LIU Pei-zhi,HAO Bing,ZHANG Hai-xia,XU Bing-she,GUO Jun-jie

（Key Laboratory of Interface Science and Engineering in Advanced Materials, Ministry of Education,Taiyuan University of Technology, Taiyuan 030024, China）

Abstract：Although carbon-based materials,such as graphene,metal-organic frameworks (MOFs),polymers and biomolecules,have aroused increasing scientific interest in the fields of physics,chemistry,materials science and molecular biology,their atomicscale observation is still a challenge due to their structural instability under the electron beam.Ambiguous atomic arrangements have critically limited the fundamental understanding on these materials and their potential applications in electronics,mechanics,thermodynamics,catalysis,bioscience and medicine.Very recently,revolutionary sub-?ngstr?m resolution achievements of transmission electron microscopy (TEM) using a low voltage,a low electron dose,or a cryogenic environment have greatly facilitated the atomicscale structural and chemical examination of electron beam sensitive materials.In particular,the ability to image light elements atom by atom gives unprecedented insight into the structures and properties of novel carbon-based materials.In this review,the recent developments in advanced TEM combined with various imaging and spectroscopy techniques,and their use in examining graphenebased materials,MOFs,polymers,and biomacromolecules are summarized and discussed.The current challenges in materials research and trends for the future design of TEM equipment are outlined,which is expected to provide a deeper understanding of structure–performance relationships and the discovery of new carbon materials.

Key words:Transmission electron microscopy；Beam sensitive carbon-based materials；Atomic resolution；Low voltage；Low dose

1 Introduction

In recent decades,carbon materials,in the form of carbon nanotubes,fullerenes,activated carbons etc.,have been research frontiers and captured tremendous attentions from the field of physics,chemistry and materials science.Particularly,the rise of graphene,which was unexpectedly produced in 2004[1],has greatly boosted the research enthusiasm on carbon materials.Novel carbon-based materials,from organics and biomacromolecules,have also played an increasing vital role in green energies and biological medicines.Graphene has been realized as building blocks for all sp2carbon allotropes along with its atomic structures resolved by transmission electron microscopy (TEM)[2–4].The subsequent upsurge studies of novel 2D materials and polymers benefit from the development of advanced TEM,by which precise atomic configuration,composition of intrinsic defects and local structures can be identified[5,6],and thus motivate the basic understanding of carbon-based materials as well as their potential electronic,mechanical,thermal and chemical applications[7–14].

Today the resolution of the spherical aberration coefficient（Cs）corrected TEM has been pushed under 0.05 nm[15],which approaches the delocalization of thermal vibration of atoms.However,structure identification of carbon-based materials is still challenging,since the fragile bonding between light elements,such as B,C,N and O,are usually electron beam sensitive.Accordingly,the development of aberration-corrected TEM with sophisticated operation modes and advanced detectors has provided a powerful tool to probe the atomic-scale structural and chemical information via a combination of various imaging and spectroscopy techniques,as shown in Fig.1.Particularly,gentle TEM techniques such as low voltage TEM,low dose TEM with single electron counting direct detection (SECDD) camera or integrated differential phase contrast (iDPC) detector,and cryo-TEM have been recently developed,which enables the atomic resolution characterization of beam sensitive carbon-based materials.

Fig.1 A summary of Cs corrected TEM techniques applied in novel carbon-based materials.

During TEM characterization,the interaction between electron beam and a specimen will cause ionization damage (radiolysis) and knock-on damage in the specimen,along with a heating effect.Knock-on damage predominates in conducting specimens,while radiolysis is the predominant damage mechanism for organic specimens and insulators[16,17].It was found that reducing the TEM accelerating voltage below some threshold value can efficiently eliminate the knock-on damage.For example,direct observations of Stone-Wales (SW) defects[3],single heteroatoms in graphene[9],and oxygen atoms in graphene multivacancies with a crown ether structure[10],have been reported using low voltage TEM.On the other hand,reducing the incident beam current (electron dose) can efficiently eliminate the radiolysis of organic materials or insulators.Atomic structures of metal-organic frameworks (MOFs),which contain metal-ligand bonds and are usually electrically insulated,have been successfully resolved with low dose TEM[18–20].Cryo-TEM can eliminate the heating effect and radiolysis from electron beam,and isolate samples from air,by which atomic structures of chemical active species,polymers[21]and biomolecules[22]can be resolved.

In this review,we summarized recent achievements in gentle TEM with low voltage,low electron dose,or cryogenic circumstance,and enumerated a series of successful applications in novel carbon-based materials for the characterization of local structures with atomic resolution.It demonstrates that it is now possible to directly identify intrinsic structures,defects,catalytic active sites,biomolecules,and give new insights into the structure–performance relationships and novel applications for beam sensitive carbon-based materials.Future possibilities for in situ TEM studies of dynamic structures are also discussed.

