Stimulated Emission Depletion Beam Research Articles

Focus image scanning microscopy for sharp and gentle super-resolved microscopy

To date, the feasibility of super-resolution microscopy for imaging live and thick samples is still limited. Stimulated emission depletion (STED) microscopy requires high-intensity illumination to achieve sub-diffraction resolution, potentially introducing photodamage to live specimens. Moreover, the out-of-focus background may degrade the signal stemming from the focal plane. Here, we propose a new method to mitigate these limitations without drawbacks. First, we enhance a STED microscope with a detector array, enabling image scanning microscopy (ISM). Therefore, we implement STED-ISM, a method that exploits the working principle of ISM to reduce the depletion intensity and achieve a target resolution. Later, we develop Focus-ISM, a strategy to improve the optical sectioning and remove the background of any ISM-based imaging technique, with or without a STED beam. The proposed approach requires minimal architectural changes to a conventional microscope but provides substantial advantages for live and thick sample imaging.

MINSTED nanoscopy enters the Ångström localization range

Super-resolution techniques have achieved localization precisions in the nanometer regime. Here we report all-optical, room temperature localization of fluorophores with precision in the Ångström range. We built on the concept of MINSTED nanoscopy where precision is increased by encircling the fluorophore with the low-intensity central region of a stimulated emission depletion (STED) donut beam while constantly increasing the absolute donut power. By blue-shifting the STED beam and separating fluorophores by on/off switching, individual fluorophores bound to a DNA strand are localized with σ = 4.7 Å, corresponding to a fraction of the fluorophore size, with only 2,000 detected photons. MINSTED fluorescence nanoscopy with single-digit nanometer resolution is exemplified by imaging nuclear pore complexes and the distribution of nuclear lamin in mammalian cells labeled by transient DNA hybridization. Because our experiments yield a localization precision σ = 2.3 Å, estimated for 10,000 detected photons, we anticipate that MINSTED will open up new areas of application in the study of macromolecular complexes in cells.

Lifetime based axial contrast enable simple 3D-STED imaging

Stimulated Emission Depletion (STED) microscopy increase spatial image resolution by laterally sharpening the illumination profile of the confocal microscope. However, it remains compromised in axial resolution. To improve axial STED resolution, constructive interference of the STED depletion beam must be formed surrounding the focal plane to turn off the fluorophores beyond the focal plane. For isotropic 3D-STED resolution, this axial STED interference pattern must be overlayed with the doughnut STED beam at nanometer accuracy. Such optical configurations can be challenging in alignment. In this current work, we introduced a straightforward lifetime based axial contrast in STED microscope by imaging the samples on an ITO coated glass coverslip. The STED laser generates surface plasmon resonance on the ITO surface that enhanced the metal induced energy transfer MIET effect on the ITO surface. The enhanced MIET effect established a lifetime gradient with ∼20% dynamic range that extend for mor than 400 nm from the ITO surface. The axial contrast based on the lifetime gradient was directly used for 3D-STED imaging of tubulin fibers inside COS-7 cells, where the vertical displacement of single tubulin fiber was revealed. Lifetime gating could be applied to further improve lateral spatial resolution. Considering that most common implementation of STED microscopes uses pulsed lasers and timing electronics, there is no optical modification of the microscope is required in the current 3D-STED approach.

Shaping the illumination beams for STED imaging through highly scattering media

Stimulated emission depletion (STED) microscopy has been widely applied in single-cell and single-molecular studies benefiting from its ability of super-resolution imaging. However, it is challenging to perform STED imaging in highly scattering media, such as biological tissues, due to high scattering, which destroys the structured illumination beams. Here, we present a technique to produce the desired illumination beams beyond high scattering for STED microscopy. A dual-wavelength transmission matrix method is employed for shaping the excitation beam and the STED beam simultaneously, achieving the temporal and spatial overlap of the two beams behind a scattering medium. Experimentally, the overlapped foci were constructed by using a digital micromirror device (DMD). The extinction ratio of the STED focus reaches −23.8 dB, which compares favorably to the oft-quoted desirable value for STED applications. In particular, raster scanning of overlapped foci and the switching of the STED focus were demonstrated by adopting the fast switching ability of the DMD. Construction of the illumination beams beyond scattering will benefit STED microscopy in deep tissues.

