Estimation-free spatial-domain image reconstruction of structured illumination microscopy

Structured illumination microscopy(SIM)achieves super-resolution(SR)by modulating the high-frequency information of the sample into the passband of the optical system and subsequent image reconstruction.The traditional Wiener-filtering-based reconstruction algorithm operates in the Fourier domain,it requires prior knowledge of the sinusoidal illumination patterns which makes the time-consuming procedure of parameter estimation to raw datasets necessary,besides,the parameter estimation is sensitive to noise or aberration-induced pattern distortion which leads to reconstruction artifacts.Here,we propose a spatial-domain image reconstruction method that does not require parameter estimation but calculates patterns from raw datasets,and a reconstructed image can be obtained just by calculating the spatial covariance of differential calculated patterns and differential filtered datasets(the notch filtering operation is performed to the raw datasets for attenuating and compensating the optical transfer function(OTF)).Experiments on reconstructing raw datasets including nonbiological,biological,and simulated samples demonstrate that our method has SR capability,high reconstruction speed,and high robustness to aberration and noise.

Multiplexed stimulated emission depletion nanoscopy(mSTED)for 5-color live-cell long-term imaging of organelle interactome

Stimulated emission depletion microscopy(STED)holds great potential in biological science applications,especially in studying nanoscale subcellular structures.However,multi-color STED imaging in live-cell remains challenging due to the limited excitation wavelengths and large amount of laser radiation.Here,we develop a multiplexed live-cell STED method to observe more structures simultaneously with limited photo-bleaching and photo-cytotoxicity.By separating live-cell fluorescent probes with similar spectral properties using phasor analysis,our method enables five-color live-cell STED imaging and reveals long-term interactions between different subcellular structures.The results here provide an avenue for understanding the complex and delicate interactome of subcellular structures in live-cell.

End-to-end computational design for an EUV solar corona multispectral imager with stray light suppression

An extreme ultraviolet solar corona multispectral imager can allow direct observation of high temperature coronal plasma,which is related to solar flares,coronal mass ejections and other significant coronal activities.This manuscript proposes a novel end-to-end computational design method for an extreme ultraviolet(EUV)solar corona multispectral imager operating at wavelengths near 100 nm,including a stray light suppression design and computational image recovery.To suppress the strong stray light from the solar disk,an outer opto-mechanical structure is designed to protect the imaging component of the system.Considering the low reflectivity(less than 70%)and strong-scattering(roughness)of existing extreme ultraviolet optical elements,the imaging component comprises only a primary mirror and a curved grating.A Lyot aperture is used to further suppress any residual stray light.Finally,a deep learning computational imaging method is used to correct the individual multi-wavelength images from the original recorded multi-slit data.In results and data,this can achieve a far-field angular resolution below 7",and spectral resolution below 0.05 nm.The field of view is±3 R_(☉)along the multi-slit moving direction,where R☉represents the radius of the solar disk.The ratio of the corona's stray light intensity to the solar center's irradiation intensity is less than 10-6 at the circle of 1.3 R_(☉).

Review of 4Pi Fluorescence Nanoscopy

Since the 1990s,continuous technical and scientific advances have defied the diffraction limit in microscopy and enabled three-dimensional(3D)super-resolution imaging.An important milestone in this pursuit is the coherent utilization of two opposing objectives(4Pi geometry)and its combination with superresolution microscopy.Herein,we review the recent progress in 4Pi nanoscopy,which provides a 3D,non-invasive,diffraction-unlimited,and isotropic resolution in transparent samples.This review includes both the targeted and stochastic switching modalities of 4Pi nanoscopy.The schematics,principles,applications,and future potential of 4Pi nanoscopy are discussed in detail.

Modulated illumination localization microscopy-enabled sub-10 nm resolution

Optical microscopy is an essential tool for exploring the structures and activities of cells and tissues.To break the limit of resolution caused by diffraction,researchers have made continuous advances and innovations to improve the resolution of optical microscopy since the 1990s.These contributions,however,still make sub-10nm imaging an obstacle.Here,we name a series of technologies as modulated illumination localization microscopy(MILM),which makes ultra-high-resolution imaging practical.Besides,we review the recent progress since 2017 when MINFLUX was proposed and became the inspiration and foundation for the follow-up devel-opment of MILM.This review divides MILM into two types:point-scanning and wide-field.The schematics,principles and future research directions of MILM are discussed elaborately.