2 Development of TEM and its operation modes

From 1933 when the first transmission electron microscope was constructed by Ernst Ruska and his advisor Max Knoll (Fritz-Haber-Institut der Max-Planck-Gesellschaft,Berlin,Federal Republic of Germany) to the year 1986 when the Nobel Prize in Physics was divided to Ruska“for his fundamental work in electron optics,and for the design of the first electron microscope”[23],more than 50 years had been passed.Commercial mass-produced transmission electron microscopes have been developed through technical improvements,and been widely used in the exploring of materials.For example,the applying of TEM in material sciences demonstrated the dislocation theory in 1950s[24]by Peter B.Hirsch,R.W.Horne and Michael J.Whelan,directly prompted the discovery of quasicrystals in 1980s[25–27]by Daniel Shechtman and Kexin Guo’s group,and the discovery of carbon nanotubes in 1991[28]by Sumio Iijima.

To date,quite a few sophisticated operation modes in TEM have been developed,and widely used in material characterization.In a transmission electron microscope,electron beam emitted from a point source is accelerated to an operation voltage,30?300 kV for instance,and then modulated by a condenser lens,to form a parallel illumination for conventional TEM (CTEM),or a converged illumination for scanning transmission electron microscopy(STEM)[29].Schematics of the optical configurations of CTEM and STEM are shown in Fig.2.The convergence semi-angle (α) for TEM mode is typically less than 0.1 mrads (0.005 7°),which is effectively a parallel beam.In contrast,αfor STEM mode is usually larger than 20 mrads,which gives a convergent beam,or an electron prob.Both bright field (BF) and dark field (DF) images can be formed in the two modes,but with complementary advantages in information acquisition.

Fig.2 Schematic of the CTEM and STEM optical configuration.

2.1 BF and DF imaging in conventional TEM

Parallel beams in CTEM pass through a thin specimen and then form diffraction patterns at the back focal plane,as shown in Fig.3.The brightest spot in center is direct beam,which is incoherent and contains most intensity of electron beams.Surrounding dots with less brightness are diffracted beams,which are coherent and tuned by lattice planes in specimen.An objective aperture can be inserted to select either the direct beam (Fig.3(a)) or a diffracted beam(Fig.3(b)) to form the corresponding BF image or DF image of CTEM.Since the DF (BF) images only contains coherent (incoherent) component of electron beam,image contrast becomes much better and defects in crystals (such as grain boundaries,dislocations and lattice distortions) can be recognized easily.

Fig.3 Diagrams illustrated (a) BF and (b) DF illumination arrangements in a conventional TEM.Faint spots denote diffraction dots,the crosshair in the center denotes the TEM optic axis,and the dark rings denote the objective aperture.

When electrons pass through a thin sample,scattering from atoms in the thin sample is relatively weak,and thus the interaction between electron and atom holds for the so-called weak phase-object approximation (WPOA).Under WPOA,atomic lattice in the thin sample gives a phase field that can tune the interference of the passing-through-electrons,and the consequent constructive interference is a 2D electrostatic potential projection of the atomic lattice,which is called a high resolution TEM (HRTEM) image.

2.2 Angular DF and angular BF imaging in STEM

As mentioned above,HRTEM image is actually a multi-electron wave interference image rather than a real image of atoms in the sample.In contrast,as shown in Fig.4,a focused electron probe under STEM mode is used to scan across the specimen in a 2D raster.Annular detectors with various radius collect electrons scattered by the specimen with different collection semi-angles (β),and form angular darkfield (ADF) and angular bright-field (ABF) images.Meanwhile,simultaneous elemental analysis can be achieved with equipped electron energy-loss spectroscopy (EELS) or X-ray energy dispersive spectroscopy (EDS).Albert V.Crewe in University of Chicago demonstrated the first successful observation of single uranium and thorium atoms in 1970,with the STEM and ADF detector designed by his team[30].Later,Stephen J.Pennycook at Oak Ridge National Laboratory (ORNL) obtained atomic-number contrast images on crystalline materials of YBa2Cu3O7-xand ErBa2Cu3O7-xwith STEM-ADF imaging[31],and developed the incoherent imaging theory[32]in the end of 1980s.

Fig.4 Schematic diagram of the common imaging and spectrum modes in a STEM,composed of ADF or ABF imaging,EDX,and EELS.

When the collection semi-angleβof the detector is greater than the convergence semi-angle α of the probe,an ADF image can be recorded.High-angle scattering is largely incoherent thermal diffuse scattering,image contrast of bright spots usually appears exactly at the locations of the atomic columns.Meanwhile,the incoherent illumination can also improve the resolution to 1/2 of that with coherent beam.Thus,the corresponding STEM-ADF images reflect real positions of atoms with better resolution,and are much more straightforward to understand than HRTEM images of phase contrast.

ADF image is powerful to image heavy atoms in the materials due to their strong ability to scatter the incoming electrons.Specially,HAADF,MAADF,or LAADF images denote the collection angles of high,medium,and low angle,respectively.HAADF image with a rather larger collection angle,mostly contributed by the Rutherford scattering of electron by the nuclei in the specimen,shows an approximate Z2dependence of atom number,which is also named Zcontrast image (Z is the element number).LAADF image,in which coherent diffraction effects contribute a large part to the recorded signal,is good at visualizing the strained or defected features.MAADF image with a medium collection angle can combine the advantages of both LAADF and HAADF imaging techniques,which can be tuned according to practical experimental requirements[33].

