In contrast to most conventional transmission electron microscopy (TEM) techniques, which only allow the spatial distribution of image intensity to be recorded, off-axis electron holography allows the phase shift of the electron wave that has passed through an electron-transparent specimen to be measured. The phase shift can, in turn, be used to provide information about local variations in magnetic induction and electrostatic potential within and around the specimen. Recent developments in the technique include the reconstruction of electrostatic potentials and magnetic fields in three dimensions, the use of advanced specimen holders with multiple electrical contacts to study nanoscale working devices, improvements in the stability of transmission electron microscopes to optimise phase sensitivity and the development of new approaches for improving temporal resolution using both direct electron detectors and double exposure electron holography. We are currently using the technique to characterize electrostatic potentials and magnetic fields in a wide variety of nanoparticles, nanostructures and thin films that are subjected to electrical biases and externally applied magnetic fields, as well as to elevated and reduced temperatures. Figure 1 shows representative results obtained from a study of the thermomagnetic behaviour of nanoscale grains of magnetite during heating in situ in the TEM. The magnetic induction maps show first a horseshoe-like magnetic state and then magnetic phase contours that flow from the bottom to top of the grain at higher temperature.

An important limitation of backprojection-based algorithms for reconstructing magnetic fields in three dimensions is the presence of artefacts resulting from incomplete tilt series of phase images and the inability to include additional constraints and known physical laws. Accordingly, one of our aims is the development of a robust model-based approach that can be used to reconstruct the three-dimensional magnetization distribution in a specimen from phase images recorded as a function of specimen tilt angle using off-axis electron holography. In order to perform each reconstruction, we generate simulated magnetic induction maps by projecting best guesses for the three-dimensional magnetization distribution in the specimen onto two-dimensional Cartesian grids. Our simulations make use of known analytical solutions for the phase shifts of simple geometrical objects, with numerical discretization performed in real space to avoid artefacts generated by discretization in Fourier space, without a significant increase in computation time (Figs 2 and 3). Our forward simulation approach is used within an iterative model-based algorithm to solve the inverse problem of reconstructing the three-dimensional magnetization distribution in the specimen from tilt series of two-dimensional phase images recorded about two independent tilt axes. Results will be presented from studies of magnetite nanocrystals, lithographically patterned magnetic elements and magnetic skyrmions examined as a function of temperature and applied magnetic field. At the same time, we are developing a similar algorithm for the reconstruction of three-dimensional charge density distributions in materials. Preliminary results will be presented from studies of charge distributions in electrically biased needle-shaped specimens, which require the analysis of differences between phase images recorded using two applied voltages, in order to subtract the mean inner potential to the phase shift.

The above studies are part of a wider program of research aimed at recording off-axis electron holograms of nanoscale materials and devices in the presence of multiple external stimuli. Further examples will be presented from studies of electrically biased resistive switching devices and two-dimensional flakes of transition metal dichalcogenides, whose electrical properties can be influenced strongly by the presence of contamination and defects, as well as by their interfaces to metal contacts. We are grateful to J. Ungermann, M. Riese, G. Pozzi, W. Williams, A. R. Muxworthy, M. Farle, M. Beleggia, T. F. Kelly and N. Kiselev for valuable contributions to this work and to the European Research Council for an Advanced Grant.

    Magnetic skyrmion is a topologically protected quantum spin texture. It is so stable and expected to be utilized for future memory devices featuring ultralow energy consumption. However, influences of structural defects in real materials remain to be elucidated in such practical applications. Since magnetic skyrmion is a nano-scale magnetic structure, visualization techniques with very high spatial resolution are essential to investigate such influences of nano-scale structural defects, such as edges, dislocations and grain boundaries on magnetic skyrmion. Here, we present the direct visualization of magnetic skyrmion lattice in a thin film specimen of FeGe_1-xSi_x by aberration-corrected differential phase-contrast scanning transmission electron microscopy (DPC-STEM) taking advantages of a segmented annular all-field (SAAF) detector [1] connected via photomultiplier tubes with a high-speed numerical processer.

