Stimulated Emission Depletion System Research Articles

Theoretical design of a single-mode fiber-based bi-order Bessel beam for a STED system

Stimulated emission depletion (STED) microscopy has attracted great research attention due to its applications to breaking diffraction limits for imaging and lithography. However, its implementation based on single-mode fibers often encounters challenges such as complex structural integration, costly fabrication processes, and the need for specific fiber designs. Herein, a low-cost bi-order Bessel beam based on one single-mode fiber integrated with a structurally simple wavelength-scale microstructure (WSM) on fiber end was proposed for STED system. Through simulation study for full-scale WSM optimization, we have successfully developed a bicolor laser beam (BLB) consisting of a zero-order Bessel beam at a wavelength of 405 nm and a donuts high-order Bessel beam at a wavelength of 532 nm. This fiber-based configuration allows us to achieve a diffraction-limited spot size with a working distance of 0.67λpump and a minimum FWHM of 0.395λpump. By combining wavelength division multiplexing technology with power modulation of the donuts beam, this work provides a promising way for achieving super-resolution imaging or lithography with only one single-mode fiber.

Low-power compact continuous-wave stimulated emission depletion microscopy.

Stimulated emission depletion (STED) microscopy can break the optical diffraction barrier and provide subdiffraction resolution. According to the STED superresolution imaging principle, the resolution of STED is positively related to the power of the depletion laser. However, high-laser power largely limits the study of living cells or living bodies. Moreover, the high complexity and high cost of conventional pulsed STED microscopy limit the application of this technique. Therefore, this paper describes a simple continuous-wave STED (CW-STED) system constructed on a 45 × 60 cm breadboard and combined with digitally enhanced (DE) technology; low-power superresolution imaging is realized, which has the advantages of reducing system complexity and cost. The low-system complexity, low cost, and low-power superresolution imaging features of CW-STED have great potential to advance the application of STED microscopy in biological research.

3D super-resolution deep-tissue imaging in living mice.

Stimulated emission depletion (STED) microscopy enables the three-dimensional (3D) visualization of dynamic nanoscale structures in living cells, offering unique insights into their organization. However, 3D-STED imaging deep inside biological tissue is obstructed by optical aberrations and light scattering. We present a STED system that overcomes these challenges. Through the combination of two-photon excitation, adaptive optics, red-emitting organic dyes, and a long-working-distance water-immersion objective lens, our system achieves aberration-corrected 3D super-resolution imaging, which we demonstrate 164 µm deep in fixed mouse brain tissue and 76 µm deep in the brain of a living mouse.

Dual-color STED super-resolution microscope using a single laser source.

STED (stimulated emission depletion) microscopy is one of the most promising super-resolution fluorescence microscopies，due to its fast imaging and ultra-high resolution. In this paper, we present a dual-color STED microscope with a single laser source. Polarization beam splitters are used to separate the output from a supercontinuum laser source into four laser beams, including two excitation beams (488, 635 nm) and two depletion beams (592, 775 nm). These four laser beams are then used to build a low cost dual-color STED system to achieve a spatial resolution of 75 nm in cell samples.

Double-cladding optical fiber design and optimization for simplifying the STED system

Abstract A special double-cladding optical fiber with three-layer step-index structure was introduced for simplifying the stimulated-emission-depletion (STED) microscopy. With this special fiber, a dark hollow beam with decent doughnut-shaped focal spot can be generated for incident light in a specific wavelength range. At the same time, a Gaussian-like beam can be generated when the wavelength of incident light is out of (either longer or shorter than) the specific range. An all-fiber STED illumination system has been achieved by this special fiber. For special cases, a semi-fiber solution based on this special fiber has also been realized.

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.

Influences of aberration on spatial resolution of STED microscope in probing a specimenwith discontinuous refraction indices.

Probing depth and system aberrations have direct impacts on the spatial resolution of stimulated emission depletion (STED) microscopes. Based on the vectorial diffraction theory, the influence of coma and astigmatism on the focal patterns of STED microscopes in probing stratified mediums with discontinuous refractive indices (e.g.,glass cover slip, solution, and biological samples, etc.) have been illustrated in detail. The spatial resolution of the STED system has been discussed by analyzing the full width at half-maximum size of the fluorescence spots. It is found that, while probing in stratified media with discontinuous refractive indices, the spatial resolution of a STED microscope can be very sensitive to the existence of aberrations, e.g., coma and astigmatism, at different probing depths, as a result of mismatched axial positions of the excitation and depletion patterns. The spatial resolution of STED can be degraded up to 1.87- and 1.95-fold compared to that without aberrations. Therefore, a careful evaluation of the influence of aberration and discontinuous refractive indices should be taken into account when applying a STED microscope to realize super-resolution images.