Whenβis smaller thanα,an ABF image can be recorded.Recently,an ABF imaging technique has been developed for its sensitivity to light elements,and the contrast interpretation of ABF images was explained by the s-state channeling model,where the contrast of atomic columns is approximately proportional to the Z1/3[34].ABF imaging of light elements yields an absorption type of gray contrast as compared to the dark contrast of heavy elements over a wide range of sample thicknesses and defocus[35].With an optimized collection semi-angle of 11 mard≤β≤ 22 mard,hydrogen atom in a crystalline solid YH2was resolved[36],and the ABF imaging has been widely used in probing oxygen octahedra rotation in perovskites and charging/discharging in lithium battery materials[37].

After successful implementation of aberration correctors in the STEM equipped with a cold-field gun in recent years,a sub-?ngstr?m probe with high current makes the atomic resolved EELS and EDX with improved signal-to-noise ratio (SNR) truly feasible in routine practice,and energy-loss near edge structure (ELNES) of EELS can provide rich information about electronic structures of materials.As a reasonable generalization,the STEM mode is good at ADF imaging coupled with spectroscopy,while CTEM is good at phase contrast imaging.

2.3 The improvement of resolution and development of a Cs corrector

Till the end of 1970s,the point resolution of TEM reaches～0.3 nm,which can distinguish lattice fringes of close packed crystal planes easily.The further improvement of resolution is mainly hindered by spherical aberration of electromagnetic lens in a TEM.Spherical aberration arises from the fact that further off-axis electron is bent more strongly back toward the axis.As a result,a point object is imaged as a disk of finite size during the imaging process,and thus the ability of an objective lens to magnify detail in a sample is greatly limited.In 1949,Otto Scherzer have prompted that this effect can be diminished by balancing the spherical aberration against a particular negative value of defocus ?f,known as“Scherzer defocus”.The point resolution limit (r) of an uncorrected HRTEM[38]can then be expressed as the following:

Here,Csis the spherical aberration coefficient of an objective lens,and λ is the wave length of electron beam.For a typical microscope operated at 200 kV,λ=0.002 51 nm.IfCs=0.65 mm,its point resolution is 0.21 nm.A resolution of 0.2 nm can distinguish close packed lattice fringes in most crystal materials.However,a real atom by atom identification calls for a sub-?ngstr?m resolution,which takes almost another 20 years to accomplish.

As early as in 1936,Scherzer proved that spherical aberrations in a rotationally symmetric electromagnetic lens cannot be fully eliminated,and prompted a design of multipole lenses to reduce spherical and chromatic aberrations in 1947[39].The multipole lenses require highly accurate machining and alignment,which was beyond the technology available at that time.Herald Rose,Scherzer’s PhD student,improved the design,and demonstrated the feasibility of eliminate theCswith a double hexapole lens and a round-lens doublet in 1990.Maximilian Haider,PhD student of Rose,together with Knut Urban,constructed a hexapole corrector and mounted it just below the objective lens of a CTEM of 200 kV Philips CM 200 ST in 1998,and the point resolution was improved from 0.24 nm to better than 0.14 nm[40].The interface of CoSi2grown epitaxially on Si (111) seen along the＜110＞ direction was successfully resolved atomically sharp with the help of thisCscorrector.This success was achieved 16 years after Prof.Scherzer passed away,and more than 60 years after his conceiving of aCscorrector.

Another design of aCscorrector with quadrupole-octupoles (QO) was accomplished by Ondrej L.Krivanek.He developed an imaging filter for Gatan company with quadrupoles and hexapoles to correct second order aberrations and distortions,which had very similar optics with an electron microscope.Then he designed the QO corrector for STEM together with Niklas Dellby in 1997.TheCscorrector for STEM is mounted before the objective lens to eliminate the stronger convergence of further off-axis beam.As a result,all the off-axis electron beams can be focused to a sub-?ngstr?m point on the back focal plane,and the resolution is determined by the intensity distribution of the illuminating probe,or more briefly,the probe size.Their design reached a resolution of 0.075 nm on a 120 kV STEM at ORNL in 2002[41],and pairs of Si columns on (444) plane with a distance of 0.078 nm was directly and clearly recognized on a 300 kV STEM at ORNL in 2004[42].

Aberration-correction had already become the new frontier in electron microscopy,and the commercializedCscorrected TEM springs up.Haider founded the CEOS GmbH (Corrected Electron Optical Systems GmbH) in 1996 together with Joachim Zach to produce hexapole correctors.Krivanek founded Nion company (“NION”is just composed of the first 2 letters of their name“Niklas”and“Ondrej”) in 1997 together with Niklas Dellby to produce dedicated STEM equipped with a QO corrector,and hold the Director of Research of Gatan company since 1985.Today the resolution record of theCscorrected TEM has been pushed under 0.05 nm[15].Meanwhile,the well-focused electron beam takes advantages of 10 times more electrons compared with that in an uncorrected TEM,which greatly benefits the atomic resolution chemical analysis with EELS and EDS.

