Capturing the non-equilibrium state in light–matter–free-electron interactions through ultrafast transmission electron microscopy

Ultrafast transmission electron microscope(UTEM) with the multimodality of time-resolved diffraction, imaging,and spectroscopy provides a unique platform to reveal the fundamental features associated with the interaction between free electrons and matter. In this review, we summarize the principles, instrumentation, and recent developments of the UTEM and its applications in capturing dynamic processes and non-equilibrium transient states. The combination of the transmission electron microscope with a femtosecond laser via the pump–probe method guarantees the high spatiotemporal resolution, allowing the investigation of the transient process in real, reciprocal and energy spaces. Ultrafast structural dynamics can be studied by diffraction and imaging methods, revealing the coherent acoustic phonon generation and photoinduced phase transition process. In the energy dimension, time-resolved electron energy-loss spectroscopy enables the examination of the intrinsic electronic dynamics of materials, while the photon-induced near-field electron microscopy extends the application of the UTEM to the imaging of optical near fields with high real-space resolution. It is noted that light–free-electron interactions have the ability to shape electron wave packets in both longitudinal and transverse directions, showing the potential application in the generation of attosecond electron pulses and vortex electron beams.

Stimulated Raman scattering microscopy with phase-controlled light focusing and aberration correction for rapid and label-free, volumetric deep tissue imaging

We report a novel stimulated Raman scattering(SRS)microscopy technique featuring phase-controlled light focusing and aberration corrections for rapid,deep tissue 3D chemical imaging with subcellular resolution.To accomplish phasecontrolled SRS(PC-SRS),we utilize a single spatial light modulator to electronically tune the axial positioning of both the shortened-length Bessel pump and the focused Gaussian Stokes beams,enabling z-scanning-free optical sectioning in the sample.By incorporating Zernike polynomials into the phase patterns,we simultaneously correct the system aberrations at two separate wavelengths(~240 nm difference),achieving a~3-fold enhancement in signal-to-noise ratio over the uncorrected imaging system.PC-SRS provides>2-fold improvement in imaging depth in various samples(e.g.,polystyrene bead phantoms,porcine brain tissue)as well as achieves SRS 3D imaging speed of~13 Hz per volume for real-time monitoring of Brownian motion of polymer beads in water,superior to conventional point-scanning SRS 3D imaging.We further utilize PC-SRS to observe the metabolic activities of the entire tumor liver in living zebrafish in cellsilent region,unraveling the upregulated metabolism in liver tumor compared to normal liver.This work shows that PCSRS provides unprecedented insights into morpho-chemistry,metabolic and dynamic functioning of live cells and tissue in real-time at the subcellular level.

PC-bzip2: a phase-space continuity-enhanced lossless compression algorithm for light-field microscopy data

Light-field fluorescence microscopy(LFM)is a powerful elegant compact method for long-term highspeed imaging of complex biological systems,such as neuron activities and rapid movements of organelles.LFM experiments typically generate terabytes of image data and require a substantial amount of storage space.Some lossy compression algorithms have been proposed recently with good compression performance.However,since the specimen usually only tolerates low-power density illumination for longterm imaging with low phototoxicity,the image signal-to-noise ratio(SNR)is relatively low,which will cause the loss of some efficient position or intensity information using such lossy compression algorithms.Here,we propose a phase-space continuity-enhanced bzip2(PC-bzip2)lossless compression method for LFM data as a high-efficiency and open-source tool that combines graphics processing unit-based fast entropy judgment and multicore-CPU-based high-speed lossless compression.Our proposed method achieves almost 10%compression ratio improvement while keeping the capability of high-speed compression,compared with the original bzip2.We evaluated our method on fluorescence beads data and fluorescence staining cells data with different SNRs.Moreover,by introducing temporal continuity,our method shows the superior compression ratio on time series data of zebrafish blood vessels.

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.

