Name: Cryogenic Scanning Electron Nanobeam Diffraction Workflows for Structural Characterization of Beam-Sensitive Battery Interfaces (Video) | JoVE Journal
Uploaded: 2026-10-01T12:30:00.000+00:00
Duration: 8 min 21 s
Description: Learn cryogenic scanning electron nanobeam diffraction for structural characterization of beam-sensitive battery interfaces. This protocol preserves native states while minimizing electron beam damage in energy storage materials.

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H.J.K. acknowledges support from the Center for Functional Nanomaterials (CFN), which is a U.S. Department of Energy Office of Science User Facility, at Brookhaven National Laboratory under Contract No. DE-SC0012704. The work was partly supported from the National Science Foundation (NSF), Division of Materials Research (DMR), Future Manufacturing Research Grant #2134715. This work was performed in part at the Singh Center for Nanotechnology at the University of Pennsylvania, a member of the National Nanotechnology Coordinated Infrastructure (NNCI) network, which is supported by the National Science Foundation (Grant NNCI-2025608). Additional support for the Nanoscale Characterization Facility at the Singh Center is provided by the NSF through the University of Pennsylvania Materials Research Science and Engineering Center (MRSEC) (DMR-2309043). H.J.K and E.A.S acknowledge the technical support of Saskia Mimietz-Oeckler from Leica Microsystems.