Rose,Haider and Urban were awarded the Wolf Prize in Physics 2011 for their fundamental and pioneer contribution in theCscorrector[43].They were also awarded the Kavli Prize in nanoscience 2020 together with Krivanek for their development of theCscorrector in TEM and sub-?ngstr?m resolution imaging and chemical analysis using electron beams[44].

3 Characterization of carbon-based materials with the Cs corrected TEM

The challenges of the atomic-scale observation exist in both traditional carbon materials,such as graphene,graphene-based single-atom catalysts(SACs) and electromagnetic wave absorption composites,and novel carbon related materials,such as MOFs,polymers and biomolecules.After the widely equippedCscorrectors,the main bottlenecks of structure identification by TEM for beam sensitive carbonbased materials is the beam damage during observation,and the simultaneous imaging of light elements(such as Li,C,N and O) and heavy ones.In the following,the application of theCscorrected TEM with low voltage,low electron dose,or cryogenic temperature in these carbon-based materials in last decade are discussed.The following paragraphs start from the characterization of intrinsic structures of graphene and alike 2D materials,such as graphene and h-BN.Then the characterization of functionalized carbon related materials,such as graphene-based SACs,MOFs,solid electrolyte interphase (SEI) layers in lithium-ion batteries (LIBs),and nanoporous carbons from biomass materials are discussed.Besides,the electron beam induced structure evolutions of graphene-based materials,and applications of cryo-TEM in biomolecules are included. Structure dynamics of graphene nanospheres and future possibilities for in situ TEM studies are outlined in the end.

3.1 Characterization of graphene and alike 2D materials

Graphene is consisted of sp2bonded carbon atoms with a honeycomb structure within a single layer,as shown in Fig.5(a),which is also the building block of fullerenes,carbon nanotubes,and graphite[8].A single graphene layer is a zero-gap semiconductor with a linear Dirac-like spectrum around the Fermi energy as shown in Fig.5(b)),while three and more stacking graphene layers show a clear semi-metallic behavior.When the stacking layers of graphene are more than 10,it will behave three-dimensional (3D)characteristics closing to graphite[45].

Fig.5 The structure of graphene:(a) Honeycomb structure of graphene,with a hexagonal unit cell and a bond length of 0.142 nm.Graphene has 2 types of common edges:the zigzag edge and armchair edge,(b) Band structure of graphene[46].

Graphene was brought to the public since Andre Geim and Konstantin Novoselov of the University of Manchester were awarded the Nobel Prize in Physics 2010 for their "groundbreaking experiments regarding the 2D material graphene"[47].Especially,the identification of 2D materials directly and experimentally benefits from the development of advanced electron microscopy. Then the unique characteristics of graphene and other 2D materials such as hexagonal boron nitride (h-BN) have been demonstrated to be the same as the theory prediction,and the upsurge study on the manufacturing of nanostructure electrical and optical devices was started up.

The threshold voltage of knock-on damage is 113 keV for carbon nanotubes,140 keV for graphite,and 74 eV for monolayer h-BN[48].Atoms at defects or edges are more active,and thus more sensitive to electron beam during TEM observation.As a consequence,low voltage imaging in combination with aberration correction is vitally necessary for direct identification of atomic structures in graphene and the alike 2D materials.

Fig.6 shows the intrinsic structure studies in graphene and alike 2D materials.Graphene lattice and Stone-Wales (SW) defects were observed with atomic resolution by Meyer et al.[3]in 2008 under a 80 kV TEM,shown as Fig.6(a),which was a start of direct experimental study of the atomic structure of 2D materials.Grain boundaries stitched intricate patchwork of graphene grains were resolved by Huang et al.under a 60 kV STEM,which explained the severely weakened mechanical but not electrical properties of polycrystalline monolayer graphene[49].Guo et al.studied oxygen atoms incorporated in graphene multivacancies with a crown ether configuration in 2014 under a 60 kV STEM[10],as shown in Fig.6(b,c),which conducted the study of heterogeneous doping and chemical applications of graphene such as high sensitivity sensing and high selectivity ion adsorption.

