Influence of fluorescence time characteristics on the spatial resolution of CW-stimulated emission depletion microscopy

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.

Stimulated emission-depletion-based point-scanning structured illumination microscopy

Wide-field linear structured illumination microscopy(LSIM)extends resolution beyond the diffraction limit by moving unresolvable high-frequency information into the passband of the microscopy in the form of moiréfringes.However,due to the diffraction limit,the spatial frequency of the structured illumination pattern cannot be larger than the microscopy cutoff frequency,which results in a twofold resolution improvement over wide-field microscopes.This Letter presents a novel approach in point-scanning LSIM,aimed at achieving higher-resolution improvement by combining stimulated emission depletion(STED)with point-scanning structured illumination microscopy(ps SIM)(STED-ps SIM).The according structured illumination pattern whose frequency exceeds the microscopy cutoff frequency is produced by scanning the focus of the sinusoidally modulated excitation beam of STED microscopy.The experimental results showed a 1.58-fold resolution improvement over conventional STED microscopy with the same depletion laser power.

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.

Silicon-substituted rhodamines for stimulated emission depletion fluorescence nanoscopy

As an essential part in the toolbox of super-resolution microscopy,stimulated emission depletion(STED)nanoscopy has been widely explored in revealing the substructure and bioactivities in fluorescence imaging.Among the applied STED fluorophores,silicon-substituted rhodamines(SiRs)belong to one of the most extensively employed fluorophores.The carboxy-SiR was favored in STED bioimaging with many advantages,including reliable photostability,cell permeability,tunable fluorogenicity,feasible structural decoration and so on.We reviewed the research of carboxy-SiR in the STED nanoscopy and hopefully this can inspire more efforts in the design and application of STED fluorophores.

Fast dual-beam alignment method for stimulated emission depletion microscopy using aggregation-induced emission dye resin

A stimulated emission depletion is capable of breaking the diffraction limit by exciting fluorescent molecules with a solid Gaussian beam and quenching the excited molecules with another donut beam through stimulated emission.The coincidence degree of these two beams in three dimensions will significantly influence the spatial resolution of the microscope.However,the conventional alignment approach based on raster scanning of gold nanoparticles by the two laser beams separately suffers from a mismatch between fluorescence and scattering modes.To circumvent the above problems,we demonstrate a fast alignment design by scanning the second beam over the fabricated sample,which is made of aggregation-induced emission(AIE)dye resin.The relative positions of solid and donut laser beams can be represented by the fluorescent AIE from the labeled spots in the dye resin.This design achieves ultra-high resolutions of 22 nm in the x/y relative displacement and 27 nm in the z relative displacement for fast spatial matching of the two laser beams.This study has potential applications in scenarios that require the spatial matching of multiple laser beams,and the field of views of different objectives,for example,in a microscope with high precision.

Photophysical Property of Photoactive Molecules with Multibranched Push-Pull Structures

The structure-property characteristics of a series of newly synthesized intramolecular charge- transfer （ICT） compounds, single-branch monomer with triphenylmethane as electron donor and 2,1,3-benzothiadiazole as acceptor, the corresponding two-branch dimer and three- branch trimer, have been investigated by means of steady-state and femtosecond time- resolved stimulated emission fluorescence depletion （FS TR-SEP FD） techniques in different polar solvents. The TD-DFT calculations are further performed to explain the observed ICT properties. The interpretation of the experimental results is based on the comparative studies of the series of compounds which have increased amount of identical branch moiety. The similarity of the absorption and fluorescence spectra as well as strong solvent-dependence of the spectral properties for the three compounds reveal that the excited state of the dimer and trimer are nearly the same with that of the monomer, which may localize on one branch. It is found that polar excited state emerged through multidimensional intramolecular charge transfer from the donating moiety to the acceptor upon excitation, and quickly relaxed to one branch before emission. Even so, the red-shift in the absorption and emission spectra and decreased fluorescence radiative lifetime with respect to their monomer counterpart still suggest some extent delocalization of excited state in the dimer and trimer upon excitation. The similar behavior of their excited ICT state is demonstrated by FS TR-SEP FD measurements, and shows that the trimer has the largest charge-separate extent in all studied three samples. Finally, steady-state excitation anisotropy measurements has further been carried out to estimate the nature of the optical excitation and the mechanism of energy redistribution among the branches, where no plateau through the ICT band suggests the intramolecular excitation transfer process between the branches in dimer and trimer.

