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Optical STEM detection for scanning electron microscopy

Kievits, A.J. (author), Duinkerken, B. H.Peter (author), Fermie, Job (author), Lane, R. (author), Giepmans, Ben N.G. (author), Hoogenboom, J.P. (author)

Recent advances in electron microscopy techniques have led to a significant scale up in volumetric imaging of biological tissue. The throughput of electron microscopes, however, remains a limiting factor for the volume that can be imaged in high resolution within reasonable time. Faster detection methods will improve throughput. Here, we have...

journal article 2024

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Robust Local Thickness Estimation of Sub-Micrometer Specimen by 4D-STEM

Skoupý, R. (author), Boltje, D.B. (author), Slouf, Miroslav (author), Mrázová, Kateřina (author), Láznička, Tomáš (author), Taisne, C.M. (author), Krzyžánek, Vladislav (author), Hoogenboom, J.P. (author), Jakobi, A. (author)

A quantitative four-dimensional scanning transmission electron microscopy (4D-STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)-milling of lamellae for (cryo-)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to...

journal article 2023

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Need for Speed: Imaging Biological Ultrastructure with the 64-beams FAST-EM

Kievits, A.J. (author), Peter Duinkerken, B. H. (author), Giepmans, Ben N.G. (author), Hoogenboom, J.P. (author)

journal article 2023

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Imaging resonant micro-cantilever movement with ultrafast scanning electron microscopy

Garming, M.W.H. (author), Kruit, P. (author), Hoogenboom, J.P. (author)

Here, we demonstrate ultrafast scanning electron microscopy (SEM) for making ultrafast movies of mechanical oscillators at resonance with nanoscale spatiotemporal resolution. Locking the laser excitation pulse sequence to the electron probe pulses allows for video framerates over 50 MHz, well above the detector bandwidth, while maintaining...

journal article 2022

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Ultrafast scanning electron microscopy with sub-micrometer optical pump resolution

Garming, M.W.H. (author), Weppelman, I.G.C. (author), Lee, M. (author), Stavenga, T. (author), Hoogenboom, J.P. (author)

Ultrafast scanning electron microscopy images carrier dynamics and carrier induced surface voltages using a laser pump electron probe scheme, potentially surpassing all-optical techniques in probe resolution and surface sensitivity. Current implementations have left a four order of magnitude gap between optical pump and electron probe...

journal article 2022

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Integrated Array Tomography for 3D Correlative Light and Electron Microscopy

Lane, R. (author), Wolters, Anouk H.G. (author), Giepmans, Ben N.G. (author), Hoogenboom, J.P. (author)

Volume electron microscopy (EM) of biological systems has grown exponentially in recent years due to innovative large-scale imaging approaches. As a standalone imaging method, however, large-scale EM typically has two major limitations: slow rates of acquisition and the difficulty to provide targeted biological information. We developed a 3D...

journal article 2022

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Electron-beam patterned calibration structures for structured illumination microscopy

Hari, S. (author), Slotman, Johan A. (author), Vos, Y. (author), Floris, Christian (author), van Cappellen, W.A. (author), Hagen, C.W. (author), Stallinga, S. (author), Houtsmuller, A.B. (author), Hoogenboom, J.P. (author)

Super-resolution fluorescence microscopy can be achieved by image reconstruction after spatially patterned illumination or sequential photo-switching and read-out. Reconstruction algorithms and microscope performance are typically tested using simulated image data, due to a lack of strategies to pattern complex fluorescent patterns with...

journal article 2022

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A cryogenic, coincident fluorescence, electron and ion beam microscope

Boltje, D.B. (author), Hoogenboom, J.P. (author), Jakobi, A. (author), Jensen, Grant J. (author), Jonker, C.T.H. (author), Kaag, Max J. (author), de Agrela Pinto, C. (author), van der Wee, E.B. (author), Hoedt, Sander Den (author)

Cryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualization and structure determination of macromolecular complexes at sub-nanometre 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,...

journal article 2022

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How innovations in methodology offer new prospects for volume electron microscopy

Kievits, A.J. (author), Lane, R. (author), Carroll, E.C.M. (author), Hoogenboom, J.P. (author)

Detailed knowledge of biological structure has been key in understanding biology at several levels of organisation, from organs to cells and proteins. Volume electron microscopy (volume EM) provides high resolution 3D structural information about tissues on the nanometre scale. However, the throughput rate of conventional electron microscopes...

review 2022

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Organelle Segmentation Facilitated by Correlative Light Microscopy Data

Lane, R. (author), Balkenende, L. (author), van Staalduine, S.E. (author), Wolters, Anouk H.G. (author), Giepmans, Ben N.G. (author), Voortman, Lennard (author), Hoogenboom, J.P. (author)

journal article 2022

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Correlative light and electron microscopy reveals fork-shaped structures at actin entry sites of focal adhesions

Legerstee, Karin (author), Sueters, J. (author), Abraham, Tsion E. (author), Slotman, Johan A. (author), Kremers, Gert Jan (author), Hoogenboom, J.P. (author), Houtsmuller, A.B. (author)