Fig.6 Atomic structures of defects in graphene and alike 2D materials.(a) HRTEM image of SW defect in graphene (Scale bar is 0.2 nm)[3].(b-c) STEM-ADF image of 3 oxygen atoms in graphene multivacancies(Scale bar is 0.2 nm)[10].(d-f) STEM-ADF image of a nitrogen anti-site NB in a suspended h-BN monolayer,and its edge reconstruction[11] (Red and green dots correspond to B and N atom).(g-i) STEM-ADF image and ELNES for threefold and fourfold coordinated Si impurities in monolayer graphene (Scale bar is 0.2 nm)[51].

h-BN,or“white graphene”,has the same atomic arrangement as graphene but is constructed with hetero elements and behaves opposite conductivity.Structure identification of h-BN is more rigid and interesting.Substitutional atoms of carbon and oxygen in h-BN were resolved atom by atom in 2010 under a 60 kV STEM[6],which is a demonstration of milestone thatCscorrected STEM-ADF imaging becomes a powerful method for both structural and chemical analyzing with atomic resolution.Intrinsic defects such as boron vacancies,nitrogen vacancies,nitrogen anti sites,dislocations[50],and edge configurations and reconstructions of the suspended h-BN monolayer were directly resolved for the first time afterward with the same instrument by Liu et al.[11],as shown in Fig.6(d-f),which implicates the semiconductor transition by self-doping of N induced by the defects and edge configuration of h-BN.

Atom by atom ELNES study at graphene edge and chemical bonding of a single Si atom in graphene as shown in Fig.6(g-i) was performed by Suenaga et al.[5]and Zhou et al.[51]respectively,which demonstrated the atomic resolution study of structure and bonding in novel 2D materials.Recently,the atomic structures of a free-standing monolayer amorphous carbon (MAC) were identified by theCscorrected TEM[52],which is a significant progress on the cognition of non-crystalline solids.These atomic resolution studies of intrinsic structures of novel 2D materials are vitally important for basic understanding of these materials as well as for potential electronic,mechanical,thermal,and chemical applications.

3.2 Characterization of graphene-based metal single atom catalysts

Metal SACs have been attracting increasing attention in recent years owing to their maximum metal atom utilization efficiency and incredible performance in several key catalytic reactions such as hydrogenation,hydrogen energy and CO2conversion[53,54].The unique one-atom-thick structure of graphene with modifiable defects by oxygen or nitrogen functional groups can be an excellent support for SACs,which also make graphene-based SACs an excellent model system to probe and quantitatively establish the correlation between the atomistic structure of the welldefined single metal active sites and the catalytic properties.ADF imaging in sub-?ngstr?m resolution with low voltage TEM gives a better contrast between metal atoms and light atoms,such as C,N and O,in the graphene support.Thus,it provides an opportunity of elucidating atomic structure and ligand field of active sites at atomic level,which is the basis of comprehensive understanding the catalytic mechanisms of graphene-based SACs with density functional theory.The following shows some of significant examples of ADF-STEM studies on the structure–activity relationships and the enhanced catalysis mechanisms of graphene-based SACs.

Isolated single Pt atoms anchored to graphene nanosheet was obtained by using the atomic layer deposition technique by Sun et al.[55](Fig.7(a)),which exhibited 10 times improved methanol oxidation catalytic activity over that of commercial Pt/C catalyst.Single Ni anchored to nanoporous graphene and single cobalt on nitrogen-doped graphene (Co-NG)catalysts for hydrogen evolution reaction (HER) were synthesized respectively by Qiu et al.[56](Fig.7(b))and Fei et al.[57]with high activity and stability in aqueous media,which demonstrated the success of SACs achieved in inorganic solid state HER catalysts.A series of monodispersed Fe,Co,Ni embedded in NG with a MN4C4structural configuration (M represents atomic transition metal) was reported again by Fei et al.[58]for oxygen evolution reaction (OER),and the excellent catalytic activity and mechanistic pathways were illustrated at atomic level.

Fig.7 STEM characterization of carbon-supported SACs.(a) ADF image of Pt/graphene catalyst for methanol oxidation[55].(b) HAADF image of single Ni doped graphene for HER[56].(c) ADF image of Nb atoms anchored in onion-like carbon shells used for ORR catalysis[9].(d) ADF image of dispersed single W atoms in high-defective graphene layers of carbon onions for ORR catalysis[59].(f) ADF image Pt atoms in carbon onions,which is catalytically active for chemoselective hydrogenation of nitroarenes[60].

Guo’s group developed a series of SACs trapped in defect-rich graphitic shells of carbon nanoonions(Fig.7(c-e)),which facilitates the loading level and stability of isolated metal atoms.The accurate structure characterization by state-of-the-art STEM elucidated that the lattice defects in graphene layers of carbon nanoonions played the key role of anchoring single metal atoms as the catalytic active sites.Thus,the high stability and catalytic activity originate from the interaction of metal atoms and the intrinsic defects in carbon onions,and highly depend on the controlling of distribution of these defects which can balance mass and charge transfer during catalysis.These low voltage STEM study demonstrates that determination of atomistic structure and its correlation with catalytic properties represents a critical step towards the rational design and synthesis of SACs with exceptional atom utilization efficiency and catalytic activities.

In addition,defective graphitic layers trapped with metal atoms or nanoparticles can also serve as an electromagnetic wave absorption material.Aberrationcorrected STEM studies revealed that the electromagnetic response originated from the intrinsic polarization of metals in the composites,and can be tuned by the coupling of metals and graphitic layers[7,61].

