Stimulated Emission Depletion Pulse Research Articles

Two-photon STED nanoscopy realizing 100-nm spatial resolution utilizing high-peak-power sub-nanosecond 655-nm pulses

We developed two-photon excitation stimulated emission depletion (STED) nanoscopy using high-peak-power sub-nanosecond 655-nm pulses. The STED pulse exhibited ideal optical properties and sufficient pulse energy to realize a 70-nm spatial resolution in the compact setup with electrically controllable components. For biological applications, we screened suitable fluorescent dyes or proteins and realized the sub-100 nm spatial resolution imaging of presynaptic protein clusters in fixed primary cultured neurons without severe photobleaching. We expect this method to enable visualization of ultrastructures and the cluster dynamics of biomolecules representing physiological functions in living cells and tissue.

Photobleaching in STED nanoscopy and its dependence on the photon flux applied for reversible silencing of the fluorophore

In STED (stimulated emission depletion) nanoscopy, the resolution and signal are limited by the fluorophore de-excitation efficiency and photobleaching. Here, we investigated their dependence on the pulse duration and power of the applied STED light for the popular 750 nm wavelength. In experiments with red- and orange-emitting dyes, the pulse duration was varied from the sub-picosecond range up to continuous-wave conditions, with average powers up to 200 mW at 80 MHz repetition rate, i.e. peak powers up to 1 kW and pulse energies up to 2.5 nJ. We demonstrate the dependence of bleaching on pulse duration, which dictates the optimal parameters of how to deliver the photons required for transient fluorophore silencing. Measurements with the dye ATTO647N reveal that the bleaching of excited molecules scales with peak power with a single effective order ~1.4. This motivates peak power reduction while maintaining the number of STED-light photons, in line with the superior resolution commonly achieved for nanosecond STED pulses. Other dyes (ATTO590, STAR580, STAR635P) exhibit two distinctive bleaching regimes for constant pulse energy, one with strong dependence on peak power, one nearly independent. We interpret the results within a photobleaching model that guides quantitative predictions of resolution and bleaching.

Precise background subtraction in stimulated emission double depletion nanoscopy.

Low-resolution background in stimulated emission depletion (STED) nanoscopy can arise from incomplete depletion or re-excitation by the STED beam. We have recently introduced stimulated emission double depletion (STEDD), a technique to efficiently suppress this background. In STEDD, the conventional, doughnut-shaped STED pulse, which depletes excited fluorophores outside the center of the focal region, is followed by a second Gaussian STED pulse, which specifically depletes the central region. The background is removed by calculating a weighted difference of photon events collected before and after the second STED pulse. Here, we present a simple, yet powerful, method to determine the weight factor, which depends on the fluorescence decay, from a direct analysis of the acquired data. We vary the weight factor to identify its optimal value as the one for which the weight of high-frequency components in the spectrum of the acquired STEDD image is maximized. This strategy is also applicable to other differential approaches for background suppression in imaging.

Background suppression in fluorescence nanoscopy with stimulated emission double depletion

Stimulated emission depletion (STED) fluorescence nanoscopy is a powerful super-resolution imaging technique based on the confinement of fluorescence emission to the central subregion of an observation volume through de-excitation of fluorophores in the periphery via stimulated emission. Here, we introduce stimulated emission double depletion (STEDD) as a method to selectively remove artificial background intensity. In this approach, a first, conventional STED pulse is followed by a second, delayed Gaussian STED pulse that specifically depletes the central region, thus leaving only background. Thanks to time-resolved detection we can remove this background intensity voxel by voxel by taking the weighted difference of photons collected before and after the second STED pulse. STEDD thus yields background-suppressed super-resolved images as well as STED-based fluorescence correlation spectroscopy data. Furthermore, the proposed method is also beneficial when considering lower-power, less redshifted depletion pulses. Stimulated emission double depletion addresses the issue of background in super-resolution imaging and quantitative microscopy through implementation of a two-pulse sequence in a modified stimulated emission depletion set-up. The measured background intensity is removed from each voxel in the acquired images thanks to time-resolved detection.

Time alignment optimization of pulsed STED microscopy

Stimulated emission depletion (STED) microscopy implemented with both pulsed excitation and STED beams (pulsed STED) is especially suitable in biological superresolution imaging as it reduces photobleaching rates and provides better resolution. Proper time alignment between the excitation and STED pulse can improve resolution. To get the optimal time alignment the fluorescence depletion effect was analysed under different time alignments in theory first. Then, the relationship between the time alignment and the position of two moveable mirrors used for changing the excitation optical path length was established. Finally, the depletion experiments were performed under different time alignments. By both simulation and experimentation, the optimal time alignment was got which was about 210ps when the widths of excitation and STED pulses were both 120ps. STED image with 60nm resolution was achieved based on the optimal time alignment. The method provides a reliable way to optimize the time alignment of pulsed STED microscopy.

