Cryogenic focused ion beam (Cryo-FIB) milling has become a standard step in the cryogenic electron tomography (Cryo-ET) workflow and is required to thin cells to electron-semitransparency. However, this destructive process removes the vast majority of the cellular material and raises a critical question: what thin section should be preserved for Cryo-ET analysis? Using a tri-coincident cryogenic FIB-SEM-LM system, we identify an interferometric optical response that can be used for targeting lamella production to fluorescently labeled structures with accuracy beyond the diffraction limit. Here we demonstrate this approach using synthetic samples of fluorescent beads embedded in micron-scale droplets of amorphous ice. We then apply the approach to capture virions inside host cells. Successful targeting is confirmed by Cryo-ET revealing clusters of virions in intracellular vesicles. The method does not require any fluorescent fiducials or axial registration and can be performed on any fluorescently labeled structure that is visible in widefield fluorescence microscopy. Fluorescence microscopy during CryoFIB milling produces an interferogram that can be used to direct lamella production to labeled structures with accuracy beyond the axial diffraction limit. The approach relies only on real-time feedback from the structure, requiring no image registration.

Cryogenic focused ion beam (FIB) milling is essential for fabricating thin lamella-shaped samples out of frozen-hydrated cells for high-resolution structure determination. Structural information can only be resolved at high resolution if the lamella thickness is between 100 and 200 nm. While the lamella fabrication workflow has improved significantly since its conception, quantitative, live feedback on lamella thickness, quality, and biological target inclusion remains lacking. Using coincident light microscopy integrated into the FIB scanning electron microscope (FIB-SEM), we present three strategies that enable accurate, live control during lamella fabrication. First, we combine four-dimensional (4D) STEM with fluorescence microscopy (FM) targeting to determine lamella thickness. Second, with reflected light microscopy (RLM), we screen target sites for ice contamination and monitor lamella thickness and protective Pt coating integrity during FIB milling. Third, we exploit thin-film interference for fine-grained feedback on thickness uniformity below 500 nm. Finally, we present a fluorescence-targeted, quality-controlled workflow for frozen-hydrated lamellae, benchmarked with excellent agreement with energy-filtered transmission electron microscopy (EFTEM) measurements and tomograms from electron cryotomography.

In volume fluorescence microscopy, refractive index matching is essential to minimize aberrations. There are, however, common imaging scenarios where a refractive index mismatch (RIM) between immersion and a sample medium cannot be avoided. This RIM leads to an axial deformation in the acquired image data. Over the years, different axial scaling factors have been proposed to correct for this deformation. While some reports have suggested a depth -dependent axial deformation, so far none of the scaling theories has accounted for a depth -dependent, non-linear scaling. Here, we derive an analytical theory based on determining the leading constructive interference band in the objective lens pupil under RIM. We then use this to calculate a depth -dependent re -scaling factor as a function of the numerical aperture (NA), the refractive indices n 1 and n 2 , and the wavelength lambda . We compare our theoretical results with wave -optics calculations and experimental results obtained using a measurement scheme for different values of NA and RIM. As a benchmark, we recorded multiple datasets in different RIM conditions, and corrected these using our depth -dependent axial scaling theory. Finally, we present an online web applet that visualizes the depth -dependent axial re -scaling for specific optical setups. In addition, we provide software that will help microscopists to correctly re -scale the axial dimension in their imaging data when working under RIM.

We present a quantitative four-dimensional scanning transmission electron microscopy (4D-STEM)imaging technique (q4STEM) for local thickness estimation across amorphous specimen such asobtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis. Our method isbased on measuring spatially resolved diffraction patterns to obtain the angular distribution of electronscattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitativeevaluation using Monte Carlo simulations. The method is independent of signal intensity calibrationsand only requires knowledge of the detector geometry, which is invariant for a given instrument.We demonstrate that the method yields robust thickness estimates for sub-micrometer amorphousspecimen using both direct detection and light conversion 2D-STEM detectors in a coincident FIBSEMand a conventional SEM. Due to its facile implementation and minimal dose requirements,we anticipate that this method will find applications for in-situ thickness monitoring during lamellafabrication of beam-sensitive materials.

We describe a cryogenic sample chamber for low energy electron microscopy (LEEM), and present first experimental results. Modifications to our IBM/SPECS aberration-corrected LEEM instrument are presented first. These include incorporation of mechanisms for cooling the sample and its surroundings, and reduction of various sources of heat load. Using both liquid nitrogen and liquid helium, we have reached sample temperatures down to about 15 K. We also present first results for low-temperature LEEM, obtained on a three-monolayer pentacene film. Specifically, we observe a reduction of the electron beam irradiation damage cross-section at 15 eV by more than a factor of five upon cooling from 300 K down to 52 K. We also observe changes in the LEEM-IV spectra of the sample upon cooling, and discuss possible causes.

Cryogenic electron tomography (cryo-ET) combined with subtomogram averaging, allows in situ visualization and structure determination of macromolecular complexes at subnanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in situ fluorescence microscopy-guided FIB fabrication of a frozen-hydrated lamella to address this problem: we built a coincident three-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing FIB scanning electron microscope. We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.

