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Chinese Physics B

Influence of fluorescence time characteristics on the spatial resolution of CW-stimulated emission depletion microscopy

Haiyun Qin, Wei Zhao, Chen Zhang, Yong Liu, Guiren Wang, Kaige Wang

Published Mar 1, 2018• 2 citations
Publisher

Abstract

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.

DOI
10.1088/1674-1056/27/3/037803

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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.
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.
Abstract Nonlinear depletion of fluorescence states by stimulated emission constitutes the basis of stimulated emission depletion (STED) microscopy. Despite significant efforts over the past decade, achieving super-resolution at low saturation intensities by STED remains a major technical challenge. By harnessing the surface quenching effect in NaGdF 4 :Yb/Tm nanocrystals, we report here high-efficiency emission depletion through surface migration. Using a dual-beam, continuous-wave laser manipulation scheme (975-nm excitation and 730-nm de-excitation), we achieved an emission depletion efficiency of over 95% and a low saturation intensity of 18.3 kW cm −2 . Emission depletion by surface migration through gadolinium sublattices enables super-resolution imaging with sub-20 nm lateral resolution. Our approach circumvents the fundamental limitation of high-intensity STED microscopy, providing autofluorescence-free, re-excitation-background-free imaging with a saturation intensity over three orders of magnitude lower than conventional fluorophores. We also demonstrated super-resolution imaging of actin filaments in Hela cells labeled with 8-nm nanoparticles. Combined with the highly photostable lanthanide luminescence, surface-migration emission depletion (SMED) could provide a powerful mechanism for low-power, super-resolution imaging or biological tracking as well as super-resolved optical sensing/writing and lithography.
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.
Stimulated emission depletion (STED) is the key optical technology enabling super-resolution microscopy below the diffraction limit. Here, we demonstrate that modulation of STED in the time domain, combined with properly designed lock-in detection, can radically enhance the contrast of fluorescent images of strongly autofluorescent biotissues. In our experiments, the temporally modulated STED technique, implemented with low-intensity continuous-wave laser sources, is shown to provide an efficient all-optical suppression of a broadband fluorescent background, allowing the contrast of fluorescent images of mammal brain tissues tagged with nitrogen-vacancy diamond to be increased by five orders of magnitude.
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.
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.