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<li>Gao, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((LiF+artifacts+in+XPS+analysis+of+the+SEI+for+lithium+metal+batteries%5BTitle%5D)+AND+%22ACS+Appl+Mater+Interfaces%22%5BJournal%5D)">LiF artifacts in XPS analysis of the SEI for lithium metal batteries.</a> ACS Appl Mater Interfaces. 17 (5), 8513-8525 (2025).</li>
<li>Zachman, M. J., Tu, Z., Choudhury, S., Archer, L. A., Kourkoutis, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-STEM+mapping+of+solid-liquid+interfaces+and+dendrites+in+lithium-metal+batteries%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Cryo-STEM mapping of solid-liquid interfaces and dendrites in lithium-metal batteries.</a> Nature. 560 (7718), 345-349 (2018).</li>
<li>Koh, H., Das, S., Zhang, Y., Detsi, E., Stach, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electron+beam-induced+artifacts+in+SEI+characterization%3A+Evidence+from+controlled-dose+diffraction+studies%5BTitle%5D)+AND+%22ACS+Energy+Lett%22%5BJournal%5D)">Electron beam-induced artifacts in SEI characterization: Evidence from controlled-dose diffraction studies.</a> ACS Energy Lett. 10, 534-540 (2025).</li>
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<li>Zuo, J. -M., Tao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Scanning+electron+nanodiffraction+and+diffraction+imaging%5BTitle%5D)+AND+%22Scanning+Transmission+Electron+Microscopy%3A+Imaging+and+Analysis%22%5BJournal%5D)">Scanning electron nanodiffraction and diffraction imaging.</a> Scanning Transmission Electron Microscopy: Imaging and Analysis. , 393-427 (2011).</li>
<li>Bustillo, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((4D-STEM+of+beam-sensitive+materials%5BTitle%5D)+AND+%22Acc+Chem+Res%22%5BJournal%5D)">4D-STEM of beam-sensitive materials.</a> Acc Chem Res. 54 (11), 2543-2551 (2021).</li>
<li>Voyles, P. M., Muller, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fluctuation+microscopy+in+the+STEM%5BTitle%5D)+AND+%22Ultramicroscopy%22%5BJournal%5D)">Fluctuation microscopy in the STEM.</a> Ultramicroscopy. 93 (2), 147-159 (2002).</li>
<li>Yousaf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Visualization+of+battery+materials+and+their+interfaces%2Finterphases+using+cryogenic+electron+microscopy%5BTitle%5D)+AND+%22Mater+Today%22%5BJournal%5D)">Visualization of battery materials and their interfaces/interphases using cryogenic electron microscopy.</a> Mater Today. 58, 238-274 (2022).</li>
<li>Wang, X., Li, Y., Meng, Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryogenic+electron+microscopy+for+characterizing+and+diagnosing+batteries%5BTitle%5D)+AND+%22Joule%22%5BJournal%5D)">Cryogenic electron microscopy for characterizing and diagnosing batteries.</a> Joule. 2 (11), 2225-2234 (2018).</li>
<li>Zachman, M. J., Asenath-Smith, E., Estroff, L. A., Kourkoutis, L. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Site-specific+preparation+of+intact+solid-liquid+interfaces+by+label-free+in+situ+localization+and+cryo-focused+ion+beam+lift-out%5BTitle%5D)+AND+%22Microsc+Microanal%22%5BJournal%5D)">Site-specific preparation of intact solid-liquid interfaces by label-free in situ localization and cryo-focused ion beam lift-out.</a> Microsc Microanal. 22 (6), 1338-1349 (2016).</li>
<li>Koh, H., Detsi, E., Stach, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Understanding+ion-beam+damage+to+air-sensitive+lithium+metal+with+cryogenic+electron+and+ion+microscopy%5BTitle%5D)+AND+%22Microsc+Microanal%22%5BJournal%5D)">Understanding ion-beam damage to air-sensitive lithium metal with cryogenic electron and ion microscopy.</a> Microsc Microanal. 29 (4), 1350-1356 (2023).</li>
<li>Creekmore, B. C., Kixmoeller, K., Black, B. E., Lee, E. B., Chang, Y. -W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultrastructure+of+human+brain+tissue+vitrified+from+autopsy+revealed+by+cryo-ET+with+cryo-plasma+FIB+milling%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Ultrastructure of human brain tissue vitrified from autopsy revealed by cryo-ET with cryo-plasma FIB milling.</a> Nat Commun. 15 (1), 2660(2024).</li>
<li>Parmenter, C. D., Nizamudeen, Z. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cryo-FIB-lift-out%3A+Practically+impossible+to+practical+reality%5BTitle%5D)+AND+%22J+Microsc%22%5BJournal%5D)">Cryo-FIB-lift-out: Practically impossible to practical reality.</a> J Microsc. 281 (2), 157-174 (2021).</li>
<li>Schaffer, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+cryo-FIB+lift-out+technique+enables+molecular-resolution+cryo-ET+within+native+Caenorhabditis+elegans+tissue%5BTitle%5D)+AND+%22Nat+Methods%22%5BJournal%5D)">A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.</a> Nat Methods. 16 (8), 757-762 (2019).</li>
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The authors declare no competing financial interest. One co-author is an employee of Leica Microsystems. Leica Microsystems did not influence the study design, data collection, analysis, or interpretation.