Evaluation of saturation intensity based on the FWHM of CW stimulated emission depletion microscopy

We propose a numerical method to evaluate the saturation intensity of fluorescence, which is a key parameter in determining the spatial resolution of a stimulated emission depletion (STED) microscopy system. The saturation intensities of four different continuous-wave (CW) STED systems reported from the studies of previous researchers (Willig et al., Nature Methods, 2007, 4, 915–918; Hein et al., PNAS, 2008, 105, 14271–14276; Hotta et al., JACS, 2010, 132, 5021–5023) have been calculated. It is found that pTDI fluorescent dye with a 633-nm excitation beam and a 780-nm depletion beam can provide the smallest saturation intensity (or the best depletion effect under the same depletion power) in all four cases. Based on the saturation intensity, we revisit the influence of wavefront modulation on the resolution improvement of the STED system. It is found that by applying azimuthally polarized light, the lateral resolution of the STED system could be increased to 26 nm with an improvement of up to 67%.

Investigation on improvement of lateral resolution of continuous wave STED microscopy by standing wave illumination.

In this paper, we report the enhancement of resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy by a novel method of structured illumination of an excitation beam. Illumination by multiple excitation beams through the specific pupil apertures with high in-plane wave vectors leads to interference of diffracted light flux near the focal plane, resulting in the contraction of the point spread function (PSF) of the excitation. Light spot reduction by the suggested standing wave (SW) illumination method contributes to make up much lower depletion efficiency of the CW STED microscopy than that of the pulsed STED method. First, theoretical analysis showed that the full width at half maximum (FWHM) of the effective PSF on the detection plane is expected to be smaller than 25% of that of conventional CW STED. Second, through the simulation, it was elucidated that both the donut-shaped PSF of the depletion beam and the confocal optics suppress undesired contribution of sidelobes of the PSF by the SW illumination to the effective PSF of the STED system. Finally, through the imaging experiment on 40-nm fluorescent beads with the developed SW-CW STED microscopy system, we obtained the result which follows the overall tendency from the simulation in the aspects of resolution improvement and reduction of sidelobes. Based on the obtained result, we expect that the proposed method can become one of the strategies to enhance the resolution of the CW STED microscopy.

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.

Sub-diffraction-limit cell imaging using a super-resolution microscope with simplified pulse synchronization

Stimulated emission depletion (STED) microscope is one of the most prominent super-resolution bio-imaging instruments, which holds great promise for ultrahigh-resolution imaging of cells. To construct a STED microscope, it is challenging to realize temporal synchronization between the excitation pulses and the depletion pulses. In this study, we present a simple and low-cost method to achieve pulse synchronization by using a condensed fluorescent dye as a depletion indicator. By using this method, almost all the confocal microscopes can be upgraded to a STED system without losing its original functions. After the pulse synchronization, our STED system achieved sub-100-nm resolution for fluorescent nanospheres and single-cell imaging.

Diffraction-Unlimited Fluorescence Imaging with an EasySTED Retrofitted Confocal Microscope.

The easySTED technology provides the means to retrofit a confocal microscope to a diffraction-unlimited stimulated emission depletion (STED) microscope.Although commercial STED systems are available today, for many users of confocal laser scanning microscopes the option of retrofitting their confocal system to a STED system ready for diffraction-unlimited imaging may present an attractive option. The easySTED principle allowing for a joint beam path of excitation and depletion light promises some advantages concerning technical complexity and alignment effort for such an STED upgrade. In the one beam path design of easySTED the use of a common laser source, either a supercontinuum source or two separate lasers coupled into the same single-mode fiber, becomes feasible. The alignment of the focal light distribution of the STED beam relative to that of the excitation beam in all three spatial dimensions is therefore omitted respectively reduced to coupling the STED laser into the common single-mode fiber. Thus, only minor modifications need to be applied to the beam path in the confocal microscope to be upgraded. Those comprise adding polarization control elements and the easySTED waveplate, and adapting the beamsplitter to the excitation/STED wavelength combination.

Coma aberrations in combined two- and three-dimensional STED nanoscopy.

Stimulated emission depletion (STED) microscopes, like all super-resolution methods, are sensitive to aberrations. Of particular importance are aberrations that affect the quality of the depletion focus, which requires a point of near-zero intensity surrounded by strong illumination. We present analysis, modeling, and experimental measurements that show the effects of coma aberrations on depletion patterns of two-dimensional (2D) and three-dimensional (3D) STED configurations. Specifically, we find that identical coma aberrations create focal shifts in opposite directions in 2D and 3D STED. This phenomenon could affect the precision of microscopic measurements and has ramifications for the efficacy of combined 2D/3D STED systems.

