Depletion Laser Research Articles

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.

New advances in the research of stimulated emission depletion super-resolution microscopy

Due to the influence of the diffraction limit, the lateral spatial resolution and axial spatial resolution of traditional optical microscopes are limited to ~200 nm and ~500 nm, respectively. In the past two decades, with the rapid development of high-intensity lasers, high-sensitivity detectors and other optoelectronic devices, there have been reported many super-resolution imaging techniques that bypass the optical diffraction limit with different methods. Among these techniques, stimulated emission depletion microscopy (STED) technology has the advantages of high imaging resolution and fast imaging speed. This technology uses two lasers for imaging, one of which is used to excite fluorescence, and the other donut-shaped depletion laser is used to suppress the emission of fluorescent molecules around the fluorescent spot, in order to reduce the fluorescence point spread function and achieve super resolution Imaging. After recent years of development, the STED system has got great progress no matter from the generation, calibration and scanning of the beam, and the final imaging. In terms of laser source, new laser sources such as continuous wave beams, supercontinuum laser, stimulated Raman scattered laser, and higher-order Bessel beams have appeared; in scanning and calibration, new efficiency technology such as parallel scanning and automatic calibration have also appeared; In imaging, new methods such as time gating and phasor analysis have emerged to improve imaging quality. These new technologies and methods are of great significance to improve the efficiency of STED system construction and imaging. In addition, this paper also focuses on the ways to expand the imaging functions of the STED system. First, for three-dimensional STED imaging, this paper mainly introduces three methods to realize three-dimensional STED imaging by wavefront non-coherent adjustment, 4Pi and structured light illumination methods. Second, for multi-color imaging, this paper introduces several dual-color and multi-color imaging techniques for special dyes. Third, this paper introduces the combination of STED technology with fluorescence correlation spectroscopy technology, cell expansion technology, scanning ion-conductance microscope, photo-activated localization microscopy/stochastic optical reconstruction microscopy and other technologies. Finally, this paper systematically discusses the new research progress of STED technology in recent years, and discusses the future development trend of STED technology.

Numerically Enhanced Stimulated Emission Depletion Microscopy with Adaptive Optics for Deep-Tissue Super-Resolved Imaging.

In stimulated emission depletion (STED) nanoscopy, the major origin of decreased signal-to-noise ratio within images can be attributed to sample photobleaching and strong optical aberrations. This is due to STED utilizing a high-power depletion laser (increasing the risk of photodamage), while the depletion beam is very sensitive to sample-induced aberrations. Here, we demonstrate a custom-built STED microscope with automated aberration correction that is capable of 3D super-resolution imaging through thick, highly aberrating tissue. We introduce and investigate a state of the art image denoising method by block-matching and collaborative 3D filtering (BM3D) to numerically enhance fine object details otherwise mixed with noise and further enhance the image quality. Numerical denoising provides an increase in the final effective resolution of the STED imaging of 31% using the well established Fourier ring correlation metric. Results achieved through the combination of aberration correction and tailored image processing are experimentally validated through super-resolved 3D imaging of axons in differentiated induced pluripotent stem cells growing under an 80 μm thick layer of tissue with lateral and axial resolution of 204 and 310 nm, respectively.

STED Direct Laser Writing of 45 nm Width Nanowire.

Controlled fabrication of 45 nm width nanowire using simulated emission depletion (STED) direct laser writing with a rod-shape effective focus spot is presented. In conventional STED direct laser writing, normally a donut-shaped depletion focus is used, and the minimum linewidth is restricted to 55 nm. In this work, we push this limit to sub-50 nm dimension with a rod-shape effective focus spot, which is the combination of a Gaussian excitation focus and twin-oval depletion focus. Effects of photoinitiator type, excitation laser power, and depletion laser power on the width of the nanowire are explored, respectively. Single nanowire with 45 nm width is obtained, which is λ/18 of excitation wavelength and the minimum linewidth in pentaerythritol triacrylate (PETA) photoresist. Our result accelerates the progress of achievable linewidth reduction in STED direct laser writing.

An indirectly stimulated emission depletion method for STED microscopy

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.

A simple empirical algorithm for optimising depletion power and resolution for dye and system specific STED imaging.