There are two critical steps in successfully carrying out these experiments. The first is to avoid frost and ice formation on the TEM samples. As discussed above, SEIs are beam-sensitive and prone to structural changes under excessive electron-beam irradiation. Ice can be removed with electron beam irradiation, but doing so may require a large electron dose, especially for a thick layer of ice, as in Figure 1B. Once ice forms, the remaining allowable electron dose before sample damage is quickly consumed during the SEND and EELS measurements. In unfortunate cases, the structures can be completely damaged before actual measurements begin. Therefore, minimizing frost and preventing the formation of large ice crystals are essential. Using our newly designed cryo-FIB/SEM shuttle, we demonstrate&#160;ice-free transfer between cryo-FIB/SEM and cryo-TEM.
The second critical step is controlling the electron beam dose. SEND is well-suited for controlling electron dose, which is not possible in high-resolution TEM techniques. By controlling convergence angles and step sizes, precise control of the electron dose is possible. Unlike TEM workflows, it does not require repeated focus/defocus adjustments or high-dose navigation of samples, both of which can expose samples to high accumulated dose, especially at high magnification. Furthermore, high-resolution TEM imaging requires atomic-scale stability. Otherwise, image quality degrades. As such, achieving good-quality images often requires taking multiple exposures, which increases the accumulated electron dose9. However, these scenarios are not well-suited for beam-sensitive SEIs. On the other hand, SEND allows all acquisitions to be performed at low magnification, with only the desired electron dose passing through the region of interest. At the end of the Discussion, we listed a troubleshooting subsection.
A limitation of SEND for SEI characterization is its spatial resolution, which is typically limited to a range of sub-nanometers to a few nanometers. This is because of the trade-off between real-space resolution and reciprocal-space signal-to-noise ratio. Increasing the convergence semi-angle reduces diffraction intensity, which is acceptable for crystalline materials, where diffraction signals are inherently strong and sometimes further enhanced by channeling effects23. However, for amorphous materials like SEIs, the convergence semi-angle must remain small enough to maintain sufficient diffraction intensity while still achieving sub-nanometer probe sizes. In our experiments, we optimized the convergence semi-angle to 2 mrad with a probe size of 1 nm.
In practice, the step size cannot be as small as the probe size. When beam damage is taken into account, the actual step size should be around a few nm, and a smaller step size exceeds the critical dose because the SEIs are destroyed. As a result, the lower spatial resolution of SEND relative to HRTEM may limit our ability to resolve nanoscale features since some SEI layers are 1-10 nm thick. The challenge becomes even greater when electrodes and vitrified electrolytes overlap in projection, making it difficult to resolve SEIs sandwiched between them. Non-uniform electrode thickness can also complicate 3D-to-2D projection during analysis. Accordingly, several challenges and methodological trade-offs must be carefully considered to optimize SEND characterization of SEIs.
Although we demonstrated this technique primarily on SEIs formed in liquid electrolytes, this approach is also applicable to interfaces between solid electrodes and solid electrolytes. Preserving the native state at these interfaces is difficult as they can be easily deformed and damaged during disassembly, and the substantial thickness of solid electrolytes makes cross-sectioning with ion beam milling difficult. Nevertheless, optimized FIB strategies may enable access to these interfaces. For example, by milling from the current collector side and precisely controlling the Li deposition thickness, it may be feasible to prepare ~20 &#181;m-deep lamellae suitable for cryo-SEND experiments.
Troubleshooting and practical guidance 
Frost or ice contamination 
Frost contamination during cryogenic transfer is a common failure mode and may appear as speckles of ice particles in STEM-HAADF images. In TEM images, ice is often not readily visible, but underfocus or overfocus can make it more apparent. The presence of diffraction patterns from hexagonal or cubic ice crystals also indicates contamination and can overlap with signals from SEIs, potentially compromising structural interpretation.
When severe ice is present, one mitigation strategy is to use a parallel electron beam at ~10 e&#8315;/&#197;2&#183;s to remove the ice particles. While this can introduce damage to beam-sensitive regions, it may still enable collection of usable diffraction data and serve as a reference for assessing SEI beam tolerance. Preventive strategies include improving cryo-transfer conditions by minimizing ambient exposure time, ensuring the shuttle is sufficiently cold, and using a dry room or dehumidified environment. A lidded cryo-FIB/SEM shuttle is highly recommended. It is also important to note that hexagonal and nanocrystalline ice typically form due to inadequate transfer control, whereas large cubic ice crystals are often introduced by impure liquid nitrogen. Therefore, clean liquid nitrogen should always be used when in contact with lamellae.
Beam-induced damage 
Structural degradation from electron beam exposure is another significant concern. Indicators of beam damage include progressive changes in diffraction features, such as a shift from amorphous to crystalline structures, particularly the formation of Li&#8322;O. These changes may become irreversible after repeated scans. EELS data may also exhibit progressive spectral shifts, and in severe cases, vitrified electrolytes can produce gas bubbles when rastered at high magnification.
To reduce the risk of beam damage, users should lower the pixel time to decrease total accumulated dose. Additional mitigation strategies include reducing dwell time, increasing step size, and using low magnification when selecting imaging parameters and defining regions of interest. These adjustments help preserve native SEI structure during data acquisition.