Nanosecond control and optical pulse shaping by stimulated emission depletion in a liquid crystal.

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.

Fluorescence Lifetime Imaging Microscopy with Subdiffraction-Limited Resolution

In this study, we demonstrate subdiffraction-limited fluorescence lifetime imaging microscopy (FLIM) by engineering the point spread function (PSF) with stimulated emission depletion (STED). The enhanced spatial resolution allows the number of fluorophores in the PSF to reduce in turn the associated heterogeneity in lifetime analysis. Moreover, time gating can be performed using time-correlated single photon counting (TCSPC) to carefully select detected fluorescence photons so as to optimize the spatial resolution and the signal-to-noise ratio in STED imaging. This flexibility also supports the removal of the unintended effects of lifetime reduction that is caused by STED pulses.

Performance improvement in nanoparticle-assisted stimulated-emission-depletion nanoscopy

We revisit stimulated-emission-depletion (STED) nanoscopy theory for the case when metal nanoparticles are used to improve the nanoscope’s performance. We show that the improved performance can be estimated in a simple way with no need for heavy computations. We then systematically study the dependence of the improved performance on the field and decay rate enhancements, as well as on the STED pulse and time-gating durations.

Strategies to maximize the performance of a STED microscope

In stimulated emission depletion (STED) microscopy, the spatial resolution scales as the inverse square root of the STED beam's intensity. However, to fully exploit the maximum effective resolution achievable for a given STED beam's intensity, several experimental precautions have to be considered. We focus our attention on the temporal alignment between the excitation and STED pulses and the polarization state of the STED beam. We present a simple theoretical framework that help to explain their influence on the performance of a STED microscope and we validate the results by imaging calibration and biological samples with a custom made STED architecture based on a supercontinuum laser source. We also highlight the advantages of using time gating detection in terms of temporal alignment.

Optimizing Parameters for Wll STED Imaging

Stimulated emission depletion (STED) nanoscopy is one of the most important recent innovations in biological imaging. This technology enables noninvasive study of biological specimens with nanometer resolution.The most powerful versions of STED nanoscopes rely on pairs of synchronized pulsed laser beams. The excitation beam is focused into the sample, producing an ordinary diffraction limited spot of excited molecules. The red shifted and doughnut shaped depletion beam follows immediately the excitation, quenching the fluorescence to the ground state by stimulated emission.Thus the diffraction barrier can be overcome narrowing the fluorescence spot by saturating the stimulated emission transition. Although the STED concept can be formulated in a simple equation[1][2], the realization of such a nanoscope needs several experimental precautions to exploit the maximum resolution achievable. We will report on detailed experimental studies focusing on the performance of the microscope in dependence on the arrival times of the corresponding excitation and STED pulses as well as the influence of the polarization of the STED beam to the quality of the donut. The latter has a direct influence to the final image quality and is of great importance.Moreover we will show the effects and the enhancement that can be achieved by applying time gating detection.[1] Hell, S. W. 2004. Strategy for far-field optical imaging and writing without diffraction limit. Physics Letters A 326:140-145.[2] Wildanger, D., E. Rittweger, L. Kastrup, and S. W. Hell. 2008. STED microscopy with a supercontinuum laser source. Optics Express 16:9614-9621.

Time-gating improves the spatial resolution of STED microscopy

Stimulated-emission depletion (STED) microscopy improves image resolution by encoding additional spatial information in a second stimulated-decay channel with a spatially-varying strength. Here we demonstrate that spatial information is also encoded in the fluorophore lifetime and that this information can be used to improve the spatial resolution of STED microscopy. By solving a kinetic model for emission in the presence of a time-varying STED pulse, we derive the effective resolution as a function of fluorophore lifetime and pulse duration. We find that the best resolution for a given pulse power is achieved with a pulse of infinitesimally short duration; however, the maximum resolution can be restored for pulses of finite duration by time-gating the fluorescence signal. In parallel, we consider time-gating in the presence of a continuous-wave (CW) STED beam and find that time-gating produces theoretically unbounded resolution with finite laser power. In both cases, the cost of this improved resolution is a reduction in the brightness of the final image. We conclude by discussing situations in which time-gated STED microscopy (T-STED) may provide improved microscope performance beyond an increase in resolution.

Absolute Optical Cross Section of Individual Fluorescent Molecules

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