Aberration correction in stimulated emission depletion microscopy to increase imaging depth in living brain tissue.

.Significance: Stimulated emission depletion (STED) microscopy enables nanoscale imaging of live samples, but it requires a specific spatial beam shaping that is highly sensitive to optical aberrations, limiting its depth penetration. Therefore, there is a need for methods to reduce optical aberrations and improve the spatial resolution of STED microscopy inside thick biological tissue.Aim: The aim of our work was to develop and validate a method based on adaptive optics to achieve an a priori correction of spherical aberrations as a function of imaging depth.Approach: We first measured the aberrations in a phantom sample of gold and fluorescent nanoparticles suspended in an agarose gel with a refractive index closely matching living brain tissue. We then used a spatial light modulator to apply corrective phase shifts and validate this calibration approach by imaging neurons in living brain slices.Results: After quantifying the spatial resolution in depth in phantom samples, we demonstrated that the corrections can substantially increase image quality in living brain slices. Specifically, we could measure structures as small as 80 nm at a depth of inside the biological tissue and obtain a 60% signal increase after correction.Conclusion: We propose a simple and robust approach to calibrate and compensate the distortions of the STED beam profile introduced by spherical aberrations with increasing imaging depth and demonstrated that this method offers significant improvements in microscopy performance for nanoscale cellular imaging in live tissue.

ISM-Assisted Tomographic STED Microscopy Allows for Sample-Gentle Superresolution Imaging

Fluorescence nanoscopy allows to non-invasively resolve three-dimensional cell structures beyond the diffraction limit. One of these high resolution imaging techniques is stimulated emission depletion (STED) microscopy. However, the practically achievable resolution of a STED microscope is often limited by photobleaching. One method to overcome this limitation is tomographic STED (tomoSTED) microscopy. In tomoSTED microscopy, excited fluorophores in the sample plane are depleted by a 0-π phase step modified and subsequently focused STED beam, which leads to an effective narrowing of the fluorescence-allowed area in a single direction. A full highly resolved spatial information of the object in two dimensions is then obtained by rotating the depletion pattern. The number of required pattern rotations depends on the ratio of the resolutions in the depleted and non-depleted directions. Since the resolution per pattern orientation is only increased along a single direction, imaging along the other direction is still diffraction-limited. Therefore, the resolution along this direction can be increased by utilizing the concept of image scanning microscopy (ISM). ISM allows to improve the resolution of a scanning microscope by a factor of square root of two. By combining tomoSTED with ISM, the number of required pattern orientations can therefore be reduced leading to a lower overall acquisition time which translates directly into a lower light dose. On simulated as well as on experimental data, we show that approximately a factor of square root of two lower number of orientations are required to achieve the same resolution and image quality as in tomoSTED microscopy without ISM assistance. Under typical recording conditions, this corresponds to a six-fold reduction of the required light dose as compared to a classical STED microscope.

Cumulative effects of photobleaching in volumetric STED imaging—artefacts and possible benefits

In stimulated emission depletion (STED) imaging, the excitation and depletion laser beams extend well beyond the focal plane in the imaged sample. We investigated how photobleaching resulting from this irradiation can affect STED images, by acquiring 3D images of fluorescent polystyrene beads using a 2D STED microscope, and applying different Z pixel sizes, scanning speeds, resulting in different laser light doses. While higher STED beam irradiances can increase the spatial resolution, they can also significantly increase photobleaching and thereby reduce signal-to-background levels. In 2D STED imaging, based on a single scan within the focal plane, scan parameters can often be selected to avoid photobleaching effects. Upon 3D optical sectioning experiments however, using the same scan parameters, additional cumulative effects of photobleaching may appear, due to the extension of the excitation and depletion laser beams beyond the focal planes being scanned. Apart from a reduction in signal-to-background levels, such photobleaching can lead to an apparent shift of the axial localization of the objects in the images, but also to an increased resolution in the axial dimension. These findings, confirmed by simulations based on a simplified model for photobleaching, suggests some caution in volumetric STED imaging experiments, but also a possibility for enhanced axial resolution in such experiments.