Optimizing battery interfaces, particularly solid-electrolyte interphases (SEIs), is critical for stable and high-performance rechargeable batteries1. SEIs form as decomposition products from the reactions between liquid or solid electrolytes and battery electrodes. Once they form, these layers regulate ion and electron transport. Insufficient ion transport imposes significant barriers for ion mobility and results in high overpotentials in battery systems, while electrolytes are continuously consumed if they are not electronically passivated. Therefore, functional SEIs must promote fast ion transport and suppress electron leakage to sustain long-term cycling.
Li metal batteries are promising because they can improve the energy density of battery systems compared to conventional graphite-based systems1, but the SEIs that form on Li metal are chemically complex and highly sensitive to electrochemical conditions2. However, the required criteria for SEI stability become more stringent as the field pushes toward the practical systems with high mass loadings and fast charging conditions. Describing SEIs simply as either &#34;good&#34; or &#34;poor&#34; offers limited insights3. Therefore, spatially resolved characterization for SEIs is essential to uncover the structural and chemical origins of SEI stability.
A wide variety of characterization techniques, such as time-of-flight secondary ion mass spectroscopy (TOF-SIMS)4,5 and X-ray photoelectron spectroscopy (XPS)6,7, are commonly used to analyze SEI composition, but their spatial resolutions are insufficient to resolve heterogeneous and buried structures. Recently, cryogenic transmission electron microscopy (cryo-TEM) has emerged as a powerful tool to elucidate SEI structures3,8. Cryo-TEM in conjunction with high-resolution (HR) imaging has superior spatial resolution when compared to other techniques. Despite this advantage, it is increasingly recognized that conventional HRTEM imaging often applies electron doses that exceed the stability limits of SEI components, leading to beam-induced crystallization or decomposition9,10. As a result, there is a growing need for electron microscopy workflows that explicitly control dose while preserving structural fidelity.
The specific goal of this protocol is to establish a low-dose, cryogenic scanning electron nanobeam diffraction (SEND) workflow tailored for battery interfaces, with an emphasis on reliable SEI structural characterization. SEND is a variant of scanning transmission electron microscopy (STEM), where the convergence semi-angle is varied to balance real space and reciprocal space resolution, and diffraction patterns are captured over a rastered region at each scan position11,12. This results in four-dimensional data sets that include real-space information and reciprocal-space information. By tuning the convergence semi-angle, probe size, and step size, the illumination conditions can be optimized to remain well below the critical dose while still resolving nanoscale ordering, crystallinity, and heterogeneity12.
This workflow is needed because existing cryo-TEM approaches primarily rely on phase-contrast imaging, which couples structural interpretability to relatively high cumulative doses. In contrast, SEND decouples structural sensitivity from direct image formation, allowing short-range and medium-range order to be quantified at doses well below those required for HRTEM10,13. At the protocol level, SEND addresses several key challenges: (i) systematic dose control during data acquisition, (ii) improved structural reliability for beam-sensitive SEI phases, and (iii) integration of cryogenic sample handling with diffraction-based analysis suitable for heterogeneous battery interfaces.
The method described here is intended for the study of beam-sensitive interphases in rechargeable batteries, including SEIs on Li metal and other reactive electrode materials, where characteristic thicknesses are on the order of tens of nanometers. SEND is particularly well-suited for probing nanoscale heterogeneity, amorphous-crystalline mixtures, and short-range ordering. However, its applicability is limited by sample thickness, signal-to-noise constraints at low electron doses, and the need for careful data interpretation in highly disordered systems.
We demonstrate, in this protocol, how to perform SEND for battery research, focusing particularly on sample preparation and data acquisition steps. We also discuss ways to minimize frost formation, especially when creating electron-transparent samples, as frost contamination can obscure SEI features and complicate diffraction analysis. These methods provide detailed guidelines for reproducible, artifact-free characterization of battery interfaces. This technique is not limited to the SEI characterization but also to other systems involving beam-sensitive materials.
Electron transparency is a prerequisite for transmission electron microscopy (TEM). In this work, we demonstrate two complementary workflows to achieve electron-transparent battery specimens: on-grid TEM preparation and cryogenic focused ion beam (cryo-FIB) milling14,15. The on-grid preparation method is effective when samples are intrinsically thin or composed of low-atomic number materials, such as lithium plates or nanoparticles, which can be directly transferred to or electrochemically deposited onto commercial TEM grids. However, this approach can compromise the original structural integrity of battery interfaces. Battery materials are inherently heterogeneous, and preserving their mechanical and interfacial structure during TEM observation is critical. In addition, on-grid protocols often require rinsing to remove liquid electrolytes, which can lead to the loss of loosely bound or fragile SEI components, thereby altering the native interfacial structure.
By contrast, the cryo-FIB method enables site-specific thinning while preserving buried interfaces and vitrified electrolytes, making it the preferred approach when maintaining the native morphology and chemistry of battery interfaces is essential. Cryo-FIB milling provides controlled, reproducible electron transparency across a wide range of battery architectures, regardless of initial sample thickness or the presence of liquid electrolytes. In this protocol, we present both preparation strategies and outline their respective advantages and limitations. We also emphasize the cryo-FIB workflow as a robust method for structural characterization of beam-sensitive battery interfaces.