In-situ imaging of proteins in cells allows to characterize their native structure, conformation, and interactions with their environment. This can be achieved through electron cryo-tomography but requires the milling of a 100-200 nm thick lamella with a focused ion beam [1]. To include the fluorescent feature of interest in the lamella correlative targeting methods are utilized [2,3]. These methods can however be laborious and prone to contamination of the sample, hindering the acquisition of high-quality electron tomograms. Integration of a light microscope into a cryo-FIB-SEM allows for a simplified workflow to target fluorescent features [4-6]. Regular wide-field fluorescence microscopy, however, does not bring sufficient resolving power to allow localization and targeting of sparse fluorescence features in the 100-200 nm thick lamella. We are therefore implementing 3D localization using astigmatic imaging in an integrated cryo-fluorescence-FIB-SEM [6,7]. The cryogenic conditions pose two unknown constraints on the localization accuracy: (1) a limited numerical aperture (NA) of the objective lens, and (2) cryogenic immobilization of the fluorescence transition dipole moment of the molecule. Using vectorial point spread function (PSF) software [8], we simulated fluorescence microscopy reflecting the conditions of the experimental integrated cryo-fluorescence-FIB-SEM in our lab [6]. This includes a limited NA of 0.85 and induced astigmatism. We evaluated different biological targeting scenarios where the features are composed either of multiple fluorescent molecules ( e.g. fluorescently labeled vesicles) or a single fluorescent

Journal Article A Cryogenic Fluorescence Microscope Retrofittable in Coincidence with a FIB/SEM Get access Daan B Boltje, Daan B Boltje Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The NetherlandsDelmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Corresponding author: boltje@delmic.com Search for other works by this author on: Oxford Academic Google Scholar Jacob P Hoogenboom, Jacob P Hoogenboom Department of Imaging Physics, Delft University of Technology, Lorentzweg 1, 2628 CJ Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Arjen J Jakobi, Arjen J Jakobi Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, 2629 HZ Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Grant J Jensen, Grant J Jensen California Institute of Technology, Pasadena, CA 91125, United States Search for other works by this author on: Oxford Academic Google Scholar Caspar TH Jonker, Caspar TH Jonker Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Abraham J Koster, Abraham J Koster Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Mart GF Last, Mart GF Last Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Jürgen M Plitzko, Jürgen M Plitzko Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, 82152 Planegg-Martinsried, Germany Search for other works by this author on: Oxford Academic Google Scholar Stefan Raunser, Stefan Raunser Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany Search for other works by this author on: Oxford Academic Google Scholar Sebastian Tacke, Sebastian Tacke Department of Structural Biochemistry, Max Planck Institute of Molecular Physiology, Otto-Hahn-Str. 11, 44227 Dortmund, Germany Search for other works by this author on: Oxford Academic Google Scholar ... Show more Roger Wepf, Roger Wepf Centre for Microscopy and Microanalysis, The University of Queensland, St. Lucia Queensland 4072, Brisbane, Australia Search for other works by this author on: Oxford Academic Google Scholar Sander den Hoedt Sander den Hoedt Delmic B.V., Kanaalweg 4, 2326 EB Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1276–1278, https://doi.org/10.1017/S1431927622005256 Published: 01 August 2022

Journal Article Reproducible lamella preparation for electron cryo-tomography by in-situ thickness estimation during fluorescence-guided FIB milling Get access Radim Skoupy, Radim Skoupy Department of Bionanoscience, Kavli Institute of Nanoscience Delft University of Technology, Delft, The NetherlandsDepartment of Imaging Physics, Delft University of Technology, Delft, The Netherlands Corresponding author: R.Skoupy@tudelft.nl Search for other works by this author on: Oxford Academic Google Scholar Daan B Boltje, Daan B Boltje Department of Imaging Physics, Delft University of Technology, Delft, The NetherlandsDelmic B.V., Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Jacob P Hoogenboom, Jacob P Hoogenboom Department of Imaging Physics, Delft University of Technology, Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Arjen J Jakobi Arjen J Jakobi Department of Bionanoscience, Kavli Institute of Nanoscience Delft University of Technology, Delft, The Netherlands Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 28, Issue S1, 1 August 2022, Pages 1210–1211, https://doi.org/10.1017/S1431927622005049 Published: 01 August 2022

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Contemporary scanning laser Kerr magnetometers are very efficient measurement devices that achieve very good signal to noise ratios and spatial resolution for magnetization measurements. When going beyond simple hysteresis loop measurements, large amounts of data can be acquired quickly and, thus, control software and automated processing becomes necessary to go beyond these simple measurements. Here, we present an open source software that realizes first-order reversal curves and magnetic property mapping based on scanning laser Kerr magnetometry. The modular software is easily extensible and provides a convenient user interface to make these more complex measurements accessible to a broader user base.