Achieving high-resolution of 21 nm for STED nanoscopy assisted by CdSe@ZnS quantum dots

Although quantum dots (QDs) show strong photoluminescence and high photostability, they are not widely used as probes for stimulated emission depletion (STED) nanoscopy because the excitation of the doughnut-shaped STED beam always generates background noise (the so-called “halo”) that hampers superresolution microscopy. In this study, we attempted to use commercially available CdSe@ZnS QDs with green emission (QD526) as a probe in STED nanoscopy. A lateral resolution of 21.0 nm for a single QD was obtained when using a 488-nm excitation laser and a 592-nm depletion laser. The high resolution achieved was mainly attributed to the fact that no halo was generated around the STED spots. This was also confirmed through spectral analysis in that no spontaneous fluorescence was detected when the QDs were irradiated by the 592-nm laser. The results indicated that the halo could be avoided effectively in QD-assisted STED nanoscopy by tuning the wavelength of the emission peak of the QDs and the depletion laser. This study provides insights into how to easily avoid the halo in QD-assisted STED nanoscopy and how to improve the nanoscopy resolution.

Efficient two-photon excitation stimulated emission depletion nanoscope exploiting spatiotemporal information.

.Stimulated emission depletion (STED) microscopy is a powerful bioimaging technique that theoretically provides molecular spatial resolution while preserving the most important assets of fluorescence microscopy. When combined with two-photon excitation (2PE) microscopy (2PE-STED), subdiffraction resolution may be achieved for thick biological samples. The most straightforward implementation of 2PE-STED microscopy entails introduction of an STED beam operating in continuous wave (CW) into a conventional Ti:sapphire-based 2PE microscope (2PE CW-STED). In this implementation, resolution enhancement is typically achieved using time-gated detection schemes, often resulting in drastic signal-to-noise/-background ratio (SNR/SBR) reductions. Herein, we employ a pixel-by-pixel phasor approach to discard fluorescence photons lacking super-resolution information to enhance image SNR/SBR in 2PE CW-STED microscopy. We compare this separation of photons by lifetime tuning approach against other postprocessing algorithms and combine it with image deconvolution to further optimize image quality.

A Fiber-Coupled Stimulated Emission Depletion Microscope for Bend-Insensitive Through-Fiber Imaging

We present results for a new type of fiber-coupled stimulated emission depletion (STED) microscope which uses a single fiber to transport STED and excitation light, as well as collect the fluorescence signal. Our method utilizes two higher-order eigenmodes of polarization maintaining (PM) fiber to generate the doughnut-shaped STED beam. The modes are excited with separate beams that share no temporal coherence, yielding output that is independent of fiber bending. We measured the resolution using 45 nm fluorescent beads and found a median bead image size of 116 nm. This resolution does not change as function of fiber bending radius, demonstrating robust operation. We report, for the first time, STED images of fixed biological samples collected in the epi-direction through fiber. Our microscope design shows promise for future use in super-resolution micro-endoscopes and in vivo neural imaging in awake and freely-behaving animals.

A special three-layer step-index fiber for building compact STED systems

Up to now, most of stimulated-emission-depletion (STED) systems were lens-based bulky systems. Exchanging some spatial light paths with optical fiber components will make the systems more flexible and will benefit various fields. A big problem to achieve this goal is that the STED beam generated by the traditional method of bulky systems cannot be maintained in an optical fiber due to its birefringence. In this article, we will introduce a type of special optical fiber. With the special fiber, a dark hollow beam with doughnut-shaped focal spot and a concentric beam with Gaussian-shaped focal spot can be generated at the same time. Parameters of a sample and a compact STED system based on it are demonstrated.

Supercritical angle fluorescence for enhanced axial sectioning in STED microscopy.