<table><tbody><tr><td>Conical tubes</td><td>Eppendorf</td><td>339652</td><td></td></tr><tr><td>Cryo FIB Stage, and associated transfer equipment and tools</td><td>Leica Microsystems&amp;nbsp;</td><td></td><td></td></tr><tr><td>DEC (Diethyl carbonate)</td><td>Sigma Aldrich</td><td>D91551</td><td></td></tr><tr><td>Direct electron detector</td><td>Gatan Metro</td><td></td><td></td></tr><tr><td>DME (Dimethoxyethane)</td><td></td><td>259527</td><td>Sigma Aldrich</td></tr><tr><td>EC (Ethylene carbonate)</td><td>Sigma Aldrich</td><td>E26258</td><td></td></tr><tr><td>Kim wipes</td><td></td><td></td><td>laboratory wipes</td></tr><tr><td>Li metal chip</td><td>Xiamen TOB New Energy Technology Co.</td><td></td><td></td></tr><tr><td>LiFSI (Li bis(fluorosulfonyl)imide)</td><td>American Elements</td><td>LI-FSI-03-P</td><td></td></tr><tr><td>LiPF6 (lithium hexafluorophosphate)</td><td></td><td>013950</td><td>Oakwood</td></tr><tr><td>LiTFSI (Lithium bis(trifluoromethanesulfonyl)imide)</td><td>Sigma Aldrich</td><td>919977</td><td></td></tr><tr><td>Mesh TEM grid</td><td>Pelco</td><td>1GC200</td><td></td></tr><tr><td>Microscope</td><td>JEOL&amp;nbsp;</td><td>F200</td><td></td></tr><tr><td>Poly propylene separators</td><td>Celgard&amp;nbsp;</td><td>3501</td><td></td></tr><tr><td>Scintillator based detector</td><td>Gatan Oneview</td><td></td><td></td></tr></tbody></table>

cryogenic scanning electron nanobeam diffraction workflows for structural characterization of beam-sensitive battery interfaces

Cryogenic Scanning Electron Nanobeam Diffraction Workflows for Structural Characterization of Beam-Sensitive Battery Interfaces

