–probe fashion, as well as to alter the fluorescence lifetime and anisotropy of organic fluorophores in solution. Furthermore, stimulated emission depletion (STED) of the excited state has been introduced in fluorescence microscopy to break the diffraction resolution barrier, but has recently also been used to measure cross sections in the bulk. We attained single molecule sensitivity with a scanning confocal setup fed by two synchronized pulse trains: a first pulse at a wavelength of l= 565 nm for excitation was followed by a red-shifted pulse at l= 778 nm for STED, both adapted to the spectrum (Figure 1b) of the xanthene dye JA 26 (1). Efficient STED demands a STED pulse duration t