We demonstrate subwavelength axial sectioning on biological samples with a stimulated emission depletion (STED) microscope combined with supercritical angle fluorescence (SAF) detection. SAF imaging is a powerful technique for imaging the membrane of the cell based on the direct exploitation of the fluorophore emission properties. Indeed, only when fluorophores are close to the interface can their evanescent near-field emission become propagative and be detected beyond the critical angle. Therefore, filtering out the SAF emission from the undercritical angle fluorescence (UAF) emission in the back focal plane of a high-NA objective lens permits nanometer axial sectioning of fluorescent emitters close to the coverslip. When combined with STED microscopy, a straightforward gain in axial resolution can be reached without any alteration of the STED beam path. Indeed, STED-SAF implementation only requires a modification in the detection path of the STED microscope and thus could be widely implemented.

Exploiting the tunability of stimulated emission depletion microscopy for super-resolution imaging of nuclear structures

Imaging of nuclear structures within intact eukaryotic nuclei is imperative to understand the effect of chromatin folding on genome function. Recent developments of super-resolution fluorescence microscopy techniques combine high specificity, sensitivity, and less-invasive sample preparation procedures with the sub-diffraction spatial resolution required to image chromatin at the nanoscale. Here, we present a method to enhance the spatial resolution of a stimulated-emission depletion (STED) microscope based only on the modulation of the STED intensity during the acquisition of a STED image. This modulation induces spatially encoded variations of the fluorescence emission that can be visualized in the phasor plot and used to improve and quantify the effective spatial resolution of the STED image. We show that the method can be used to remove direct excitation by the STED beam and perform dual color imaging. We apply this method to the visualization of transcription and replication foci within intact nuclei of eukaryotic cells.

Three dimensional live-cell STED microscopy at increased depth using a water immersion objective.

Modern fluorescence superresolution microscopes are capable of imaging living cells on the nanometer scale. One of those techniques is stimulated emission depletion (STED) which increases the microscope's resolution many times in the lateral and the axial directions. To achieve these high resolutions not only close to the coverslip but also at greater depths, the choice of objective becomes crucial. Oil immersion objectives have frequently been used for STED imaging since their high numerical aperture (NA) leads to high spatial resolutions. But during live-cell imaging, especially at great penetration depths, these objectives have a distinct disadvantage. The refractive index mismatch between the immersion oil and the usually aqueous embedding media of living specimens results in unwanted spherical aberrations. These aberrations distort the point spread functions (PSFs). Notably, during z- and 3D-STED imaging, the resolution increase along the optical axis is majorly hampered if at all possible. To overcome this limitation, we here use a water immersion objective in combination with a spatial light modulator for z-STED measurements of living samples at great depths. This compact design allows for switching between objectives without having to adapt the STED beam path and enables on the fly alterations of the STED PSF to correct for aberrations. Furthermore, we derive the influence of the NA on the axial STED resolution theoretically and experimentally. We show under live-cell imaging conditions that a water immersion objective leads to far superior results than an oil immersion objective at penetration depths of 5-180 μm.

Influence of fluorescence time characteristics on the spatial resolution of CW-stimulated emission depletion microscopy**Project supported by the National Natural Science Foundation of China (Grant Nos. 11672229 and 61378083), International Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2011DFA12220), Major Research Plan of the National Natural Science Foundation

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.

Light needles in scattering media using self-reconstructing beams and the STED principle

Stimulated emission depletion (STED) microscopy generates super-resolved images of single cells by point-wise depletion of fluorescence around a small focal volume. Scanned light sheet microscopy, on the other side, generates images line-wise by scanning a weakly focused laser beam through thousands of scattering cells. Here we address the question of whether fluorescence from an excitation beam can be depleted by a STED beam over tens of micrometers while propagating through scattering material. Therefore, we use two self-reconstructing Bessel beams in continuous wave mode with a different angular momentum for fluorescence excitation and depletion along a distance of 110 μm. We show that despite significant scattering at various arrangements of microspheres embedded in agarose gel and despite strong losses in spatial coherence, it is possible to generate a sufficiently good overlap of both beam intensities. Without affecting the self-healing capability of the illumination photons in the Bessel beam’s ring system, the emission of fluorescence photons thereof can be strongly suppressed. This results in a needle-like fluorescence distribution inside scattering media, providing new perspectives for fundamental principles and applications in microscopy and metrology.