    Polycrystalline FeGe_1-xSi_x was grown from FeGe_0.8Si_0.2 ingot by conventional solid-state reaction annealed at 900 °C for 100 hours.  A thin film specimen was fabricated from a bulk crystal by using an Ion Slicer (EM-9100IS, JEOL, Ltd.). For DPC STEM observations, we used a STEM (JEM-2100F, JEOL, Ltd.) equipped with a probe-forming aberration corrector (CEOS, GmbH) and a Schottky field emission gun operated at 200 kV.  This microscope was equipped with a SAAF detector.  We used a double-tilt liquid-nitrogen cooling specimen holder (Model 636, Gatan, Inc.).  Analysis of DPC STEM images was done either online by using a direct reconstruction system [2] implemented as an application of LabView software (National Instruments, Inc.) running on Windows or offline by using a program written in Digital Micrograph Scripting language (Gatan, Inc.).

    Figure 1a shows a schematic diagram of electron-optical system of DPC STEM used in the present study.  In-plane magnetization can be mapped by analyzing the Lorentz deflection of electron beam using segmented annular detector as schematically shown in Fig. 1b.  As shown in Fig. 2a, a set of four images is selected from sixteen images obtained by the segmented detector.  Such images are first converted into two images corresponding to a horizontal and a vertical component image of Lorentz deflection (Fig. 2b).  Finally, the two Lorentz deflection images are analyzed to show a in-plane magnetization vector, intensity, and magnetic helicity images as shown in Fig. 2c.  Figure 3 demonstrates the live reconstruction of in-plane magnetic field and intensity of a magnetic skyrmion lattice.  A unique structural relaxation mechanism in a magnetic Skyrmion domain boundary core, revealed by the technique for the first time [3], will be presented in detail.

References

[1] N. Shibata et al., J. Electron Microsc. 59 (2010) 473.

[2] N. Shibata et al., Sci. Rep. 5 (2015) 10040.

[3] T. Matsumoto et al., Sci. Adv. 2 (2016) e1501280.

[4] This work was supported by the Japan Science and Technology Agency SENTAN and Precursory Research for Embryonic Science and Technology. A part of this work was conducted at the Research Hub for Advanced Nano Characterization, The University of Tokyo, supported under “Nanotechnology Platform” (project No. 12024046) sponsored by MEXT, Japan. T.M. acknowledges support from GRENE from MEXT and N.S. acknowledges supports from the JSPS KAKENHI Grant number 26289234 and the Grant-in-Aid for Scientific Research on Innovative Areas "Nano Informatics" (grant number 25106003) from JSPS.

For off-axis electron holography, an electrostatic biprism is usually located close to the selected area (SA) aperture plane of the transmission electron microscope (TEM). The application of a voltage to the biprism results in overlap of two parts of an incident electron beam and allows both the amplitude and phase of the electron wavefunction that has passed through a specimen to be recovered. The quality of the reconstructed electron wave depends directly on the information contained in the hologram. An off-axis electron hologram is characterized by its interference fringe spacing, contrast and overlap width. The interference fringe spacing and overlap width are determined by the electron optics of the TEM and by the deflection angle at the biprism. The interference fringe spacing is inversely proportional to the deflection angle, while the overlap width is influenced by the width of the biprism. In order to achieve as narrow a fringe spacing as possible with high fringe contrast, the biprism should be as narrow and stable as possible. Previous attempts to make ultra-narrow biprisms have included glass fibres coated with metal or patterned SiNx with focused ion beam. None of these attempts have provided a reproducible method of making ultra-narrow biprisms with perfect control over their dimensions.

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Here, we illustrate an approach that can be used to fabricate a biprism that has a rectangular cross-section and is located between two counter electrodes that are at the same height. We pattern the biprism in the top Si layer of a Si-on-insulator (SOI) wafer. The wafer consists of a micron-thick single-crystalline Si layer that is isolated electrically from its substrate and can be left free-standing using an etching process. When combined with microelectromechanical systems (MEMS) processes, structures can be patterned down to nm scale in three dimensions. In this way, the width of the biprism and the distance to the counter-electrodes can be chosen to have dimensions down to ~100 nm. A further advantage of using an SOI wafer to fabricate a biprism is the large Young’s modulus of the single-crystalline Si biprism (170 GPa), when compared with that of a conventional biprism made from glass (~70 GPa). In addition, the two counter-electrodes can be biased independently. A schematic diagram and scanning electron microscopy (SEM) images of a biprism on an electrically-contacted MEMS chip are shown in Fig. 1, alongside a three-dimensional design drawing of a custom-made aperture rod.