Unraveling the morphological evolution mechanism of solid sulfur species in lithium-sulfur batteries with operando light microscopy

Solid-liquid phase conversion between various sulfur species in lithium-sulfur(Li-S)batteries is a fundamental reaction of the sulfur cathode.Disclosing the morphological evolution of solid sulfur species upon cycling is of great significance to achieving high energy densities.However,an in-depth investigation of the internal reaction is still lacking.In this work,the evolution process of solid sulfur species on carbon substrates is systematically studied by using an operando light microscope combined with in situ electrochemical impedance spectra technology.The observation of phenomena such as bulk solid sulfur species can form and dissolve independently of the conductive substrates and the transformation of supercooled liquid sulfur to crystalline sulfur.Based on the phenomena mentioned above,a possible mechanism was proposed in which the dissolution reaction of solid sulfur species is a spatially free reaction that involves isotropic physical dissolution,diffusion of molecules,and finally the electrochemical reaction.Correspondingly,the formation of solid sulfur species tends to be a form of crystallization in a saturated solution rather than electrodeposition,as is commonly believed.Our findings offer new insights into the reaction of sulfur cathodes and provide new opportunities to design advanced sulfur cathodes for Li-S batteries.

Laterally swept light-sheet microscopy enhanced by pixel reassignment for photon-efficient volumetric imaging

In light-sheet fluorescence microscopy,the axial resolution and field of view are mutually constrained.Axially swept light-sheet microscopy(ASLM)can decouple the trade-off,but the confocal detection scheme using a rolling shutter also rejects fluorescence signals from the specimen in the field of interest,which sacrifices the photon efficiency.Here,we report a laterally swept light-sheet microscopy(LSLM)scheme in which the focused beam is first scanned along the axial direction and subsequently laterally swept with the rolling shutter.We show that LSLM can obtain a higher photon efficiency when similar axial resolution and field of view can be achieved.Moreover,based on the principle of image scanning microscopy,applying the pixel reassignment to the LSLM images,hereby named iLSLM,improves the optical sectioning.Both simulation and experimental results demonstrate the higher photon efficiency with similar axial resolution and optical sectioning.Our proposed scheme is suitable for volumetric imaging of specimens that are susceptible to photobleaching or phototoxicity.

Internal Structure of Cambrian Fossil Embryo Markuelia Revealed in the Light of Synchrotron Radiation X-ray Tomographic Microscopy

In the light of Synchrotron Radiation X-ray Tomographic Microscopy （SRXTM）, the internal structure of Markuelia hunanensis is revealed. In one example, vitrification and peeling show the annuli hidden under the chorion. Sectioning and 3-D reconstruction display an intact digestive tract from the inverted introvert to the terminal anus. The inverted introvert forms a rugby cavum. The following digestive tract is rope-like coiling, parallel to the body axis, about 650 μm in length, and uniform in diameter （-80 μm）. An exquisitely preserved pipe-like structure is hidden in the middle of the rope-like structure, diameter 20--40 lam, with a length of -120 μm. We interpret this pipe-like structure as the possible epidermis of the gut and its surroundings as the possible residue of musculature, similar to that in Priapulans. The two symmetrical rod-shape structures connecting the body wall and digestive tract are interpreted as the possible retractor muscles. After comparing the well preserved Left-form and Right-form Body of Markuelia, we suggest that they may represent a dimorphism. Counted directly, one sample of Markuelia hunanensis possesses 62 annulations and the other 68.

Three-dimensional,isotropic imaging of mouse brain using multi-view deconvolution light sheet microscopy

We present a threedimensional(3D)isotropic imaging of mouse brain using light-sheet fuo-rescent microscopy(LSFM)in conjumction with a multi-view imaging computation.Unlike common single view LSFM is used for mouse brain imaging,the brain tissue is 3D imaged under eight views in our study,by a home-built selective plane ilumination microscopy(SPIM).An output image containing complete structural infornation as well as significantly improved res olution(~4 times)are then computed based on these eight views of data,using a bead-guided multi-view registration and deconvolution.With superior imaging quality,the astrocyte and pyrarmidal neurons together with their subcellular nerve fbers can be clearly visualized and segmented.With further incuding other computational methods,this study can be potentially scaled up to map the conectome of whole mouse brain with a simple light.sheet microscope.