3D fluorescence emission difference microscopy based on spatial light modulator

We report three-dimensional fluorescence emission difference(3D-FED)microscopy using a spatial light modulator(SLM).Zero phase,0–2
vortex phase and binary 0-pi phase are loaded on the SLM to generate the corresponding solid,doughnut and z-axis hollow excitation spot,respectively.Our technique achieves super-resolved image by subtracting three di®erently acquired images with proper subtractive factors.Detailed theoretical analysis and simulation tests are proceeded to testify the performance of 3D-FED.Also,the improvement of lateral and axial resolution is demonstrated by imaging 100 nm°uorescent beads.The experiment yields lateral resolution of 140 nm and axial resolution of approximate 380 nm.

Fluorescence emission difference microscopy based on polarization modulation

In this paper,we propose a new fluorescence emission difference microscopy(FED)technique based on polarization modulation.An electro-optical modulator(EOM)is used to switch the excitation beam between the horizontal and vertical polarization states at a high frequency,which leads to solid-and donut-shaped beams after spatial light modulation.Experiment on the fluorescent nanoparticles demonstrates that the proposed method can achieve~λ=4 spatial resolution.Using the proposed system,the dynamic imaging of subcellular structures in living cells over time is achieved.

Super-resolution microscopy based on parallel detection

Image scanning microscopy based on pixel reassignment can improve the confocal resolution limit without losing the image signal-to-noise ratio(SNR)greatly[C.J.R.Sheppard,"Super resolution in confocal imaging,"Optik 80(2)53-54(1988).C.B.Miller,E.Jorg,"Image scanning microscopy,"Phys.Reu.Lett.104(19)198101(2010).C.J.R.Sheppard,s.B.Mehta,R Heintzmann,"Superresolution by image scanning microscopy using pixel reassignment,"Opt.Lett.38(15)28892892(2013)].Here,we use a tailor-made optical fiber and 19 avalanche pho-todiodes(APDs)as parallel detectors to upgrade our existing confocal microscopy,termed as parallel-detection super resolution(PDSR)microscopy.In order to obtain the correct shift value,we use the normalized 2D cross correlation to calculate the shifting value of each image.We characterized our system performance by imaging fuorescence beads and applied this system to observing the 3D structure of biological specimen.

Effect of spatial spectrum overlap on Fourier ptychographic microscopy

Fourier ptychographic microscopy(FPM)is a newly developed imaging technique which stands out by virtue of its high-resolution and wide FOV.It improves a microscope's imaging perfor-mance beyond the diffraction limit of the employed optical components by illuminating the sample with oblique waves of different incident angles,similar to the concept of synthetic aperture.We propose to use an objective lens with high-NA to generate oblique illuminating waves in FPM.We demonstrate utilizing an objective lens with higher NA to iluminate the sample leads to better resolution by simulations,in which a resolution of 0.28 pum is achieved by using a high-NA illuminating objective lens(NA=1.49)and a low-NA collecting objective lens(NA=0.2)in coherent imaging(λ=488 nm).We then deeply study FPM's exact relevance of convergence speed to spatial spectrum overlap in frequency domain.The simulation results show that an overlap of about 60%is the optimal choice to acquire a high-quality recovery(520*520 pixels)with about 2 min's computing time.In addition,we testify the robustness of the algorithm of FPM to additive noises and its suitability for phase objects,which further proves FPM's potential application in biomedical imaging.