Here we show an easy method for determining an effective dye saturation factor ('PSTED ') for STED (Stimulated Emission Depletion) microscopy. We define PSTED to be a combined microscope system plus dye factor (analogous to the traditional ground truth Is measurement, which is microscope independent) that is functionally defined as the power in the depletion beam that provides a resolution enhancement of 41% compared to confocal, according to the modified Abbe's formula for STED resolution enhancement. We show that the determination of PSTED provides insight not only into the suitability of a particular dye and the best imaging parameters to be used for an experiment, but also sets the critical value for correctly determining the point spread function (PSF) used in deconvolution of STED images. PSTED can be a function of many experimental variables, both microscope and sample related. Here we show the utility of doing PSTED determinations by (1) exploiting the simple relationship between width and a threshold-defined area provided by a Gaussian PSF, for either linear or spherical objects and (2) linearising the normally inverse hyperbolic function of resolution versus power that can determine PSTED . We show that this rearrangement allows us to determine PSTED using only a few measurements: either at a few relatively low depletion powers, on traditional bead size measurements or by finding the total area of a naturally occurring sub-limit sized biological feature (in this case, microtubules). We show the derivation of these equations and methods and the utility of its use by characterising several dyes and a local imaging parameter relevant to STED microscopy. This information is used to predict the enhancement of resolution of the point spread function necessary for post-processing deconvolution. LAY DESCRIPTION: Stimulated Emission Depletion (STED) microscopy is a fluorescence imaging superresolution technique that achieves tens of nanometres resolution. This is done by utilising a depletion laser to effectively quench (deplete) fluorescence in a donut shape overlapping the normally excited fluorescence spot. The size of the remaining (undepleted) central fluorescence spot is power dependent allowing 'tunable' resolution with the power of the STED depletion laser. This depletion power versus resolution relationship is dye and instrument dependent. We have developed a method for quickly measuring this relationship to optimise experiments based on individual dyes and microscope specific parameters. This allows for quickly optimising microscope settings and for correctly postprocessing images.

Transition Dipole Moment Measurements of Ultracold Photoassociated85Rb133Cs Molecules by Depletion Spectroscopy**Supported by the National Key Research and Development Program of China under Grant No 2017YFA0304203, the National Natural Science Foundation of China under Grant Nos 61675120 and 11434007, the National Natural Science Foundation of China for Excellent Research Team under Grant No 61121064, the Shanxi Scholarship Council of China, the

The transition dipole moments (TDMs) of ultracold 85Rb133Cs molecules between the lowest vibrational ground level, X1Σ+ (v = 0, J = 1), and the two excited rovibrational levels, 23Π0+ (v′ = 10, J′ = 2) and 21Π1 (v′ = 22, J′ = 2), are measured using depletion spectroscopy. The ground-state 85Rb133Cs molecules are formed from cold mixed component atoms via the 23Π0− (v = 11, J = 0) short-range level, then detected by time-of-flight mass spectrum. A home-made external-cavity diode laser is used as the depletion laser to couple the ground level and the two excited levels. Based on the depletion spectroscopy, the corresponding TDMs are then derived to be 3.5(2)×10−3ea0 and 1.6(1)×10−2ea0, respectively, where ea0 represents the atomic unit of electric dipole moment. The enhance of TDM with nearly a factor of 5 for the 21Π1 (v′ = 22, J′ = 2) excited level means that it has stronger coupling with the ground level. It is meaningful to find more levels with much more strong coupling strength by the represented depletion spectroscopy to realize direct stimulated Raman adiabatic passage transfer from scattering atomic states to deeply molecular states.

Low‐Saturation‐Intensity, High‐Photostability, and High‐Resolution STED Nanoscopy Assisted by CsPbBr3 Quantum Dots

Stimulated emission depletion (STED) nanoscopy is one of the most promising super-resolution imaging techniques for microstructure imaging. Commercial CdSe@ZnS quantum dots are used as STED probes and ≈50 nm lateral resolution is obtained. Compared with other quantum dots, perovskite CsPbBr3 nanoparticles (NPs) possess higher photoluminescence quantum yield and larger absorption cross-section, making them a more effective probe for STED nanoscopy. In this study, CsPbBr3 NPs are used as probes for STED nanoscopy imaging. The fluorescence intensity of the CsPbBr3 sample is hardly weakened at all after 200 min irradiation with a 39.8 mW depletion laser, indicating excellent photobleaching resistance of the CsPbBr3 NPs. The saturation intensity of the CsPbBr3 NPs is extremely low and estimated to be only 0.4 mW (0.126 MW cm-2 ). Finally, an ultrahigh lateral resolution of 20.6 nm is obtained for a single nanoparticle under 27.5 mW STED laser irradiation in CsPbBr3 -based STED nanoscopy imaging, which is a tenfold improvement compared with confocal microscopy. Because of its high fluorescence stability and ultrahigh resolution under lower depletion power, CsPbBr3 -assisted STED nanoscopy has great potential to investigate microstructures that require super-resolution and long-term imaging.

