Continuous Wave Stimulated Emission Depletion Research Articles

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.

A shape-switch-block method for confocal light-sheet microscopy with sectioned Bessel beams and stimulated emission depletion

Microscopy seeks to simultaneously maximize optical resolution, contrast, speed, volume size, and probe tolerability, which is possible by combining different complementary imaging techniques with their specific strengths. Here, we show how to combine three physical concepts to increase resolution and contrast in light-sheet microscopy by making the effective light-sheet thinner through phase shaping, fluorophores-switching, and dynamic blocking of fluorescence. This shape-switch-block principle is realized by illumination with two holographically shaped, sectioned Bessel beams. Second, by switching off fluorophores in the proximity of the excitation center using continuous-wave stimulated emission depletion (STED). And third, by blocking fluorescence outside the switching region by confocal line detection. Thereby, we reduce the light-sheet thickness by 35%, achieving an isotropic resolution with beads in a 300 × 70 × 50 µm³ volume. Without STED, we obtain 0.37 µm resolution in cell clusters at improved sectioning and penetration depth. The shape-switch-block concept promises high potential, also for other microscopy techniques.

Photobleaching reduction in modulated super-resolution microscopy.

Important breakthroughs in far-field imaging techniques have been made since the first demonstrations of stimulated emission depletion (STED) microscopy. To date, the most straightforward and widespread deployment of STED microscopy has used continuous wave (CW) laser beams for both the excitation and depletion of fluorescence emission. A major drawback of the CW STED imaging technique has been photobleaching effects due to the high optical power needed in the depletion beam to reach sub-diffraction resolution. To overcome this hurdle, we have applied a synchronous detection approach based on modulating the excitation laser beam, while keeping the depletion beam at CW operation, and frequency filtering the collected signal with a lock-in amplifier to record solely the super-resolved fluorescence emission. We demonstrate here that such approach allows an important reduction in the optical power of both laser beams that leads to measurable decreases in photobleaching effects in STED microscopy. We report super-resolution images with relatively low powers for both the excitation and depletion beams. In addition, typical unwanted scattering effects and background signal generated from the depletion beam, which invariably arises from mismatches in refractive index in the material composing the sample, are largely reduced by using the modulated STED approach. The capability of acquiring super-resolution images with relatively low power is quite relevant for studying a variety of samples, but particularly important for biological species as exemplified in this work.

Study on self‐assembly of colloidal particles at high ionic strength with stimulated emission depletion microscopy

AbstractUnderstanding the spatial organization of nano‐ and micro‐sized particle is very important in the fabrication of complex structures having unprecedented properties. Study on self‐assembly of submicroscopic colloidal particles at high ionic strength solution at single particle resolution can provide new insight into the nanoscale interactions. In this study, we studied the self assembly behavior of negatively charged 0.2 and 1 μm colloidal particles at high ionic strength on glass‐solution interface that is, in situ environment. The self‐assembled 0.2 μm particles could not be resolved with conventional confocal and epi‐fluorescent microscopy, so a home‐built continuous wave stimulated emission depletion (STED) microscope was used for the study. We found that particles self‐assemble into ordered and disordered structures at higher and lower ionic strength solution, respectively. The optical imaging methods allowed us to measure inter‐particle gap at second energy minimum directly. Interestingly, we found that the inter‐particle gap in the wet self‐assembly higher than the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) theory predicted. The in situ investigation of particle self‐assembly at high ionic strength will provide more insight for the understanding nanoscale interactions.

Enhanced fluorescence quenching rate of Coumarin 102 in stimulated emission depletion

The fluorescence quenching rate (KQ) of fluorescence dye in continuous-wave stimulated emission depletion (CW-STED) system is experimentally investigated by utilizing a commercial dye (Coumarin 102). With the plotting of a power-dependence curve, it is found that the quenching rate is increasing rather than staying steady with the raising of depletion beam power in CW STED. Moreover, increasing fluorescence dye concentration and excitation power could also result in the increasing of KQ, which gives rise to the negative influence on spatial resolution of CW STED microscope. The results are helpful in understanding the influence of quenching on CW STED, improving the performance of CW STED microscope and developing new STED-compatible fluorescent dye.

Inside Cover: Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam (J. Biophotonics 9/2019)

STED microscopy is a tool that enables superresolution fluorescence imaging by overcoming the diffraction limitation, and has become more useful in various fields such as biology and material science. STED resolution enhancement can be useful in resolving and visualizing sophisticated details of structures of a sample. For this, the excitation focal spot reduction of CW STED microscopy is achieved by PSF engineering using radial polarization and annular aperture, and improved lateral resolution is obtained by STED effect. This leads to a performance improvement that can lower the depletion beam power required to achieve the same superresolutionFurther details can be found in the article by Geon Lim, Wan‐Chin Kim, Seunghee Oh, Hyungsuk Lee, No‐Cheol Parket (e201900060). image

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Enhanced lateral resolution in continuous wave stimulated emission depletion microscopy using tightly focused annular radially polarized excitation beam.