Three-dimensional nanoscale vortex line visualization and chiral nanostructure fabrication of tightly focused multi-vortex beams via direct laser writing

Optical singularity is pivotal in nature and has attracted wide interest from many disciplines nowadays,including optical communication,quantum optics,and biomedical imaging.Visualizing vortex lines formed by phase singularities and fabricating chiral nanostructures using the evolution of vortex lines are of great significance.In this paper,we introduce a promising method based on two-photon polymerization direct laser writing(2PP-DLW)to record the morphology of vortex lines generated by tightly focused multi-vortex beams(MVBs)at the nanoscale.Due to Gouy phase,the singularities of the MVBs rotate around the optical axis and move towards each other when approaching the focal plane.The propagation dynamics of vortex lines are recorded by 2PP-DLW,which explicitly exhibits the evolution of the phase singularities.Additionally,the MVBs are employed to fabricate stable three-dimensional chiral nanostructures due to the spiral-forward property of the vortex line.Because of the obvious chiral features of the manufactured nanostructures,a strong vortical dichroism is observed when excited by the light carrying orbital angular momentum.A number of applications can be envisioned with these chiral nanostructures,such as optical sensing,chiral separation,and information storage.

Fluorescence interference structured illumination microscopy for 3D morphology imaging with high axial resolution

Imaging three-dimensional,subcellular structures with high axial resolution has always been the core purpose of fluorescence microscopy.However,trade-offs exist between axial resolution and other important technical indicators,such as temporal resolution,optical power density,and imaging process complexity.We report a new imaging modality,fluorescence interference structured illumination microscopy(FI-SIM),which is based on three-dimensional structured illumination microscopy for wide-field lateral imaging and fluorescence interference for axial reconstruction.FI-SIM can acquire images quickly within the order of hundreds of milliseconds and exhibit even 30 nm axial resolution in half the wavelength depth range without z-axis scanning.Moreover,the relatively low laser power density relaxes the requirements for dyes and enables a wide range of applications for observing fixed and live subcellular structures.

Method for active spatial alignment and stabilization of laser beams in multi-beam systems

We propose a new method for the development of multi-beam systems for the spatial alignment and stability of beams based on the error separation technique.This method avoids alignment errors caused by coupling effect of piezoelectric devices,inaccurate correction calculations,and detection mode of the angular deviation.According to the results by external detectors,the error value of spatial alignment and the root mean square[RMS]of deviations under control during 1 h can be equivalent to approximately 0.87 and 1.06 nm at the sample plane under an oil immersion lens[focal length f=2 mm].The RMS of deviations is less than one-third of those currently reported for multi-beam systems;therefore,higher alignment and stability accuracy can be achieved with our proposed method.

Direct laser writing breaking diffraction barrier based on two-focus parallel peripheral photoinhibition lithography(Erratum)

In section 3.2,a reference(Ref.33)was missing in the first sentence.It was already listed in the References list and correctly cited in another portion of the text.Section 3.2,the second sentence incorrectly referred to the"pattern in Fig.2";the pattern was specific to Fig.S6 in the Supplemental Material.

From microscopy to nanoscopy via visible light

The resolution of conventional optical equipment is always restricted by the diffraction limit,and improving on this was previously considered improbable.Optical super-resolution imaging,which has recently experienced rapid growth and attracted increasing global interest,will result in applications in many domains,benefiting fields such as biology,medicine and material research.This review discusses the contributions of different researchers who identified the diffractive barrier and attempted to realize optical super-resolution.This is followed by a personal viewpoint of the development of optical nanoscopy in recent decades and the road towards the next generation of optical nanoscopy.

Direct laser writing breaking diffraction barrier based on two-focus parallel peripheralphotoinhibition lithography

Direct laser writing(DLW)enables arbitrary three-dimensional nanofabrication.However,the diffraction limit poses a major obstacle for realizing nanometer-scale features.Furthermore,it is challenging to improve the fabrication efficiency using the currently prevalent single-focal-spot systems,which cannot perform high-throughput lithography.To overcome these challenges,a parallel peripheral-photoinhibition lithography system with a sub-40-nm two-dimensional feature size and a sub-20-nm suspended line width was developed in our study,based on two-photon polymerization DLW.The lithography efficiency of the developed system is twice that of conventional systems for both uniform and complex structures.The proposed system facilitates the realization of portable DLW with a higher resolution and throughput.