Observation of battery interfaces is critical, as they govern electron and ion transport and influence electrochemical processes in energy storage. Despite this importance, they are challenging to characterize due to their nanometer-scale thickness and extreme sensitivity to electron beam damage. This paper presents a dose-controlled cryogenic scanning electron nanobeam diffraction (cryo-SEND) protocol that enables structural analysis of beam-sensitive battery interfaces while preserving their native state. The central advance of this workflow is the integration of ice-free cryogenic handling with diffraction-based analysis that decouples structural sensitivity from high-dose image formation. The workflow begins with air-free sample preparation inside an inert-atmosphere glovebox, followed by cryogenic focused ion beam/scanning electron microscopy (cryo-FIB/SEM) milling to produce electron-transparent lamellae. We describe optimized cryogenic transfer procedures from the FIB to the transmission electron microscope, emphasizing the use of a cryo-shuttle equipped with a protective lid to suppress ice contamination during transport. Within the TEM, SEND is performed at cryogenic temperatures to probe interfacial structures at vitrified electrolyte-lithium metal interfaces. SEND enables crystallographic and structural analysis while significantly reducing total accumulated electron dose compared to conventional imaging. Overall, this protocol provides an approach for studying battery interfaces and is broadly applicable to cryogenic electron microscopy investigations of beam-sensitive materials.