3.3 Electron beam induced in situ characterization of graphene-based materials

Although TEM operated under a certain threshold voltage can significantly eliminate the knock-on damage of graphene-based materials,a well-controlled beam energy can provide an energy bath for the materials and then drive a defect reconstruction or movement.This dynamic observation with a real-time atomic resolution starts the in situ nanoengineering and in situ TEM,which benefits greatly for the structure dynamics of carbon materials.Dynamics of edge reconstruction and hole growth were studied with aCscorrected HRTEM at 80 kV,and the results demonstrated that electron beam irradiation induced the unsaturated carbon atoms sputtered from graphene,by which holes began to grow from initial vacancies/defects in the lattice with a preferred zigzag edge[62,63].The edge evolution of h-BN under an electron beam of 60 kV,as mentioned above in Fig.6(e,f),demonstrated clearly the preferred zigzag edge with N terminations in the h-BN monolayer.Dynamics of dislocation pairs in graphene was recorded with atom resolution[64].The stepwise dislocation movement along the zigzag lattice direction mediated either by a single bond rotation or through the loss of two carbon atoms with strain field localized around the dislocation core.Single Ni atoms on a graphene monolayer was also studied atom by atom dynamically,as shown in Fig.8,where Ni atoms were active and behaved as an“atomic knife”to cut the graphene sheet to form a graphene nanomesh[65],which implicated the precise structure control of graphene for future applications such as sensing,selective separations,biotechnology and catalysis.

Fig.8 Dynamic structure evolutions of graphene-based materials.(a-c) STEM-ADF images show dynamics of a Ni atom at t=1 s,2 s,and 3 s in a graphene nanomesh,respectively[65].

Recently,the in situ TEM technique makes it possible to investigate the evolution of carbon-based materials under heat,strain,magnetic field,electric field or chemical reaction environments[66,67],which is essentially beneficial for understanding the relationships between the atomic structures and their properties.A HER electrocatalyst of graphene nanospheres supported Ru nanoparticles (Ru@GNs) has been observed with an in situ heating TEM,as shown in Fig.9 (a-f)[68],where the defective GNs with tunable defect distribution and graphitization degree were accurately regulated by annealing at various temperatures,and adopted to support Ru nanoparticles towards the optimal HER electrocatalyst.The results agree with the previous conclusion that defects in the graphene shell enhance the stability and dispersion of enclosed metals,and the interaction between metals and graphene shells contribute to the excellent activity of the catalyst.The atomic resolution structureactivity relationship studies open up the possibility for developing novel industrial catalysts.

Fig.9 HRTEM image series demonstrate structure evolutions of (a-c)graphitic shells and (d-f) aggregation of Ru nanoparticles in Ru@GNs catalyst during in situ heating[68].

3.4 Characterization of biomass materials

Biomass materials are mainly composed of cellulose,hemicellulose and lignin,which have the most abundant natural aromatic polymers in the world[69,70].Converting biomass into carbon materials is of tremendous interest in energy storage and conversion in batteries and supercapacitors,environment remediation,gas separation and storage,oil absorption,drug delivery,catalyst,etc[70–72].TEM characterization has also been applied in biomass materials[69,70,73],and low voltage STEM is more necessarily required for their beam sensitive local structure elucidation.Guo et al.studied the atomic structures of a wood-based nanoporous carbon,as shown in Fig.10.Topological defects of connected five-and seven-atom rings in the nanoporous carbon induce curvature of graphene sheets and thus abundant nanopores,which lead to an enhanced adsorption of H2molecules[73].STEM observation indicates that carbonized cellulose nanocrystals (C-CNC) are composed of random disordered carbon while carbonized lignin (C-lignin) are composed of large domains of well-ordered carbon with a graphite sheet structure,which interpreted the high surface area and porosity of C-CNC[70].

Fig.10 (a,b) ADF STEM images of a wood-based nanoporous carbon with large areas of hexagonal lattice (marked in blue) and a few five-and seven-atom ring defects (marked in red).(c) Segment of a simulated defective graphene sheet,with five to seven dislocation structures arranged similarly to microscopy observations.

3.5 Characterization of MOFs

MOFs are a class of highly porous materials whose chemistry and structure can be tuned for potential applications in gas storage,separations and catalysis[18–20,74].They contain a hybrid of inorganic and organic materials and are usually electrically insulated.Since crystallinity of MOFs is dependent on the metal-ligand bonds that can easily be damaged by radiolysis,amorphization will occur rapidly even with relatively low electron doses of～50 e???2.Thus,low electron dose is necessary,and the sensitivity and SNR must be increased.A single electron counting direct detection (SECDD) camera launched by Gatan can operate in the electron-counting mode with the dose-fractionation function.The hundreds of frame rate enhances both contrast and resolution for low dose TEM.In STEM mode,the movement of the center of mass (COM) of a convergent beam electron diffraction pattern is linearly related to the projected electrical field in a thin sample,which can be measured by using a differential phase contrast (DPC) detector consisting of 4 segmented quadrants.The resulting integrated DPC (iDPC) image enables the linear imaging of the projected electrostatic potential in specimen,whereas ADF images its square.In addition,the integration process will naturally reject the nonintegrable vector field,such as the noises,to obtain the corresponding iDPC-STEM image.Thus a much lower dynamic range as well as a much lower dose is needed to image light elements together with heavy elements[75].These new techniques enable the atomic imaging of bulk and local structures of MOFs with low dose TEM operated at a normal accelerating voltage,and the progress in recent years is shown in Fig.11.