Cholesterol crystal binding of biliary immunoglobulin A: visualization by fluorescence light microscopy

AIM: To assess potential contributions of biliary IgA for crystal agglomeration into gallstones, we visualized cholesterol crystal binding of biliary IgA. METHODS: Crystal binding biliary proteins were extracted from human gallbladder bile using lectin affinity chromatography.Biliary IgA was isolated from the bound protein fraction by immunoaffinity chromatography. Pure cholesterol monohydrate crystals were incubated with biliary IgA and fluoresceine isothiocyanate (FITC)conjugated anti IgA at 37 degree. Samples were examined under polarizing and fluorescence light microscopy with digital image processing. RESULTS: Binding of biliary IgA to cholesterol monohydrate crystals could be visualized with FITC conjugated anti IgA antibodies.Peak fluorescence occurred at crystal edges and dislocations. Controls without biliary IgA or with biliary IgG showed no significant fluorescence. CONCLUSION: Fluorescence light microscopy provided evidence for cholesterol crystal binding of biliary IgA. Cholesterol crystal binding proteins like IgA might be important mediators of crystal agglomeration and growth of cholesterol gallstones by modifying the evolving crystal structures in vivo.

Single-cell volumetric imaging with light field microscopy: Advances in systems and algorithms

Single-cell volumetric imaging is essential for researching individual characteristics of cells.As a nonscanning imaging technique,lighteld microscopy(LFM)is a critical tool to achieve realtime three-dimensional imaging with the advantage of single-shot.To address the inherent limits including nonuniform resolution and block-wise artifacts,various modied LFM strategies have been developed to provide new insights into the structural and functional information of cells.This review will introduce the principle and development of LFM,discuss the improved approaches based on hardware designs and 3D reconstruction algorithms,and present the applications in single-cell imaging.

Variable Temperature Laser Light Scattering Microscopy (VTLLSM) Studies on 10-100 μm Size High Purity Gold and Commercial Grade Zinc Grains

The VTLLS microscopy studies were made on high purity gold and commercial grade zinc grains in a temperature range of 30-230?C. Differential area ω and surface activity Sa were estimated from photomicrographs. The ω vs dT/dt (rate of heating) curve was seen to differ from those of silver and titanium. The nature of curve between normalized ω and dT/dt was seen to be non-exponential. The characteristic relation between sectorized differential area ωsec and mean temperature was examined. The present study further establishes the simplicity and versatility of the VTLLS technique, in studying the defect-sub-structure of metal particles such as Au and Zn in presence of an imposed temperature gradient in a reasonable way. As such an attempt was made to connect the ω and defect-sub-structure related parameters.

Enhanced 3D Point Cloud Reconstruction for Light Field Microscopy Using U-Net-Based Convolutional Neural Networks

This article describes a novel approach for enhancing the three-dimensional(3D)point cloud reconstruction for light field microscopy(LFM)using U-net architecture-based fully convolutional neural network(CNN).Since the directional view of the LFM is limited,noise and artifacts make it difficult to reconstruct the exact shape of 3D point clouds.The existing methods suffer from these problems due to the self-occlusion of the model.This manuscript proposes a deep fusion learning(DL)method that combines a 3D CNN with a U-Net-based model as a feature extractor.The sub-aperture images obtained from the light field microscopy are aligned to form a light field data cube for preprocessing.A multi-stream 3D CNNs and U-net architecture are applied to obtain the depth feature fromthe directional sub-aperture LF data cube.For the enhancement of the depthmap,dual iteration-based weighted median filtering(WMF)is used to reduce surface noise and enhance the accuracy of the reconstruction.Generating a 3D point cloud involves combining two key elements:the enhanced depth map and the central view of the light field image.The proposed method is validated using synthesized Heidelberg Collaboratory for Image Processing(HCI)and real-world LFM datasets.The results are compared with different state-of-the-art methods.The structural similarity index(SSIM)gain for boxes,cotton,pillow,and pens are 0.9760,0.9806,0.9940,and 0.9907,respectively.Moreover,the discrete entropy(DE)value for LFM depth maps exhibited better performance than other existing methods.

A Workable Solution for Reducing the Large Number of Vehicle and Pedestrian Accidents Occurring on a Yellow Light