Figure 1&#160;shows examples of good and poor sample preparations for cryo-SEND experiments. When preparing battery samples by cryogenic FIB/SEM, avoiding frost contamination is essential to achieve sufficient electron transparency without introducing artifacts. After lamellae are created in the cryo-FIB/SEM stage, the samples are moved to ambient environments. The lamellae are highly vulnerable to ice formation until it is fully submerged in liquid nitrogen. To mitigate this issue, we developed a cryogenic FIB/SEM shuttle with a cover attachment to prevent ice contamination.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/70455/70455fig1.jpg" /> 
Figure 1: Good and poor examples for cryo-SEND preparations. (A) SEM image of a cryo-liftout of the Li metal deposits formed in a conventional carbonate electrolyte (1 M LiPF6 in ethylene carbonate/DMC (1:1 v/v ratio). (B) Cryo-SEM image of the same lamella after the transfer from the cryo-FIB/SEM stage. The lamella is severely contaminated with heavy frost. (C) Another cryo-liftout from the Li metal depositions formed in a high-concentration ether electrolyte (4 M LiFSI in DME). (D) Cryo-TEM image of the lamella after the transfer through the shuttle attached with a cover. No ice contamination was visible after the transfer. The 110 reflection of Li is clearly visible, and no diffraction from ice was observed. (E) A magnified STEM image with a well-preserved feature of battery morphology, suitable for further cryo-SEND experiments. Abbreviations: SEND = scanning electron nanobeam diffraction; DMC = dimethyl carbonate; DME = dimethoxyethane. <a href="https://www.jove.com/files/ftp_upload/70455/70455fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 1A,B illustrate a case where ice contamination is severe, and subsequent analysis is heavily impaired by the presence of ice layers. Although little ice is observed at the time of milling in the FIB/SEM chamber, heavy frost layers were deposited on the lamella after the sample transfer in the TEM column, as demonstrated in the HAADF-STEM image. In this case, the transfer procedure was performed using a standard SEM stub without a protective cover. When the shuttle equipped with the anticontaminator cover is used, the ice contamination level was markedly reduced, as demonstrated in Figure 1C-E. In detail, a lift-out lamella was prepared in the cryo-FIB/SEM stage (Figure 1C) and transferred back into liquid nitrogen. During this step, when the vacuum was released, the protective cover prevented exposure to ambient moisture, keeping the sample ice-free. Figure 1D shows a clean lift-out lamella with the diffraction shown in the inset, displaying a 110&#160;reflection of Li. This indicates the absence of signatures for hexagonal close-packed (HCP) or cubic ice phases. Although the contrast of ice is more noticeable in STEM mode, the lamella was entirely free of ice in Figure 1E.
While this new SEM/FIB shuttle substantially prevents frost contamination, it is still possible that residual large ice crystals suspended in liquid nitrogen can adhere to lamellae during handling. This cannot be fully eliminated in cryo-EM workflows. However, it can be minimized by handling samples in a dry room (10-15% humidity) and using clean liquid nitrogen that has not been exposed to air for more than 20 min. One may consider removing ice with electron irradiation. However, the battery electrolytes and SEIs are as beam-sensitive as ice, and such an attempt is likely to damage the structures prior to subsequent characterization. The on-grid method has been routinely carried out in multiple studies3,15,17, with minimal frost contamination. This is because it does not require additional sample preparation steps from cryo-FIB/SEM to a TEM column. However, frost formation and large ice crystal attachment can still be problematic in the workflow if experiments are not carefully controlled. As the method inherently creates many regions of interest, ice contamination is less problematic overall.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/70455/70455fig2.jpg" /> 
Figure 2: SEM images of the Li metal grains, vitrified electrolytes, and SEI. (A) Cryo-lift-out lamella of electrochemically deposited lithium metal from an ether electrolyte (a mixture of 4.6 M LiFSI and 2.3 M LiTFSI in DME). Large Li grains were shown, showing that the electrolyte and the resultant SEI played a key role in the creation of dense Li grains. (B) SEI at the interface between the Li metal deposition and the electrolyte imaged by high-angle annular dark-field STEM (HAADF-STEM) image. The generated lamella was thin enough (~100 nm) to resolve all the features of Li metal grains, electrolytes, and SEIs. The magnified STEM image shows that the vitrified electrolytes exhibited few bubbles, which represents an ideal condition for continuous SEND acquisition. Otherwise, the electrolytes released gas bubbles. Scale bars = 5 &#181;m (A), 1 &#181;m (B). The figures were adapted with permission from Ref.9. Copyright 2025 American Chemical Society. Abbreviations: LiFSI = (Lithium bis(fluorosulfonyl)imide) LiTFSI = Lithiumbis(trifluoromethanesulfonyl)imide, DME = Dimethoxyethane, SEI = solid-electrolyte interphase, SEND = scanning electron nanobeam diffraction <a href="https://www.jove.com/files/ftp_upload/70455/70455fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 2A shows an SEM image of the battery components from an electron-transparent cryo-lift-out lamella. A vitrified electrolyte was well preserved, and a corresponding HAADF-STEM in Figure 2B reveals a 40 nm-thick battery interface layer between the Li metal deposition and the vitrified electrolyte,&#160;where cryo-SEND experiments will be performed. Again, the lamella is free of ice, allowing us to maximize the number of usable regions for structural characterization. The presence of the SEI layer between the electrolyte and the Li metal grains indicates that the sample preparation protocol was successful.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/70455/70455fig3.jpg" /> 
Figure 3: Diffraction analysis of the SEIs formed in the two battery electrolytes. The high concentration in the figure refers to the mixture of 4.6 M LiFSI and 2.3 M LiTFSI in DME. (A) HAADF-STEM images from the Li metal depositions formed in each electrolyte (left) and the diffraction patterns of the SEI from their corresponding electrolytes. (B) Diffraction profiles from the SEI reveal that SEIs are structurally distinct. Scale bars in A are 100 nm for the STEM images and 5 nm-1 for the diffraction patterns, respectively. The structures of SEIs are identified as amorphous, which is the key result of the careful dose selection described in the protocol. In addition, the lamella without ice provided diffraction patterns free from frost-related diffraction, allowing us to isolate the diffraction patterns of the SEIs. The figures were adapted with permission from Ref.10. Copyright 2025 American Chemical Society. Abbreviations: LiFSI = (Lithium bis(fluorosulfonyl)imide) LiTFSI = Lithiumbis(trifluoromethanesulfonyl)imide, SEI = solid-electrolyte interphase, HAADF-STEM = high-angle annular dark-field scanning transmission&#160;electron microscopy. <a href="https://www.jove.com/files/ftp_upload/70455/70455fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Figure 3A compares the structure of SEIs formed in a low-concentration ether electrolyte (1 M LiFSI in dimethoxyethane (DME)) and a high-concentration ether electrolyte (4.6 M LiFSI and 2.3 M LiTFSI in DME). After cryo-SEND at a controlled electron dose, the structures appear amorphous, as indicated by the isotropic features in the SEIs. Within the ~1 nm probe-size resolution, the amorphous features are smaller than the length scale of the probe, resulting in the isotropic amorphous features. The SEI structures are distinct: the SEI from the mild-concentration electrolyte did not show a detectable structural correlation, whereas the SEI from the high-concentration electrolyte exhibits short-range order. The short-range order exhibits two characteristic bond features, with maxima at approximately 0.3 and 0.65 &#197;-1, as shown in Figure 3B. This indicates that SEIs from the two distinct electrolyte systems form structurally distinct SEIs, which may influence their mechanical and chemical properties. These conclusions are based on diffraction measurements acquired from multiple regions (typically 10-20 regions) within the same lamella, and were reproduced across at least two independently prepared lamellae for each electrolyte system. The corresponding diffraction mapping associated with these measurements is reported in Ref.10.
Furthermore, rigorous regulation of accumulated dose is essential to improve the data reliability and obtain the chemical and structural information in native states (Figure 4). We found that the native structures of SEIs can be severely damaged by excessive beam dose. For example, when the dose is limited to ~500 e-/&#197;2, the SEI structure formed in the high-concentration electrolyte remained amorphous, but began to crystallize when the accumulated dose exceeded the threshold. The EELS spectra in Figure 4A,B show the progressive formation of Li2O with electron dose. Repetitive SEND measurements confirm that the beam exposure drives SEIs to form Li2O (Figure 4C). The critical electron dose for different electrolytes was studied using selected-area electron diffraction9. The high-concentration ether-based electrolyte shows nanocrystal nucleation at doses above 500 e-/&#197;2. In contrast, the critical dose for 1 M-concentrated electrolytes was approximately 1000 e-/&#197;2.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/70455/70455fig4.jpg" /> 
Figure 4: Beam damage study of SEIs. (A) Li K-edge and (B) O-K edge of the SEIs from the high concentration electrolytes with electron dose. (C) Diffraction profiles of the SEI before and after the excessive electron dose. The native SEIs are free of Li2O with broad spectra in both Li and O K-edge, but they can easily form Li2O nanocrystals when excessive doses above the threshold are used. Therefore, the use of the recommended dose is important. Otherwise, the SEIs are readily damaged, leading to structural change. The figures were adapted with permission from Ref.9,10. Copyright 2025 American Chemical Society. <a href="https://www.jove.com/files/ftp_upload/70455/70455fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes a dose-controlled cryogenic scanning electron nanobeam diffraction (cryo-SEND) workflow for characterizing beam-sensitive battery interfaces. It preserves native solid-electrolyte interphases by combining ice-free cryogenic handling, electron-dose control, and diffraction-based imaging. The workflow includes guidance for sample preparation, cryogenic transfer, beam alignment, and data acquisition.