Dual-modulation difference stimulated emission depletion microscopy to suppress the background signal

Stimulated emission depletion(STED)nanoscopy is one of the most well-developed nanoscopy techniques that can provide subdiffraction spatial resolution imaging.Here,we introduce dual-modulation difference STED microscopy(dmdSTED)to suppress the background noise in traditional STED imaging.By applying respective time-domain modulations to the two continuous-wave lasers,signals are distributed discretely in the frequency spectrum and thus are obtained through lock-in demodulation of the corresponding frequencies.The background signals can be selectively eliminated from the effective signal without compromise of temporal resolution.We used nanoparticle,fixed cell,and perovskite coating experiments,as well as theoretical demonstration,to confirm the effectiveness of this method.We highlight dmdSTED as an idea and approach with simple implementation for improving the imaging quality,which substantially enlarges the versatility of STED nanoscopy.

Chip-compatible wide-field 3D nanoscopy through tunable spatial frequency shift effect

Spatial frequency shift(SFS) microscopy with evanescent wave illumination shows intriguing advantages, including large field of view(FOV), high speed, and good modularity. However, a missing band in the spatial frequency domain hampers the SFS superresolution microscopy from achieving resolution better than 3 folds of the Abbe diffraction limit. Here, we propose a novel tunable large-SFS microscopy, making the resolution improvement of a linear system no longer restricted by the detection numerical aperture(NA). The complete wide-range detection in the spatial frequency domain is realized by tuning the illumination spatial frequency actively and broadly through an angle modulation between the azimuthal propagating directions of two evanescent waves. The vertical spatial frequency is tuned via a sectional saturation effect, and the reconstructed depth information can be added to the lateral superresolution mask for 3D imaging. A lateral resolution of λ/9, and a vertical localization precision of ~λ/200(detection objective NA = 0.9) are realized with a gallium phosphide(GaP) waveguide. Its unlimited resolution enhancing capability is demonstrated by introducing a designed metamaterial chip with an unusual large refractive index. Besides the great resolution enhancement, this method shows better anti-noise capability than classical structured illumination microscopy without SFS tunability. This method is chip-compatible and can potentially provide a massproducible illumination chip module achieving the fast, large-FOV, and deep-subwavelength 3D nanoscopy.

Airy-like field under high numerical aperture optical system

The tightly focused field of an incident light beam through cubic phase modulation has been investigated by vectorial diffraction theory.For different modulation index of cubic phase and polarization states of the incident light,the focused fields have been presented.The results show that the Airy-like field can be produced by cubic phase modulation under high numerical aperture(NA)optical system.Intensity pattern and length of the main lobe are depended on modulation index for the spatial uniform polarization,and the Airy-like field is affected by polarization state for the spatial nonuniform polarization.It is helpful to structure new optical fields in optical manipulation,optical imaging,and surface plasma controlling.

Sub-diffraction dark spot localization microscopy

Single molecular localization microscopy(SMLM)is a useful tool in biological observation with sub-10-nm resolution.However,SMLM is incapable of discerning two molecules within the diffraction-limited region unless with the help of a stochastic on–off switching scheme which yet entails time-consuming processes.Here,we produce a novel kind of focal spot pattern,called sub-diffraction dark spot(SDS),to localize molecules within the sub-diffraction region of interest.In our proposed technique nominated as sub-diffracted dark spot localization microscopy(SDLM),multiple molecules within the diffraction-limited region could be distinguished without the requirement of stochastic fluorescent switches.We have numerically investigated some related impacts of SDLM,such as detection circle diameter,collected photon number,background noise,and SDS size.Simulative localization framework has been implemented on randomly distributed and specifically structured samples.In either two-or three-dimensional case,SDLM is evidenced to have2 nm localization accuracy.

Towards extremely high-order optical nonlinearity at the nanoscale

Nonlinear optics lie in the center of many optical technologies.As the superposition principle no longer holds,optical nonlinear response from materials frees many of the constraints in classical optics and has enabled a range of practical applications,from laser processing to quantum optics.Yet,irrespective of the advantages,the nonlinear response is typically observable at very high laser intensity.The photon avalanche(PA)effect is therefore exceptional,since it allows sufficiently weaker lasers for excitation.Unfortunately,PAs are mostly restricted to bulk materials and rely on cryogenic conditions,1 hindering their wide application.