Focal adhesions (FAs) are the main cellular structures to link the intracellular cytoskeleton to the extracellular matrix. FAs mediate cell adhesion, are important for cell migration and are involved in many (patho)-physiological processes. Here we examined FAs and their associated actin fibres using correlative fluorescence and scanning...

journal article 2022

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Structured illumination microscopy with noise-controlled image reconstructions

Smith, C.S. (author), Slotman, Johan A. (author), Schermelleh, Lothar (author), Chakrova, N. (author), Hari, S. (author), Vos, Y. (author), Hagen, C.W. (author), Houtsmuller, A.B. (author), Hoogenboom, J.P. (author), Stallinga, S. (author)

Super-resolution structured illumination microscopy (SIM) has become a widely used method for biological imaging. Standard reconstruction algorithms, however, are prone to generate noise-specific artifacts that limit their applicability for lower signal-to-noise data. Here we present a physically realistic noise model that explains the...

journal article 2021

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Optimization of negative stage bias potential for faster imaging in large-scale electron microscopy

Lane, R. (author), Vos, Y. (author), Wolters, A.E. (author), van Kessel, L.C.P.M. (author), Chen, S. Elisa (author), Liv, Nalan (author), Klumperman, Judith (author), Giepmans, Ben N.G. (author), Hoogenboom, J.P. (author)

Large-scale electron microscopy (EM) allows analysis of both tissues and macromolecules in a semi-automated manner, but acquisition rate forms a bottleneck. We reasoned that a negative bias potential may be used to enhance signal collection, allowing shorter dwell times and thus increasing imaging speed. Negative bias potential has previously...

journal article 2021

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Electron-Beam Induced Luminescence and Bleaching in Polymer Resins and Embedded Biomaterial

Srinivasa Raja, A. (author), de Boer, Pascal (author), Giepmans, Ben N.G. (author), Hoogenboom, J.P. (author)

Electron microscopy is crucial for imaging biological ultrastructure at nanometer resolution. However, electron irradiation also causes specimen damage, reflected in structural and chemical changes that can give rise to alternative signals. Here, luminescence induced by electron-beam irradiation is reported across a range of materials widely...

journal article 2021

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Retarding Field Integrated Fluorescence and Electron Microscope

Vos, Y. (author), Lane, R. (author), Peddie, Chris J. (author), Wolters, A.E. (author), Hoogenboom, J.P. (author)

The authors present the application of a retarding field between the electron objective lens and sample in an integrated fluorescence and electron microscope. The retarding field enhances signal collection and signal strength in the electron microscope. This is beneficial for samples prepared for integrated fluorescence and electron...

journal article 2020

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Beam displacement and blur caused by fast electron beam deflection

Zhang, L. (author), Garming, M.W.H. (author), Hoogenboom, J.P. (author), Kruit, P. (author)

Electrostatic beam blankers are an alternative to photo-emission sources for generating pulsed electron beams for Time-resolved Cathodoluminescence and Ultrafast Electron Microscopy. While the properties of beam blankers have been extensively investigated in the past for applications in lithography, characteristics such as the influence of...

journal article 2020

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Lock-in Ultrafast Electron Microscopy Simultaneously Visualizes Carrier Recombination and Interface-Mediated Trapping

Garming, M.W.H. (author), Bolhuis, M. (author), Conesa Boj, S. (author), Kruit, P. (author), Hoogenboom, J.P. (author)

Visualizing charge carrier flow over interfaces or near surfaces meets great challenges concerning resolution and vastly different time scales of bulk and surface dynamics. Ultrafast or four-dimensional scanning electron microscopy (USEM) using a laser pump electron probe scheme circumvents the optical diffraction limit, but disentangling...

journal article 2020

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Photoemission sources and beam blankers for ultrafast electron microscopy

Zhang, L. (author), Hoogenboom, J.P. (author), Cook, B.J. (author), Kruit, P. (author)

Observing atomic motions as they occur is the dream goal of ultrafast electron microscopy (UEM). Great progress has been made so far thanks to the efforts of many scientists in developing the photoemission sources and beam blankers needed to create short pulses of electrons for the UEM experiments. While details on these setups have typically...

journal article 2019

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Pulse length, energy spread, and temporal evolution of electron pulses generated with an ultrafast beam blanker

Weppelman, I.G.C. (author), Moerland, R.J. (author), Zhang, L (author), Kieft, E. (author), Kruit, P. (author), Hoogenboom, J.P. (author)

Crucial for the field of ultrafast electron microscopy is the creation of sub-picosecond, high brightness electron pulses. The use of a blanker to chop the beam that originates from a high brightness Schottky source may provide an attractive alternative to direct pulsed laser illumination of the source. We have recently presented the concept...

journal article 2019

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Concept and design of a beam blanker with integrated photoconductive switch for ultrafast electron microscopy

Weppelman, I.G.C. (author), Moerland, R.J. (author), Hoogenboom, J.P. (author), Kruit, P. (author)

We present a new method to create ultrashort electron pulses by integrating a photoconductive switch with an electrostatic deflector. This paper discusses the feasibility of such a system by analytical and numerical calculations. We argue that ultrafast electron pulses can be achieved for micrometer scale dimensions of the blanker, which are...

journal article 2018