In this paper, we address the issue of aging of oxide sputtering targets, using the example of La0.7Ca0.3MnO3 (LCMO), a material which is quite sensitive to the amount of oxygen. After prolonged use we find that the morphology of the films becomes poor: holes appear, the size of the steps between terraces becomes larger, the roughness increases, and electrical conductance in the metallic state at temperatures below the metal-insulator transition becomes smaller. We have performed experiments on reactive sputtering with water vapor in order to reverse their degradation. We discuss the growth and properties of films of LCMO on flat SrTiO3 substrates before and after the target treatment. We study both the morphological and structural changes in these films as well as the transport properties. The results indicate that a correct concentration of oxygen in the targets is important, and that a deficiency can be compensated by the water treatment, thus increasing the usable life time of targets.

We investigate thin films of La0.7Sr0.3MnO3 grown on SrTiO3 in which bridges of 100μm long and 1μm or 300nm wide (distance between voltage contacts was 25μm) were defined lithographically. In bridges of 1μm, we find the current–voltage characteristics to be perfectly linear (ohmic) over the full temperature range between 400K and 10K. Strong non-linearities however are observed for a 300nm wide bridge in the temperature regime below the transition temperature. The non-linearities are present even when applying a high magnetic field of 9T.

Nucleation processes of mixed-phase states are an intrinsic characteristic of first-order phase transitions, typically related to local symmetry breaking. Direct observation of emerging mixed-phase regions in materials showing a first-order metal-insulator transition (MIT) offers unique opportunities to uncover their driving mechanism. Using photoemission electron microscopy, we image the nanoscale formation and growth of insulating domains across the temperature-driven MIT in NdNiO3 epitaxial thin films. Heteroepitaxy is found to strongly determine the nanoscale nature of the phase transition, inducing preferential formation of striped domains along the terraces of atomically flat stepped surfaces. We show that the distribution of transition temperatures is a local property, set by surface morphology and stable across multiple temperature cycles. Our data provide new insights into the MIT of heteroepitaxial nickelates and point to a rich, nanoscale phenomenology in this strongly correlated material.

Sputter deposition of oxide materials in a high-pressure oxygen atmosphere is a well-known technique to produce thin films of perovskite oxides in particular. Also interfaces can be fabricated, which we demonstrated recently by growing LaAlO3 on SrTiO3 substrates and showing that the interface showed the same high degree of epitaxy and atomic order as is made by pulsed laser deposition. However, the high pressure sputtering of oxides is not trivial and number of parameters are needed to be optimized for epitaxial growth. Here we elaborate on the earlier work to show that only a relatively small parameter window exists with respect to oxygen pressure, growth temperature, radiofrequency power supply and target to substrate distance. In particular the sensitivity to oxygen pressure makes it more difficult to vary the oxygen stoichiometry at the interface, yielding it insulating rather than conducting.

We have grown epitaxial Sm-Co thin films by sputter deposition from a single alloy target with a nominal SmCo5 composition on Cr(100)-buffered MgO(100) single-crystal substrates. By varying the Ar gas pressure, we can change the composition of the film from a SmCo5-like to a Sm2Co7-like phase. The composition, crystal structure, morphology, and magnetic properties of these films have been determined using Rutherford Backscattering, X-ray diffraction, and magnetization measurements. We find that we can grow films with, at room temperature, coercive fields as high as 3.3 T, but with a remanent magnetization which is lower than can be expected from the texturing. This appears to be due to the Sm content of the films, which is higher than expected from the content of the target, even at the lowest possible sputtering pressures. Moreover, we find relatively large variations of film properties using targets of nominally the same composition. At low temperatures, the coercive fields increase, as expected for these hard magnets, but in the magnetization, we observe a strong background signal from the paramagnetic impurities in the MgO substrates.

We have investigated the properties of interfaces between LaAlO3 films grown on SrTiO3 substrates singly terminated by TiO2. We used RF sputtering in a high-pressure oxygen atmosphere. The films are smooth, with flat surfaces. Transmission electron microscopy shows sharp and continuous interfaces with some slight intermixing. The elemental ratio of La to Al, measured by the energy dispersive X-ray technique, is found to be 1.07. Importantly, we find these interfaces to be non-conducting, indicating that the sputtered interface is not electronically reconstructed in the way reported for films grown by pulsed laser deposition because of the different interplays among stoichiometry, mixing, and oxygen vacancies.

We grow epitaxial Sm-Co thin films by sputter deposition from an alloy target with a nominal SmCo5 composition on Cr(100)-buffered MgO(100) single-crystal substrates. By varying the Ar gas pressure, we can change the composition of the film from a SmCo5-like to a Sm2Co7-like phase. The composition, crystal structure, morphology and magnetic properties of these films have been determined using Rutherford Backscattering, X-ray diffraction and magnetization measurements. We find that the various properties are sensitive to the sputter background pressure in different ways. In particular, the lattice parameter changes in a continuous way, the coercive fields vary continuously with a maximum value of 3.3 T, but the saturation magnetization peaks when the lattice parameter is close to that of Sm2Co7. Moreover, we find that the Sm content of the films is higher than expected from the expected stoichiometry.