Application of super-resolution fluorescence microscopy in hematologic malignancies

Hematologic malignancies are one of the most common malignant tumors caused by the clonal proliferation and differentiation of hematopoietic and lymphoid stem cells.The examination of bone marrow cells combined with immunodeficiency typing is of great significance to the diagnostic type,treatment and prognosis of hematologic malignancies.Super-resolution fluorescence microscopy(SRM)is a special kind of optical microscopy technology,which breaks the resolution limit and was awarded the Nobel Prize in Chemistry in 2014.With the development of SRM,many related technologies have been applied to the diagnosis and treatment of clinical diseases.It was reported that a major type of SRM technique,single molecule localization microscopy(SMLM),is more sensitive than flow cytometry(FC)in detecting cell membrane antigens'expression,thus enabling better chances in detecting antigens on hematopoietic cells than traditional analytic tools.Furthermore,SRM may be applied to clinical pathology and may guide precision medicine and personalized medicine for clone hematopoietic cell diseases.In this paper,we mainly discuss the application of SRM in clone hematological malignancies.

STED microscopy based on axially symmetric polarized vortex beams

A stimulated emission depletion （STED） microscopy scheme using axially symmetric polarized vortex beams is pro- posed based on unique focusing properties of such kinds of beams. The concept of axially symmetric polarized vortex beams is first introduced, and the basic principle about the scheme is described. Simulation results for several typical beams are then shown, including radially polarized vortex beams, azimuthally polarized vortex beams, and high-order axi- ally symmetric polarized vortex beams. The results indicate that sharper doughnut spots and thus higher resolutions can be achieved, showing more flexibility than previous schemes based on flexible modulation of both phase and polarization for incident beams.

E®ect of light polariztion on pattern illumination super-resolution imaging

Far-¯eld°uorescence 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 di®raction 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¯xed 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.

Structural basis for the regulatory mechanism of mammalian mitochondrial respiratory chain megacomplex-Ⅰ_(2)Ⅲ_(2)Ⅳ_(2)

Mammalian mitochondrial electron transport chain complexes are the most important and complicated protein machinery in mitochondria.Although this system has been studied for more than a century,its composition and molecular mechanism are still largely unknown.Here we report the high-resolution cryo-electron microscopy(Cryo-EM)structures of porcine respiratory chain megacomplex-Ⅰ_(2)Ⅲ_(2)Ⅳ_(2)(MCⅠ_(2)Ⅲ_(2)Ⅳ_(2))in five different conformations,including State 1,State 2,Mid 1,Mid 2,and Mid 3.High-resolution Cryo-EM imaging,combined with super-resolution gated stimulated emission depletion microscopy(gSTED),strongly supports the formation of MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)in live cells.Each MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)structure contains 141 subunits(70 different kinds of peptides,2.9 MDa)in total with 240 transmembrane helices.The mutual influence among CⅠ,CⅢ,and CⅣshown in the MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)structure suggests this megacomplex could act as an integral unit in electron transfer and proton pumping.The conformational changes from different states suggest a plausible regulatory mechanism for the MCⅠ_(2)Ⅲ_(2)Ⅳ_(2)activation/deactivation process.

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.

Low-power STED nanoscopy based on temporal and spatial modulation

Stimulated emission depletion(STED)nanoscopy enables the visualization of subcellular organelles in unprecedented detail.However,reducing the power dependency remains one of the greatest challenges for STED imaging in living cells.Here,we propose a new method,called modulated STED,to reduce the demand for depletion power in STED imaging by modulating the information from the temporal and spatial domains.In this approach,an excitation pulse is followed by a depletion pulse with a longer delay;therefore,the fluorescence decay curve contains both confocal and STED photons in a laser pulse period.With time-resolved detection,we can remove residual diffraction-limited signals pixel by pixel from STED photons by taking the weighted difference of the depleted photons.Finally,fluorescence emission in the periphery of an excitation spot is further inhibited through spatial modulation of fluorescent signals,which replaced the increase of the depletion power in conventional STED.We demonstrate that the modulated STED method can achieve a resolution of<100 nm in both fixed and living cells with a depletion power that is dozens of times lower than that of conventional STED,therefore,it is very suitable for long-term superresolution imaging of living cells.Furthermore,the idea of the method could open up a new avenue to the implementation of other experiments,such as light-sheet imaging,multicolor and three-demensional(3D)super-resolution imaging.