Traffic intersections are incredibly dangerous for drivers and pedestrians. Statistics from both Canada and the U.S. show a high number of fatalities and serious injuries related to crashes at intersections. In Canada, during 2019, the National Collision Database shows that 28% of traffic fatalities and 42% of serious injuries occurred at intersections. Likewise, the U.S. National Highway Traffic Administration (NHTSA) found that about 40% of the estimated 5,811,000 accidents in the U.S. during the year studied were intersection-related crashes. In fact, a major survey by the car insurance industry found that nearly 85% of drivers could not identify the correct action to take when approaching a yellow traffic light at an intersection. One major reason for these accidents is the “yellow light dilemma,” the ambiguous situation where a driver should stop or proceed forward when unexpectedly faced with a yellow light. This situation is even further exacerbated by the tendency of aggressive drivers to inappropriately speed up on the yellow just to get through the traffic light. A survey of Canadian drivers conducted by the Traffic Injury Research Foundation found that 9% of drivers admitted to speeding up to get through a traffic light. Another reason for these accidents is the increased danger of making a left-hand turn on yellow. According to the National Highway Traffic Safety Association (NHTSA), left turns occur in approximately 22.2% of collisions—as opposed to just 1.2% for right turns. Moreover, a study by CNN found left turns are three times as likely to kill pedestrians than right turns. The reason left turns are so much more likely to cause an accident is because they take a driver against traffic and in the path of oncoming cars. Additionally, most of these left turns occur at the driver’s discretion—as opposed to the distressingly brief left-hand arrow at busy intersections. Drive Safe Now proposes a workable solution for reducing the number of accidents occurring during a yellow light at intersections. We believe this fairly simple solution will save lives, prevent injuries, reduce damage to public and private property, and decrease insurance costs.

A promising approach for quantifying focal stroke modeling and assessing stroke progression:optical resolution photoacoustic microscopy photothrombosis

To investigate the mechanisms underlying the onset and progression of ischemic stroke,some methods have been proposed that can simultaneously monitor and create embolisms in the animal cerebral cortex.However,these methods often require complex systems and the effect of age on cerebral embolism has not been adequately studied,although ischemic stroke is strongly age-related.In this study,we propose an optical-resolution photoacoustic microscopy-based visualized photothrombosis methodology to create and monitor ischemic stroke in mice simultaneously using a 532 nm pulsed laser.We observed the molding process in mice of different ages and presented age-dependent vascular embolism differentiation.Moreover,we integrated optical coherence tomography angiography to investigate age-associated trends in cerebrovascular variability following a stroke.Our imaging data and quantitative analyses underscore the differential cerebrovascular responses to stroke in mice of different ages,thereby highlighting the technique's potential for evaluating cerebrovascular health and unraveling age-related mechanisms involved in ischemic strokes.

Combining machine learning algorithms with traditional methods for resolving the atomic-scale dynamic structure of monolayer MoS_(2) in high-resolution transmission electron microscopy

High-resolution transmission electron microscopy(HRTEM)promises rapid atomic-scale dynamic structure imaging.Yet,the precision limitations of aberration parameters and the challenge of eliminating aberrations in Cs-corrected transmission electron microscopy constrain resolution.A machine learning algorithm is developed to determine the aberration parameters with higher precision from small,lattice-periodic crystal images.The proposed algorithm is then validated with simulated HRTEM images of graphene and applied to the experimental images of a molybdenum disulfide(MoS_(2))monolayer with 25 variables(14 aberrations)resolved in wide ranges.Using these measured parameters,the phases of the exit-wave functions are reconstructed for each image in a focal series of MoS_(2)monolayers.The images were acquired due to the unexpected movement of the specimen holder.Four-dimensional data extraction reveals time-varying atomic structures and ripple.In particular,the atomic evolution of the sulfur-vacancy point and line defects,as well as the edge structure near the amorphous,is visualized as the resolution has been improved from about 1.75?to 0.9 A.This method can help salvage important transmission electron microscope images and is beneficial for the images obtained from electron microscopes with average stability.

Effects of Different Light Qualities on the Growth Characteristics of Populus trinervis

Populus trinervis is native to China and plays an irreplaceable role in maintaining the ecological balance of boreal and temperate forests.P.trinervis mainly grows in high-altitude areas.At present,there are limited studies on the response of P.trinervis to different light qualities,so it is necessary to investigate the photosynthetic physiological changes of P.trinervis in different light environments.In our study,P.trinervis was grown for 8 months under light filtered by three different colored films.The three treatments were blue film,green film,and white plastic film.The effects of blue(B),green(G),and white(W)light on photosynthetic pigment content,absolute growth,photosynthetic parameters,soluble sugar content,and chlorophyll fluorescence parameters were studied,respectively.Compared to the Wtreatment,the chlorophyll a and b,carotenoids,total chlorophyll content(a+b),absolute growth of seedling height,net photosynthetic rate(PN),water use efficiency(WUE),total soluble sugars,sucrose,and nonphotochemical quenching(NPQ)of P.trinervis were significantly increased under B treatment.Meanwhile,chlorophyll a and b,carotenoids,total chlorophyll(a+b),transpiration rate(Tr),intercellular CO_(2) concentration(Ci),stomatal conductance(gs),absolute growth of seedling height and leaf length,reducing sugar,total soluble sugar content,and NPQ were significantly increased under G treatment.The results showed that the absolute growth and chlorophyll content of P.trinervis were increased under B light,while the sugar and photosynthetic parameters were increased under G light.Additional studies may look into how B light impacts absolute growth and promotional mechanisms,as well as how G light affects the accumulation of sugar levels.