Surface structures of MIL-101,a typical MOF material,were revealed using low dose iDPC-STEM and low dose TEM with a SECDD camera,as shown in Fig.11 (a-f)[20]and Fig.11(g,h)[19],respectively.Atomic resolution imaging of MIL-101 crystals with visible giant cages and super tetrahedrons consisting of Cr nodes and 1,4-benzene dicarboxylate (BDC)linkers have been achieved. Individual atomic columns of Zn and organic linkers in the framework of ZIF-8 was resolved by a low dose TEM with a SECDD camera[18].ZnN4tetrahedra displayed as a dark triplet,and the void channels of ZIF-8 displayed a bright contrast in the center of imidazole rings in the contrast transfer function corrected (CTF-corrected)and denoised image.These local structural features and evolutions of these self-assembled MOF crystals allow a better understanding of their crystal growth and structure-performance relations.

Fig.11 Characterization of MOFs with low dose electron microscopy.(a,b) iDPC-STEM image of MIL-101 (111) surface with different termination cages (Scale bars:5 nm).(c,d) The structures of single unit cells at the two types of surface terminations (Scale bars:3 nm).(e,f) The structural models show the (111) surfaces terminated by different cages[20].(g) HRTEM image of sublayer (ī11) surface of MIL-101 (Scale bar:5 nm).(h) Magnified image showing the evolution from sublayer to stable (111) surface(Scale bar:2 nm).[19](i) Cryo-TEM image of CO2-filled ZIF-8 particle along the ＜111＞ projection,with Zn clusters and adsorbed CO2 molecules being identified.[74] In the referred work above,total electron dose for iDPCSTEM is 40 e-?–2,and electron dose for low dose TEM is usually less than 10.4 e-?–2 accumulated from tens of dose fractionation frames.

Another strategy of further reducing electron beam radiolysis for MOFs is cryo-TEM,where samples are plunged into liquid nitrogen (or liquid helium) and isolated from air.Cryogenic temperatures in cryo-TEM can significantly reduce radiolysis damage without needing to lower the accelerating voltage.Surface structures of ZIF-8 and guest molecules CO2adsorbed within its sub-nanometer pore cavities were stabilized and captured with a SECDD camera for the first time by Yi Cui’s group[74],and the result are shown in Fig.11(i).Contrast of the CTF-corrected image was inversed and thus bright regions corresponded to mass density.Bright dots of the hexagonal honeycomb window correspond to Zn clusters at the unit cell vertices,and density at the center of the unit cell likely corresponds to CO2adsorbed within ZIF-8.Not only the bonding between the Zn2+and 2-methylimidazole of the ZIF-8 framework,but also the weak interaction between CO2and framework are preserved in cryo-TEM.These stark observations provide molecular scale insight for particle growth and gas adsorption.

3.6 Characterization of SEI in LIBs

Cryo-TEM has been introduced to researches of lithium-ion batteries (LIBs) since 2017[21].A typical LIB consists of a negative electrode (anode),a positive electrode (cathode),a polymer separator,and an organic electrolyte.Many components of the LIB,such as some lithium-containing electrode materials,organic electrolytes,solid electrolyte interphase (SEI)layer,are chemically reactive and sensitive to electron beam irradiation,which severely hinders their structure characterization and restrains the research of LIB materials in a low-efficiency trial-and-error paradigm.The emergence of cryo-TEM has enabled the nondestructive characterization of electron beam sensitive energy materials in the microscale and nanoscale,and even at atomic scale resolution,affording deep insights into the primary chemistry and physics of working batteries.Applications of cryo-TEM on the research of LIBs have been summarized in recent reviews[76,77].Here,the contribution of cryo-TEM on the structure determination of carbon-based SEI layers are discussed.

A SEI layer is formed during organic electrolyte decomposition on the surfaces of battery anodes,which is composed of organics and inorganics.The fragile SEI influences the structure of Li dendrites at the anode in carbonate-based electrolytes and the lifespan of LIBs.Recently,the atomic structures of SEI have been resolved at Yi Cui’s group by cryo-TEM.The SEI layer,which was formed in the widely used carbonate-based electrolyte ethylene carbonatediethyl carbonate (EC-DEC),contains small crystalline domains with a diameter of～3 nm (LiO2and Li2CO3) dispersed randomly throughout an amorphous polymer matrix that coats the Li metal.The SEI,which was formed in a carbonate-based electrolyte with a volume fraction of 10% fluoroethylene carbonate (FEC),had a more ordered multilayer structure of Li oxide with clear lattice fringes in the outer and the amorphous polymer matrix in the inner.It is convinced that the ordered multilayer structure could offer increased mechanical durability and battery cycling life.Study on the interface between the solid polymer electrolyte (SPE) and the Li anode with cryo-TEM was reported by Ouwei Sheng et al.[78]as shown in Fig.12.The clearly resolved mosaic structure of SEI with crystalline Li (red),Li2O (yellow),LiOH(white) and Li2O3(blue) nanocrystals opened up a new avenue for modifying the Li/SPE interface to realize a stable all-solid-state LMBs.