Rotational Population Measurement of Ultracold 85Rb133Cs Molecules in the Lowest Vibrational Ground State**Supported by the National Key Research and Development Program of China under Grant No 2017YFA0304203, the National Natural Science Foundation of China under Grant Nos 61675120, 11434007 and 61378015, the Program for Changjiang Scholars and Innovative Research Team in University under Grant No IRT13076, and the Applied Basic

We measure the rotational populations of ultracold 85Rb133Cs molecules in the lowest vibrational ground state by a depletion spectroscopy and quantify the molecular production rate based on the measurement of single ion signal area. The 85Rb133Cs molecules in the are formed from the short-range molecular state. A home-made external-cavity diode laser is used as the depletion laser to measure the rotational populations of the formed molecules. Based on the determination of single ion signal, the production rates of molecules in the J = 0 and J = 2 rotational levels are derived to be 4800 mole/s and 7200 mole/s, respectively. The resolution and quantification of molecules in rotational states are facilitative for the manipulation of rotational quantum state of ultracold molecules.

Stimulated Emission Depletion Microscopy Resolves Nanoparticle Assembly on a Porous Membrane Surface

Stimulated emission depletion microscopy—depending on the nature of fluorophore and sample, depletion laser power, and other experimental conditions—provides diffraction unlimited lateral resolution in the range of 20- 100 nm. In this paper, a detailed description of such a microscope along with one of the possible imaging schemes is provided. Application of such a microscope in resolving self assembly of polymeric nanoparticles on porous alumina membrane in wet condition is demonstrated. STED microscopy resolves formation of loose linear to quasi polygonal self-assembly patterns on the membrane surface. Possible reasons for the formation of such self assembly patterns are provided.

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm−2 depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

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.

Upgrade of a Scanning Confocal Microscope to a Single-Beam Path STED Microscope.

By overcoming the diffraction limit in light microscopy, super-resolution techniques, such as stimulated emission depletion (STED) microscopy, are experiencing an increasing impact on life sciences. High costs and technically demanding setups, however, may still hinder a wider distribution of this innovation in biomedical research laboratories. As far-field microscopy is the most widely employed microscopy modality in the life sciences, upgrading already existing systems seems to be an attractive option for achieving diffraction-unlimited fluorescence microscopy in a cost-effective manner. Here, we demonstrate the successful upgrade of a commercial time-resolved confocal fluorescence microscope to an easy-to-align STED microscope in the single-beam path layout, previously proposed as “easy-STED”, achieving lateral resolution < λ/10 corresponding to a five-fold improvement over a confocal modality. For this purpose, both the excitation and depletion laser beams pass through a commercially available segmented phase plate that creates the STED-doughnut light distribution in the focal plane, while leaving the excitation beam unaltered when implemented into the joint beam path. Diffraction-unlimited imaging of 20 nm-sized fluorescent beads as reference were achieved with the wavelength combination of 635 nm excitation and 766 nm depletion. To evaluate the STED performance in biological systems, we compared the popular phalloidin-coupled fluorescent dyes Atto647N and Abberior STAR635 by labeling F-actin filaments in vitro as well as through immunofluorescence recordings of microtubules in a complex epithelial tissue. Here, we applied a recently proposed deconvolution approach and showed that images obtained from time-gated pulsed STED microscopy may benefit concerning the signal-to-background ratio, from the joint deconvolution of sub-images with different spatial information which were extracted from offline time gating.

Reducing Photobleaching in STED Microscopy with Higher Scanning Speed

Photobleaching is a major limitation of super-resolution STED microscopy. Using our custom-built resonant-scanning STED microscope with a large (50 x 50 µm) field of view, we now show that the photobleaching rate in STED microscopy can be slowed down by scanning with high linear speed. The effect of scanning speed on photobleaching is more remarkable at higher levels of depletion laser irradiance. With a depletion irradiance of 0.4 GW/cm2 (time average) and a 7-fold faster scanning (speed increased from 0.17 m/s to 1.38 m/s), we were able to slow down the photobleaching of the Atto 647N dye by 80%. Photobleaching is primarily caused by the depletion light acting upon the fluorophores in the excited states. Experimental data qualitatively agree with a theoretical model. Our results encourage to increase linear scanning speed further or to reduce continuous light exposure time to significantly reduce photobleaching in STED microscopy.