Electromagnetic modeling of interference,confocal,and focus variation microscopy

We present a unified electromagnetic modeling of coherence scanning interferometry,confocal microscopy,and focus variation microscopy as the most common techniques for surface topography inspection with micro-and nanometer resolution.The model aims at analyzing the instrument response and predicting systematic deviations.Since the main focus lies on the modeling of the microscopes,the light–surface interaction is considered,based on the Kirchhoff approximation extended to vectorial imaging theory.However,it can be replaced by rigorous methods without changing the microscope model.We demonstrate that all of the measuring instruments mentioned above can be modeled using the same theory with some adaption to the respective instrument.For validation,simulated results are confirmed by comparison with measurement results.

Super-resolution microscopy and its applications in neuroscience

Optical microscopy promises researchers to soe most tiny substances directly.However,the resolution of conventional microscopy is resticted by the diffraction limit.This makes it a challenge to observe subcellular processes happened in nanoscale.The development of super-resolution microscopy provides a solution to this challenge.Here,we briefly review several commonly used super-resolution techniques,explicating their basic principles and applications in biological science,especially in neuroscience.In addition,characteristics and limitations of each techrique are compared to provide a guidance for biologists to choose the most suitable tool.

SOFFLFM:Super-resolution optical fluctuation Fourierlight-field microscopy

Fourier light-field microscopy(FLFM)uses a microlens aray(MLA)to segment the Fourierplane of the microscopic objective lens to generate multiple two-dimensional perspective views,thereby reconstructing the threedimensional(3D)structure of the sample using 3D deconvo-lution calculation without scanning.However,the resolution of FLFM is stil limited by dif-fraction,and furthermore,it is dependent on the aperture division.In order to improve itsresolution,a super-resolution opticai fuctuation Fourier light-field microscopy(SOFFLFM)wasproposed here,in which the super-resolution optical fluctuation imaging(SOFI)with the abilityof super-resolution was introduced into FLFM.SOFFLFM uses higher-order cumulants statis-tical analysis on an image sequence collected by FLFM,and then carries out 3D deconvolutioncalculation to reconstruct the 3D structure of the sample.The theoretical basis of SOFFLFM onimproving resolution was explained and then verified with the simulations.Simulation resultsdemonstrated that SOFFLFM improved the lateral and axial resolution by more than V2 and 2times in the second-and fourth-order accumulations,compared with that of FLFM.

Advancing respirable coal mine dust source apportionment:a preliminary laboratory exploration of optical microscopy as a novel monitoring tool

Exposure to respirable coal mine dust(RCMD)can cause chronic and debilitating lung diseases.Real-time monitoring capabilities are sought which can enable a better understanding of dust components and sources.In many underground mines,RCMD includes three primary components which can be loosely associated with three major dust sources:coal dust from the coal seam itself,silicates from the surrounding rock strata,and carbonates from the inert‘rock dust’products that are applied to mitigate explosion hazards.A monitor which can reliably partition RCMD between these three components could thus allow source apportionment.And tracking silicates,specifically,could be valuable since the most serious health risks are typically associated with this component-particularly if abundant in crystalline silica.Envisioning a monitoring concept based on field microscopy,and following up on prior research using polarized light,the aim of the current study was to build and test a model to classify respirable-sized particles as either coal,silicates,or carbonates.For model development,composite dust samples were generated in the laboratory by successively depositing dust from high-purity materials onto a sticky transparent substrate,and imaging after each deposition event such that the identity of each particle was known a priori.Model testing followed a similar approach,except that real geologic materials were used as the source for each dust component.Results showed that the model had an overall accuracy of 86.5%,indicating that a field-microscopy based moni-tor could support RCMD source apportionment and silicates tracking in some coal mines.