The lateral resolution of continuous wave (CW) stimulated emission depletion (STED) microscopy is enhanced about 12% by applying annular-shaped amplitude modulation to the radially polarized excitation beam. A focused annularly filtered radially polarized excitation beam provides a more condensed point spread function (PSF), which contributes to enhance effective STED resolution of CW STED microscopy. Theoretical analysis shows that the FWHM of the effective PSF on the detection plane is smaller than for conventional CW STED. Simulation shows the donut-shaped PSF of the depletion beam and confocal optics suppress undesired PSF sidelobes. Imaging experiments agree with the simulated resolution improvement.

Parametric investigations on the saturation intensity of Coumarin 102 for stimulated emission depletion application.

Stimulated emission depletion (STED) microscopy performed using continuous-wave (CW) lasers has been investigated and developed by Willig etal. (Nature Methods, 2007, 4(11):915) for nearly a decade. Kuang etal. (Review of Scientific Instruments, 2010, 81:053709) developed the CW STED microscopy technique with 405 nm excitation and 532 nm depletion beams. In their research, Coumarin 102 dye was adopted and was found to be depletable. In this study, a parametric investigation of the depletion of Coumarin 102 dye is carried out experimentally. The influence of the excitation and depletion beam intensities and dye concentrations on the depletion efficiency are studied in detail. The results indicate the following: (1) The highest depletion occurs for the 100 μM Coumarin 102 solution, with a 1.4 μW excitation beam and a 115.3 mW depletion beam. (2) The minimum saturation intensity (Is) of STED, that is 13 MWcm-2 , is observed when the Coumarin 102 solution concentration is 10 μM. (3) Is values calculated directly from the depletion power derived with the cross-sectional area due to the full-width-at-half-maximum (FWHM) of the depletion beam show poor accuracy, where Is may be overestimated. Thus, a correction factor for the cross-sectional area is proposed. We also find that Is is not exactly constant for a fixed excitation beam power and dye concentration. This trend indicates that the conventional suppression function η(x)=e- ln (2)ISTED(x)/Is derived from picosecond STED may cause errors in evaluating the depletion process in CW STED microscopy.

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.

Superresolution imaging of telomeres with continuous wave stimulated emission depletion (STED) microscope

The significant role of telomeres in cells has attracted much attention since they were discovered. Fluorescence imaging is an effective method to study subcellular structures like telomeres. However, the diffraction limit of traditional optical microscope hampers further investigation on them. Recent progress on superresolution fluorescence microscopy has broken this limit. In this work, we used stimulated emission depletion (STED) microscope to observe fluorescence-labeled telomeres in interphase cell nuclei. The results showed that the size of fluorescent puncta representing telomeres under the STED microscope was much smaller than that under the confocal microscope. Two adjacent telomeres were clearly separated via STED imaging, which could hardly be discriminated by confocal microscopy due to the diffraction limit. We conclude that STED microscope is a more powerful tool that enable us to obtain detailed information about telomeres.

Advances in Gated CW STED Microscopy: Toward a Versatile Implementation

Stimulated emission depletion (STED) microscopy based on time-gated detection is a straightforward implementation, which substantial reduces the STED beam (peak) intensity to obtain effective sub-diffraction imaging. Furthermore, when combined with STED beam operating in continuous-wave (CW) lowers the complexity and the cost with respect to the early STED implementations, the so called gated CW-STED implementation (gCW-STED). However, the time-gated detection reduces the fluorescence signal that forms the images. Hence, in a situation of high background and/or weak signal, the images degrade in terms of signal-to-noise/background ratio (SNR and SBR) and the benefits of time-gated detection could vanish.In this work we reported about synergetic combinations of hardware and software methods able to compensate for the SNR and SBR reduction. In particular we demonstrate the benefits of (i) low intensity-noise laser to further reduce the STED beam intensity [1]; (ii) dedicated time gating electronics to optimize the fluorescence collection efficiency [2]; (iii) synchronous detection methods to remove the anti-Stokes emission background caused by the direct excitation from the STED beam [3]; (iv) tailored image deconvolution algorithms to improve the contrast. The combination of the proposed methods with two-photon-excitation (TPE) gCW-STED microscopy are also demonstrated.[1]I. Coto Hernandez, M. D’Amora, a Diaspro, and G. Vicidomini, “Influence of laser intensity noise on gated CW-STED microscopy,” Laser Phys. Lett., vol. 11, no. 9, p. 095603, 2014.[2]I. C. Hernandez, M. Buttafava, G. Boso, A. Diaspro, A. Tosi, and G. Vicidomini, “Gated STED microscopy with time-gated single-photon avalanche diode,” Biomed. Opt. Express, vol. 6, no. 6, p. 2258, 2015.[3]I. Coto Hernandez, C. Peres, F. C. Zanacchi, M. Amora, S. Christodoulou, P. Bianchini, A. Diaspro, and G. Vicidomini, “A new filtering technique for removing anti-Stokes emission background in gated CW-STED microscopy,” J. Biophotonics, 1-5, 2014.

Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide

STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.

Low power super resolution fluorescence microscopy by lifetime modification and image reconstruction.