Multidimensional fluorescence imaging and super-resolution exploiting ultrafast laser and supercontinuum technology

This thesis centres on the development of multidimensional fluorescence imaging tools, with a particular emphasis on fluorescence lifetime imaging (FLIM) microscopy for application to biological research. The key aspects of this thesis are the development and application of tunable supercontinuum excitation sources based on supercontinuum generation in microstructured optical fibres and the development of stimulated emission depletion (STED) microscope capable of fluorescence lifetime imaging beyond the diffraction limit. The utility of FLIM for biological research is illustrated by examples of experimental studies of the molecular structure of sarcomeres in muscle fibres and of signalling at the immune synapse. The application of microstructured optical fibre to provide tunable supercontinuum excitation source for a range of FLIM microscopes is presented, including wide-field, Nipkow disk confocal and hyper-spectral line scanning FLIM microscopes. The application of supercontinuum generation to the first super-resolved FLIM microscope is then described. This novel microscope exploited the concept of STED with a femtosecond mode-locked Ti:Sapphire laser providing a tunable excitation beam by pumping microstructured optical fibre for supercontinuum generation and directly providing the (longer wavelength) STED beam. This STED microscope was implemented in a commercial scanning confocal microscope to provide compatibility with standard biological imaging, and exploited digital holography using a spatial light modulator (SLM) to provide the appropriate phase manipulation for shaping the STED beam profile and to compensate for aberrations. The STED microscope was shown to be capable of recording superresolution images in both the lateral and axial planes, according to the settings of the SLM.

Removal of anti-Stokes emission background in STED microscopy by FPGA-based synchronous detection.

In stimulated emission depletion (STED) microscopy, the role of the STED beam is to de-excite, via stimulated emission, the fluorophores that have been previously excited by the excitation beam. This condition, together with specific beam intensity distributions, allows obtaining true sub-diffraction spatial resolution images. However, if the STED beam has a non-negligible probability to excite the fluorophores, a strong fluorescent background signal (anti-Stokes emission) reduces the effective resolution. For STED scanning microscopy, different synchronous detection methods have been proposed to remove this anti-Stokes emission background and recover the resolution. However, every method works only for a specific STED microscopy implementation. Here we present a user-friendly synchronous detection method compatible with any STED scanning microscope. It exploits a data acquisition (DAQ) card based on a field-programmable gate array (FPGA), which is progressively used in STED microscopy. In essence, the FPGA-based DAQ card synchronizes the fluorescent signal registration, the beam deflection, and the excitation beam interruption, providing a fully automatic pixel-by-pixel synchronous detection method. We validate the proposed method in both continuous wave and pulsed STED microscope systems.

A protocol for registration and correction of multicolour STED superresolution images.

Multicolour fluorescence imaging by STimulated Emission Depletion (STED) superresolution microscopy with doughnut-shaped STED laser beams based on different wavelengths for each colour channel requires precise image registration. This is especially important when STED imaging is used for co-localisation studies of two or more native proteins in biological specimens to analyse nanometric subcellular spatial arrangements. We developed a robust postprocessing image registration protocol, with the aim to verify and ultimately optimise multicolour STED image quality. Importantly, this protocol will support any subsequent quantitative localisation analysis at nanometric scales. Henceforth, using an approach that registers each colour channel present during STED imaging individually, this protocol reliably corrects for optical aberrations and inadvertent sample drift. To achieve the latter goal, the protocol combines the experimental sample information, from corresponding STED and confocal images using the same optical beam path and setup, with that of an independent calibration sample. As a result, image registration is based on a strategy that maximises the cross-correlation between sequentially acquired images of the experimental sample, which are strategically combined by the protocol. We demonstrate the general applicability of the image registration protocol by co-staining of the ryanodine receptor calcium release channel in primary mouse cardiomyocytes. To validate this new approach, we identify user-friendly criteria, which - if fulfilled - support optimal image registration. In summary, we introduce a new method for image registration and rationally based postprocessing steps through a highly standardised protocol for multicolour STED imaging, which directly supports the reproducibility of protein co-localisation analyses. Although the reference protocol is discussed exemplarily for two-colour STED imaging, it can be readily expanded to three or more colours and STED channels.

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.