Observation of battery interfaces is critical, as they govern electron and ion transport and influence electrochemical processes in energy storage. ...

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JoVE Journal

Engineering

We describe the use of synchrotron X-ray absorption spectroscopy (XAS) and X-ray diffraction (XRD) techniques to probe details of intercalation/deintercalation processes in electrode materials for Li-ion and Na-ion batteries. Both in situ and ex situ experiments are used to understand structural behavior relevant to the operation of...

Characterization of Electrode Materials for Lithium Ion and Sodium Ion Batteries Using Synchrotron Radiation Techniques

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In Situ Detection and Single Cell Quantification of Metal Oxide Nanoparticles Using Nuclear Microprobe Analysis

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Workflow and Tools for Crystallographic Fragment Screening at the Helmholtz-Zentrum Berlin

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A Sample Preparation Pipeline for Microcrystals at the VMXm Beamline

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Workflow Using a Cryogenic Coincident Fluorescence, Electron, and Ion Beam Microscope for Targeted Milling of Cells

Learn cryogenic scanning electron nanobeam diffraction for structural characterization of beam-sensitive battery interfaces. This protocol preserves native states while minimizing electron beam damage in energy storage materials.

Cryogenic Scanning Electron Nanobeam Diffraction Workflows for Structural Characterization of Beam-Sensitive Battery Interfaces

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