Fig.12 Atomic resolution cryo-TEM of SEI.(a-c) HRTEM images of the interface between crystalline Li,Li2O,LiOH and amorphous SPE.(d) Schematic diagram for the identified mosaic structure of SEI.

3.7 Characterization of biomacromolecules

More widely,cryo-TEM are used in structural biology,which has moved biochemistry into a new era of atomic scale.The Nobel Prize in Chemistry 2017 was awarded jointly to Jacques Dubochet,Joachim Frank and Richard Henderson "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solutions"[79].In cryo-TEM,everything is frozen ultrarapidly in amorphous ice and the position of individual atoms in a biomacromolecule can be unambiguously discern at a resolution of around 0.12 nm to data[22].Fig.13 demonstrates an example of structural elucidation of SARS-CoV-2 virus,which is responsible for COVID-19 pandemic,by Sai Li’s group using cryo-TEM[80].The architecture and assembly of SARS-CoV-2 provide an opportunity for efficient vaccine design and neutralizing antibody development at molecular level with the most details to date.Spliceosome Bactcomplex,which carries out the splicing of a precursor messenger RNA in exceptional detail,was resolved at an atomic resolution of 0.25 nm by Yigong Shi’s group[81].The elucidation of Bactcomplex and its remodeling mechanism is a first success in this field.These two achievements demonstrate the leading contributions of Chinese scientists in molecular biology.Undoubtedly,researchers will be able to understand how biomacromolecules work in unprecedented detail in health and disease,and lead to better drugs with fewer side effects by achieving atomic resolution.

Fig.13 Atomic architecture of the authentic SARS-CoV-2 virus,which is responsible for COVID-19 pandemic,resolved by cryo-TEM[80].

4 Summary and perspective

In this review,remarkable progresses of theCscorrected TEM with new imaging and spectroscopy techniques are summarized,and their successful applications with respect to beam sensitive novel carbon-based materials are discussed.Atomic resolution local structure and bonding state analysis on delicate materials,such as graphene,h-BN,MAC,carbon-supported SACs,MOFs,SEI in LIBs,biomass materials,and biomacromolecules has been achieved by low voltage,low doseCscorrected TEM equipped with advanced detectors,which could overcome the fundamental key problems pertaining to crystal growth and structure-performance relations of novel materials and devices.

With the help ofCscorrectors,atoms can be measured in TEM with a precision of a few pm,and resolution is limited by the delocalization of thermal vibration and the fragile bonding in beam sensitive materials.Low voltage TEM can reduce the knock-on damage predominant in conducting materials,low electron dose can reduce the radiolysis in insulators and organic specimens,and cryo-TEM can eliminate the heating effect from electron beam,reduce radiolysis and isolate samples from air.The SECDD camera and iDPC detector enable the ultrafast sequential imaging with high SNR.These techniques can be expanded to other novel beam sensitive materials such as organic-inorganic perovskites,Li metals and polymers in LIBs,transition metal carbide/carbonitride(MXene),covalent organic frameworks (COFs),and so on.Functional groups with light elements,molecules or adatoms are more beam sensitive and delocalized,and thus more challenging for directly capture under electron beam.Combination of these new techniques would be able to determine the structure of extremely beam sensitive,and even non-periodic matter such as complicated molecules directly by imaging them atom-by-atom.

On the other hand,in situ TEMs is a spring up field of exploring materials’ true atomic structures under their operating conditions dynamically with a high temporal resolution.Compared with low dose TEM and cryo-TEM,low voltage TEM is expected to exhibit additional advantages on the study of carbonbased materials dynamically and environmentally.Sample holders for dedicated in situ TEM with force,heat,laser pulse,electric field,gas and liquid environments,have already been developed.The dynamic structural evolution under working environments could bring new insights into the better understanding of the structure–performance relationships and benefit to design better catalysts and devices.In summary,the tremendous progresses in gentle TEM enable a new understanding of the atomic scale origins of properties with applications in the materials,chemical,and nanosciences.TEM characterization techniques for beam sensitive materials with ease of use are still changeling and need further development.The future for microscopy has never looked more exciting.

Acknowledgements

National Natural Science Foundation of China(52001222);Natural Science Foundation of Shanxi Province (20191102004,201901D111107,201901D211046);Scientific and Technological Innovation Programs of Higher Education Institutions in Shanxi (STIP,2019L0120,2019L0253,2019L0346);Program for the Innovative Talents of Higher Education Institutions in Shanxi;Special Foundation for Youth SanJin Scholars;Overseas High-Level Talents Introduction Innovation Team Project.