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In order to test its performance, the biprism was mounted close to the SA plane in a Philips CM20 TEM. The electron deflection was measured by recording the shift of a diffraction spot as function of applied voltage. The measured deflections are compared with predicted deflections and with similar measurements made using a conventional biprism on an FEI Titan TEM in Fig. 2. The deflection is a factor two greater for the new rectangular biprism for the same applied voltage. The measured interference fringe spacing, contrast and overlap width achieved using the new biprism are also shown in Fig. 2. Here, the maximum voltage that can be applied is limited by the distance between the biprism and the counter-electrodes, which can be increased in future designs.

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In order to demonstrate the imaging capabilities of the new biprism, an off-axis electron hologram of a MoS₂ flake was recorded in a Philips CM20 TEM. The hologram and the resulting reconstructed amplitude and phase are shown in Fig. 3. In the future, the biprism will be mounted in an image-aberration-corrected FEI Titan TEM, in which the electron optics offers greater flexibility in both normal and Lorentz imaging modes.

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The authors acknowledge financial support from the European Union under the Seventh Framework Programme under a contract for an Integrated Infrastructure Initiative (Reference 312483 ESTEEM2), the European Research Council for an Advanced Grant (Reference 320832 IMAGINE) and for Starting Grant (Reference 306535 HOLOVIEW) for financial support. We thank David Cooper and Helmut Soltner for valuable discussions and support.

It is well known that a magnetic field produces a phase change proportional to the in plane magnetic field B [1]. Alas the out of plane magnetic field is more elusive. The only measurement that so far seems to produce  any out of plane information is the magnetic dichroism [2] as it actually measures the out of plane magnetization.
Unfortunately magnetic dichroism experiment is still a complicated experiment that requires a considerable control of the condition and of the material; moreover the measures of the magnetization , often with the objective on, is not directly a measure of the magnetic field itself.
However while a plane wave along the optical axis has no significant interaction with a magnetic field along z , vortex beams  with winding number l do feel such field through a Larmor phase effect .
We will demonstrate here that through the use of large vortex beams [3][4][5] we are able, for the first time, to measure the out of plane magnetic field generated by a magnetic pillar of Co [6].
We placed the vortex around the pillar ensuring as much as possible an axial symmetry and observed the phase effect.  The result is in good agreement with the typical magnetization expected for the pillar with an average field at surface of about 2T.
Fig 1 illustrates the A and B field produced by a magnetic pillar. The actual magnetic pillar fabricated by EBID deposition is illustrated in fig 1b. Fig 2 depicts the vortex beam density with L=200 as imaged in its focal plane. Thanks to an interferometric approach we were able to measure the magnetic phase contribution and bind it to the magnetic field in proximity of the sample. The magnetic phase is visible in the inset of fig 2 showing approximate axial symmetry.
The technique has been carried on in Low Mag mode with the main objective  lens off but there are no principle limitation to the application in conventional (S) TEM mode.
Moreover this promises  to be one of the best recognition of the importance of vortex beams in microscopy for material science.
[1] R E. Dunin-Borkowski, M. R. McCartney et al Science 282 (1998) 1868
[2] P. Schattschneider, S. Rubino Nature 441 (2006) 486
[3] J. Verbeeck, H. Tian  P. Schattschneider Nature 467 (2010) 301
[4] B. J. McMorran,  A. Agrawal  et al. Science 331 (2011) 192
[5] V. Grillo et al .  Phys Rev Lett 114, 034801 (2015)
[6] G. Pozzi, C. B. Boothroyd Appl. Phys. Lett. 108, 083108 (2016)