We demonstrate a new method for obtaining sub-diffraction resolution in fluorescence microscopy. The technique involves the analysis of the time evolution of fluorescence images in the presence of weak and unstructured (fundamental Gaussian) continuous wave stimulated emission depletion. A reduced point spread functions (PSF) is obtained by the recombination of time segments of the evolving image. A significant reduction in the PSF for 20 nm fluorescent beads (ca. 240 nm to 125 nm) is obtained with an on-sample power of 7.5 mW (17 MW/cm2) - substantially lower than that required for spatially structured stimulated emission depletion microscopy.

Time-gated stimulated emission depletion nanoscopy

We have designed and built a time-gated continuous wave stimulated emission depletion (CW-STED) nanoscopy to visualize microstructures beyond the diffraction limit. An off-line time-gating detection was performed with the help of time-correlated single-photon counting technique. Experimental results showed that before time-gating, the resolution of our system was about 75 nm with a depletion beam (592 nm) power of 200 mW. By using the off-line time-gating detection, the resolution was further improved to 38 nm. Biological samples were also used to test the performance of our time-gated CW-STED, and a resolution of 70 nm was achieved with a depletion beam (592 nm) power of 85 mW. Detailed principles of time-gated CW-STED were discussed in the text. The time-gated STED provides a better resolution with finite laser power.

Tuning donut profile for spatial resolution in stimulated emission depletion microscopy

In stimulated emission depletion (STED)-based or up-conversion depletion-based super-resolution optical microscopy, the donut-shaped depletion beam profile is of critical importance to its resolution. In this study, we investigate the transformation of the donut-shaped depletion beam focused by a high numerical aperture (NA) microscope objective, and model STED point spread function (PSF) as a function of donut beam profile. We show experimentally that the intensity profile of the dark kernel of the donut can be approximated as a parabolic function, whose slope is determined by the donut beam size before the objective back aperture, or the effective NA. Based on this, we derive the mathematical expression for continuous wave (CW) STED PSF as a function of focal plane donut and excitation beam profiles, as well as dye properties. We find that the effective NA and the residual intensity at the center are critical factors for STED imaging quality and the resolution. The effective NA is critical for STED resolution in that it not only determines the donut shape but also the area the depletion laser power is dispersed. An improperly expanded depletion beam will have negligible improvement in resolution. The polarization of the depletion beam also plays an important role as it affects the residual intensity in the center of the donut. Finally, we construct a CW STED microscope operating at 488 nm excitation and 592 nm depletion with a resolution of 70 nm. Our study provides detailed insight to the property of donut beam, and parameters that are important for the optimal performance of STED microscopes. This paper will provide a useful guide for the construction and future development of STED microscopes.

STED Nanoscopy with Time-Gated Detection: Theoretical and Experimental Aspects

In a stimulated emission depletion (STED) microscope the region in which fluorescence markers can emit spontaneously shrinks with continued STED beam action after a singular excitation event. This fact has been recently used to substantially improve the effective spatial resolution in STED nanoscopy using time-gated detection, pulsed excitation and continuous wave (CW) STED beams. We present a theoretical framework and experimental data that characterize the time evolution of the effective point-spread-function of a STED microscope and illustrate the physical basis, the benefits, and the limitations of time-gated detection both for CW and pulsed STED lasers. While gating hardly improves the effective resolution in the all-pulsed modality, in the CW-STED modality gating strongly suppresses low spatial frequencies in the image. Gated CW-STED nanoscopy is in essence limited (only) by the reduction of the signal that is associated with gating. Time-gated detection also reduces/suppresses the influence of local variations of the fluorescence lifetime on STED microscopy resolution.

Dual channel STED nanoscopy of lytic granules on actin filaments in natural killer cells

Natural killer (NK) cells are innate immune effectors that eliminate diseased and tumorigenic targets through the directed secretion of specialized secretory lysosomes, termed lytic granules. This directed secretion is triggered following the formation of an immunological synapse (IS), which is characterized by actin re-modeling and receptor organization at the interface between the NK cell and its susceptible target. Actin at the IS has been described to be permissive to secretion by forming a large central clearance through which lytic granules are released. However we, and others, have recently shown that the actin network in NK cells at the IS is dynamic yet pervasive. These efforts used multiple high resolution imaging techniques to demonstrate that the actin network does not act as a barrier to secretion, but instead enables the secretion of lytic granules through minimally sized clearances. In our recent publication we visualized actin using continuous wave stimulated emission depletion (CW-STED) and lytic granules using the confocal modality. Here we report for the first time dual channel STED nanoscopy of NK cell lytic granules on actin filaments.

Sharper low-power STED nanoscopy by time gating

Applying pulsed excitation together with time-gated detection improves the fluorescence on-off contrast in continuous-wave stimulated emission depletion (CW-STED) microscopy, thus revealing finer details in fixed and living cells using moderate light intensities. This method also enables super-resolution fluorescence correlation spectroscopy with CW-STED beams, as demonstrated by quantifying the dynamics of labeled lipid molecules in the plasma membrane of living cells.