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Fluorescence Depletion by Stimulated Emission in Single-Molecule Spectroscopy

Lars Kastrup

Published Jan 1, 2004• 1 citation
Publisher

Abstract

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.

DOI
10.11588/heidok.00005033

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arXiv:1907.08604•ACS Photonics•Sep 9, 2019•17 citations
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.
As one of the most critical methods for optical super-resolved microscopy, stimulated emission depletion (STED) microscopy has been widely applied in biological and chemical fields, leading to the Nobel prize of 2014 in chemistry. In cold atomic systems, fast and high-resolution microscopy of individual atoms is crucial since it can provide direct information on the dynamics and correlations of the system. Here, we demonstrate nanosecond two-dimensional snapshots of a single trapped ion beyond the optical diffraction limit, by combining the main idea of STED with the quantum state transition control in cold atoms. We achieve a spatial resolution up to 175 nm and a time resolution up to 50 ns simultaneously using a NA=0.1 objective in the experiment, which is improved over ten times compared to direct fluorescence imaging. To show the potential of this method, we applied it to record the motion of the trapped ion and observe one cycle of the secular motion of the ion with a displacement detection sensitivity of 10 nm. Our method provides a powerful tool for probing particles' positions, momenta and correlations, as well as their dynamics in cold atomic systems.
The spatial resolution and fluorescence signal amplitude in stimulated emission depletion (STED) microscopy is limited by the photostability of available fluorophores. Here, we show that negatively-charged silicon vacancy (SiV) centers in diamond are promising fluorophores for STED microscopy, owing to their photostable, near-infrared emission and favorable photophysical properties. A home-built pulsed STED microscope was used to image shallow implanted SiV centers in bulk diamond at room temperature. The SiV stimulated emission cross section for 765-800 nm light is found to be (4.0 ± 0.3)×10^−17 cm^2, which is approximately 2-4 times larger than that of the negatively-charged diamond nitrogen vacancy center and approaches that of commonly-used organic dye molecules. We performed STED microscopy on isolated SiV centers and observed a lateral full-width-at-half- maximum spot size of 89 ± 2 nm, limited by the low available STED laser pulse energy (0.4 nJ). For a pulse energy of 5 nJ, the resolution is expected to be ∼20 nm. We show that the present microscope can resolve SiV centers separated by �� < 150 nm that cannot be resolved by confocal microscopy.
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.
Abstract In stimulated emission depletion (STED) microscopy, the resolution can be improved by directly depleting electrons from the target excitation state to the ground state at the doughnut area. In the study, an indirectly STED method for STED microscopy was proposed. A dynamic model based on state transition process for STED microscopy with triexciton fluorescence quantum dots was firstly developed. The depletion efficiencies of both direct and indirect ways were then theoretically investigated. Compared with direct depletion of quantum dots from their triexciton state to the ground state, indirect depletion of quantum dots from their biexciton state to the ground state was more efficient for triexciton depletion. This indirect depletion method offers a new route to reduce the depletion laser intensity for STED microscopy in order to achieve a nanometer imaging resolution.
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 spatial resolution and fluorescence signal amplitude in stimulated emission depletion (STED) microscopy is limited by the photostability of available fluorophores. Here, we show that negatively charged silicon vacancy (SiV) centers in diamond are promising fluorophores for STED microscopy, owing to their photostable, near-infrared emission and favorable photophysical properties. A home-built pulsed STED microscope was used to image shallow implanted SiV centers in bulk diamond at room temperature. The SiV stimulated emission cross section for 765–800 nm light is found to be (4.0 ± 0.3) × 10 –17 cm 2, which is approximately 2–4 times larger than that of the negatively charged diamond nitrogen vacancy center and approaches that of commonly used organic dye molecules. We performed STED microscopy on isolated SiV centers and observed a lateral full-width-at-half-maximum spot size of 89 ± 2 nm, limited by the low available STED laser pulse energy (0.4 nJ). For a pulse energy of 5 nJ, the resolution is expected to be ∼20 nm. We show that the present microscope can resolve SiV centers separated by ≲150 nm that cannot be resolved by confocal microscopy.