Exploring the Mechanisms in STED‐Enhanced Direct Laser Writing

Stimulated‐emission‐depletion direct laser writing allows for performing 3D optical lithography beyond the Abbe diffraction limit. However, the underlying mechanisms and limitations are poorly understood. In order to clarify for the case of 7‐diethylamino‐3‐thenoylcoumarin (DETC) as photoinitiator in pentaerythritol triacrylate, transient photoluminescence experiments as well as lithography experiments with variable repetition rate are performed. In addition, several coinitiators and DETC derivatives are investigated. While the photoluminescence of the DETC photoresist exhibits a two‐photon excitation behavior and can be largely depleted, lithography under the same conditions surprisingly shows an effective three‐photon excitation behavior at high repetition rates and an effective four‐photon behavior at low repetition rates. From the comprehensive investigation, it is concluded that at high repetition rates, residual absorption of the depletion laser is the limiting mechanism, whereas at low repetition rates an effective four‐photon process leads to direct radical generation that cannot be depleted.

ChemInform Abstract: 4‐Trifluoromethyl‐Substituted Coumarins with Large Stokes Shifts: Synthesis, Bioconjugates, and Their Use in Super‐Resolution Fluorescence Microscopy.

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

Focus on photonics and biomedical optics

Focus on photonics and biomedical optics

Dual Color STED Microscopy with Ultrafast Photon Counting

To more precisely quantify association between cardiac proteins, we built a dual-channel super-resolution stimulation emission depletion (STED) microscope with an ultrafast photon-counting acquisition system. STED microscopy is unique among super-resolution technologies due to its capability of optical sectioning and fast imaging speed. A major limitation of STED is the aggravated photobleahching, to which fast scanning speed is an excellent solution. In order to keep up with fast scanning speed and to reduce counting nonlinearity, we invented a photon counting acquisition system based on ultra-fast analog-to-digital conversion (ADC), pushing the readout rate to the gigahertz range. Incorporated into a resonant scanning confocal microscope, this system can acquire images at 16,000 lines per second and 58,000 pixels per line. By adding a continuous wave (CW) depletion laser and photon arrival time control elements, the confocal microscope was converted to a time-gated STED microscope. A second STED channel was built with picosecond depletion pulses. The dual-channel STED microscope is able to achieve a resolution of ∼40 nm in biological samples which allows a more stringent analysis of colocalization in native systems.

4‐Trifluoromethyl‐Substituted Coumarins with Large Stokes Shifts: Synthesis, Bioconjugates, and Their Use in Super‐Resolution Fluorescence Microscopy

Bright and photostable fluorescent dyes with large Stokes shifts are widely used as sensors, molecular probes, and light-emitting markers in chemistry, life sciences, and optical microscopy. In this study, new 7-dialkylamino-4-trifluoromethylcoumarins have been designed for use in bioconjugation reactions and optical microscopy. Their synthesis was based on the Stille reaction of 3-chloro-4-trifluoromethylcoumarins and available (hetero)aryl- or (hetero)arylethenyltin derivatives. Alternatively, the acylation of 2-trifluoroacetyl-5-dialkylaminophenols with available (hetero)aryl- or (hetero)arylethenylacetic acids followed by intramolecular condensation afforded coumarins with 3-(hetero)aryl or 3-[2-(hetero)aryl]ethenyl groups. Hydrophilic properties were provided by the introduction of a sulfonic acid residue or by phosphorylation of a primary hydroxy group attached at C-4 of the 2,2,4-trimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore. For use in immunolabeling procedures, the dyes were decorated with an (activated) carboxy group. The positions of the absorption and emission maxima vary in the ranges 413-480 and 527-668 nm, respectively. The phosphorylated dye, 9,CH=CH-2-py,H, with the 1-(3-carboxypropyl)-4-hydroxymethyl-2,2-dimethyl-1,2-dihydroquinoline fragment fused to the coumarin fluorophore bearing the 3-[2-(2-pyridyl)ethenyl] residue (absorption and emission maxima at 472 and 623 nm, respectively) was used in super-resolution light microscopy with stimulated emission depletion and provided an optical resolution better than 70 nm with a low background signal. As a result of their large Stokes shifts, good fluorescence quantum yields, and adequate photostabilities, phosphorylated coumarins enable two-color imaging (using several excitation sources and a single depletion laser) to be combined with subdiffractional optical resolution.

Auto-aligning stimulated emission depletion microscope using adaptive optics

Stimulated emission depletion (STED) microscopy provides diffraction-unlimited resolution in fluorescence microscopy. Imaging at the nanoscale, however, requires precise alignment of the depletion and excitation laser foci of the STED microscope. We demonstrate here that adaptive optics can be implemented to automatically align STED and confocal images with a precision of 4.3 ± 2.3 nm.