Effect of light polariztion on pattern illumination super-resolution imaging

Far-field fluorescence microscopy has made great progress in the spatial resolution, limited by light diffraction, since the super-resolution imaging technology appeared. And stimulated emission depletion (STED) microscopy and structured illumination microscopy (SIM) can be grouped into one class of the super-resolution imaging technology, which use pattern illumination strategy to circumvent the diffraction limit. We simulated the images of the beads of SIM imaging, the intensity distribution of STED excitation light and depletion light in order to observe effects of the polarized light on imaging quality. Compared to fixed linear polarization, circularly polarized light is more suitable for SIM on reconstructed image. And right-handed circular polarization (CP) light is more appropriate for both the excitation and depletion light in STED system. Therefore the right-handed CP light would be the best candidate when the SIM and STED are combined into one microscope. Good understanding of the polarization will provide a reference for the patterned illumination experiment to achieve better resolution and better image quality.

A software tool for tomographic axial superresolution in STED microscopy.

A method for generating three-dimensional tomograms from multiple three-dimensional axial projections in STimulated Emission Depletion (STED) superresolution microscopy is introduced. Our STED< method, based on the use of a micromirror placed on top of a standard microscopic sample, is used to record a three-dimensional projection at an oblique angle in relation to the main optical axis. Combining the STED< projection with the regular STED image into a single view by tomographic reconstruction, is shown to result in a tomogram with three-to-four-fold improved apparent axial resolution. Registration of the different projections is based on the use of a mutual-information histogram similarity metric. Fusion of the projections into a single view is based on Richardson-Lucy iterative deconvolution algorithm, modified to work with multiple projections. Our tomographic reconstruction method is demonstrated to work with real biological STED superresolution images, including a data set with a limited signal-to-noise ratio (SNR); the reconstruction software (SuperTomo) and its source code will be released under BSD open-source license.

A 3D aligning method for stimulated emission depletion microscopy using fluorescence lifetime distribution.

Optimal resolution by stimulated emission depletion (STED) microscopy requires precise alignment of the donut-shaped depletion focus to the excitation focus. In this article, we demonstrate that fluorescence lifetime distribution can be implemented to align the STED system. Different from the traditional aligning methods in which a scattering imaging module is often equipped, the lifetime-based method is free from probable mismatches between the scattering mode and the fluorescent mode, drift errors caused by separate imaging and complex fitting methods. Based on this method, a spatial resolution of 38 nm by time-gated detection has been achieved.

Effects of polarization on the de-excitation dark focal spot in STED microscopy

The size of the dark focal spot directly determines the resolution and stability of stimulatedemission depletion (STED) microscopy. This paper investigates the relationship betweenthe size of the dark focal spot and the polarization of the input light beam. The types offundamental polarization are discussed, their effects on the dark focal spot are comparedand the optimized mode for each kind of polarization is proposed. The results of theanalysis provide the theoretical basis and reference for designing a STED system.

Far-field optical nanoscopy based on continuous wave laser stimulated emission depletion

Stimulated emission depletion (STED) microscopy is one of the breakthrough technologies that belong to far-field optical microscopy and can achieve nanoscale spatial resolution. We demonstrate a far-field optical nanoscopy based on continuous wave lasers with different wavelengths, i.e., violet and green lasers for excitation and STED, respectively. Fluorescent dyes Coumarin 102 and Atto 390 are used for validating the depletion efficiency. Fluorescent nanoparticles are selected for characterizing the spatial resolution of the STED system. Linear scanning of the laser beams of the STED system along one line of a microscope slide, which is coated with the nanoparticles, indicates that a spatial resolution of about 70 nm has so far been achieved. A two-dimensional image of the particle pattern of the STED system is constructed and compared with scanning confocal microscope. The present work has further extended the application of the STED microscopy into the blue regime.

Breaking the Barriers in Bio Research

More than 130 years ago, Ernst Abbe set up the law that far-field optical microscopes are limited in resolution to approximately half of the wavelength of light. Today, these limits have been broken by two new concepts of microscopy: 4Pi and STED (Stimulated Emission Depletion) microscopy. Both techniques are revolutionizing the biomedical research environment, by providing resolution beyond what is possible with best light microscopes today, while maintaining all advantages of structure-specific fluorescence tagging. The principles have been invented by Stefan Hell of the Max Planck Institute for Biophysical Chemistry in Goettingen, Germany, and are commercialized by Leica Microsystems CMS. While the Leica TCS 4Pi is already available, the Leica TCS STED system will be introduced to the market in 2007.