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Proceedings of SPIE, the International Society for Optical Engineering/Proceedings of SPIE

Control of single-molecule fluorescence dynamics by stimulated emission depletion

Richard J. Marsh, Mark A. Osborne, Angus J. Bain

Published Nov 20, 2003• 1 citation
Publisher

Abstract

The feasibility of manipulating the single molecule absorption-emission cycle using picosecond stimulated emission depletion (STED) is investigated using a stochastic computer simulation. In the simulation the molecule is subjected to repeated excitation and depletion events using time delayed pairs of excitation (PUMP) and depletion (DUMP) pulses derived from a high repetition rate pulsed laser system. The model is used to demonstrate that a significant and even substantial reduction in the occurrence of 'dark states' in the fluorescence emission can be achieved using stimulated emission depletion. Variation in the PUMP-DUMP window allows precise control of the fluorescence yield with substantial increases in the fluorescence intensity observed at early PUMP-DUMP delays.

DOI
10.1117/12.505861

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The feasibility of manipulating the single molecule absorption-emission cycle using picosecond stimulated emission depletion (STED) is investigated using a stochastic computer simulation. In the simulation the molecule is subjected to repeated excitation and depletion events using time delayed pairs of excitation (PUMP) and depletion (DUMP) pulses derived from a high repetition rate pulsed laser system. The model is used to demonstrate that a significant and even substantial reduction in the occurrence of 'dark states' in the fluorescence emission can be achieved using stimulated emission depletion. Variation in the PUMP-DUMP window allows precise control of the fluorescence yield with substantial increases in the fluorescence intensity observed at early PUMP-DUMP delays.
The technique of stimulated emission depletion of fluorescence (STED) from a two photon excited molecular population is demonstrated in the S1 excited state of fluorescein in ethylene glycol and methanol. Two photon excitation (pump) is achieved using the partial output of a regeneratively amplified Ti:Sapphire laser in conjunction with an optical parametric amplifier whose tuneable output provides a synchronous depletion (dump) pulse. Time resolved fluorescence intensity and anisotropy measurements of the fluorescein emission are made using picosecond time-correlated single photon counting. Pump-dump time delayed fluorescence intensity measurements are used to characterise the response of the system and to provide additional data on saturation dynamics of the dump transition. Two photon STED is modelled using both approximate analytical techniques in the weak dump limit and by numerical solutions to the appropriate rate equations. The latter are used to fit experimental data from which it is possible to determine the cross-section for the stimulated transition and lifetime of the upper vibrational levels of the ground state.
The imaging technology of stimulated emission depletion (STED) utilizes the nonlinearity relationship between the fluorescence saturation and the excited state stimulated depletion. It implements three-dimensional (3D) imaging and breaks the diffraction barrier of far-field light microscopy by restricting fluorescent molecules at a sub-diffraction spot. In order to improve the resolution which attained by this technology, the computer simulation on temporal behavior of population probabilities of the sample was made in this paper, and the optimized parameters such as intensity, duration and delay time of the STED pulse were given.
This thesis presents the first application of the stimulated emission depletion (STED) technique to the field of single-molecule fluorescence spectroscopy. It is demonstrated that fluorescence quenching induced by STED is reversible and can be repeated a large number of cycles on a single molecule. Being ideal point-like probes, single molecules can therefore be used to characterize the resolution of STED microscopes. In a spectroscopic study, two simplifed models of the photophysical processes involved in STED are analyzed and applied to the experimental determination of the stimulated emission cross sections on a single-molecule level. In addition, the STED concept as applied in subdiffraction-resolution microscopy is transferred to fluorescence fluctuation spectroscopy. A successful implementation promises to expand the possibilities particularly of fluorescence correlation spectroscopy (FCS) which is already the most widely used fluctuation technique today but is restricted to concentrations on the nanomolar scale. Reducing the detection volume beyond the diffraction limit could render the micromolar range accessible and could thus open up new applications in the life sciences.
We study anisotropic Stimulated Emission Depletion (STED) from dye molecules, which are collectively ordered in a host liquid crystal. Due to the ordering of fluorescent emitters, the STED efficiency depends on the polarization of the depletion beam and time-delay of the STED pulse. The depletion efficiency is highest at lower temperatures in the highly ordered smectic-A phase and deteriorates in the higher temperature nematic and isotropic phases. We demonstrate by temporal tuning of STED that it is possible to generate an arbitrary sequence of nanosecond fluorescent pulses with variable width and variable delay. Our results show that the STED mechanism in principle allows for very fast (GHz) and efficient control of light by light, which could in the future be used for all-optical control of the flow of light in photonic microdevices based on liquid crystals. Using STED anisotropy and time-control, new modalities of STED imaging in liquid crystals could be developed.
As one of the most important realizations of stimulated emission depletion (STED) microscopy, the continuous-wave (CW) STED system, constructed by using CW lasers as the excitation and STED beams, has been investigated and developed for nearly a decade. However, a theoretical model of the suppression factors in CW STED has not been well established. In this investigation, the factors that affect the spatial resolution of a CW STED system are theoretically and numerically studied. The full-width-at-half-maximum (FWHM) of a CW STED with a doughnut-shaped STED beam is also reanalyzed. It is found that the suppression function is dominated by the ratio of the local STED and excitation beam intensities. In addition, the FWHM is highly sensitive to both the fluorescence rate (inverse of fluoresce lifetime) and the quenching rate, but insensitive to the rate of vibrational relaxation. For comparison, the suppression function in picosecond STED is only determined by the distribution of the STED beam intensity scaled with the saturation intensity. Our model is highly consistent with published experimental data for evaluating the spatial resolution. This investigation is important in guiding the development of new CW STED systems.