In this presentation we will compare differential phase contrast (DPC) [1] and off-axis electron holography [2] for the measurement of electrostatic potentials in semiconductor devices. DPC uses the lateral shifts of a convergent electron beam to determine the field in the sample whereas for electron holography the changes in potential is encoded in interference fringes that are formed using a biprism [1]. To fairly assess the relative sensitivity of the different techniques on the same specimen, a symmetrically doped p-n junction with a dopant concentration of 1 x 10¹⁹ cm^-3 has been measured as a function of reverse bias applied in situ in the TEM.  Figure 1(a) and (b) shows maps acquired by DPC of the p-n junction with 0V and 4V reverse bias applied. At 0V, it is difficult to see the presence of the junction whereas for a reverse bias of 4 V the space charge region is now visible. Profiles acquired from across the junction for various reverse bias voltages can be seen in Figure 1(c).  Electron holograms were acquired and Figure 1(d) and (e) show reconstructed phase images of the junction at zero bias and 4 V reverse bias. Even though a low magnification has been used to obtain a large field of view at the expense of sensitivity, the junction is clearly visible in both of the phase images. Corresponding electric field profiles that have been calculated from the potential maps are shown in Figure 1(f).  These results show that off-axis electron holography has a significantly better sensitivity than DPC. However  the advantages of using DPC is that a large field of view has been obtained and it is not necessary to examine a region close to vacuum.

These techniques have also been applied to an InGaN/GaN system. Figure 2(a) shows a HAADF STEM image of the specimen. Figure 2(b) shows a potential map and (c) profile acquired by off-axis electron holography with a spatial resolution of 5 nm.  When using DPC, sub-nanometer spatial resolution is expected and maps of the electric field in the InGaN layers can be observed in Figures 2(d) and (e). Here the specimen has been tilted onto and away from a zone axis and a large variation in the measured signal is observed which is visible in Figure 2(f). In this presentation will discuss the effects of diffracted beams on measured DPC signal. We will also discuss the advantages and disadvantages of using DPC and electron holography for the measurement of electrostatic fields in a range of different doped and III/V semiconductor specimens and show improvements that have been applied.

Acknowledgements : This work has been funded by the ERC Starting Grant 306365 « Holoview ». The experiments have been performed on the platform nanocharacterisation at Minatec.

References

[1] N.H. Dekkers, Optik, 30 (1974) 452

[2] A. Tonomura, Reviews of Modern Physics, 59 (1987) 1

Electron interferometric techniques have progressed in the last years thanks to the development of multiple biprisms microscopes. Here, we will discuss some recent developments in the field of strain measurement carried out with the I2TEM microscope (In-situ Interferometry Transmission Electron Microscope) installed in Toulouse in 2012. The I2TEM is a Hitachi HF-3300 equipped with one pre-specimen electrostatic biprism, three post-specimen biprisms, an image corrector (CEOS B-COR for correcting off-axial aberrations) and two stages (objective stage and Lorentz stage above the objective lens).

In the dark-field off-axis scheme [1], an electron beam diffracted by an epitaxially grown region is interfered with a beam diffracted by the substrate thanks to a post-specimen biprism (Fig. 1(a)).  Fig. 1(b-d) is an example obtained on a p-MOSFET like transistor with SiGe source/drain. The deformation is recorded as a frequency modulation (FM) in the hologram (Fig. 1(c)) and it can be calculated from the gradient of the reconstructed phase image (Fig 1(d)).

In a recently proposed variant called differential phase contrast dark-field holography (DPC-DFEH) [2], a pre-specimen biprism is used to create two overlapping waves on the sample (Fig. 1(e)). The interference of beams diffracted by slightly distant regions is acquired in a defocused plane. The deformation is recorded as a phase modulation (PM) in the hologram (Fig. 1(f)) and the DPC phase is directly proportional to the deformation (Fig. 1(g)).

Another option is the 4-wave dark-field setup where two biprisms oriented perpendicularly are used to interfere three reference waves and one object wave (Fig. 1(h-j)). The holographic fringes are modulated in amplitude (AM) and each amplitude contour corresponds to a given displacement of the lattice planes [3].  It can provide live information if a sufficient fringe contrast is achieved.

In all cases, strain measurement requires a reference wave diffracted by a region of known lattice parameter (usually the substrate). One solution is the “tilted reference wave” (TRW) where a pre-specimen biprism and the condenser system are used to create an object-independent reference wave with an adjustable tilt angle [4]. Fig. 2(a,b) is an example acquired in the vacuum and Fig. 2(c,d) shows the dark-field configuration for strain measurement.

Finally, a pre-specimen biprism can also be useful for electron diffraction techniques. For instance, one can create two parallel half cones on the specimen (splitting convergent beam electron diffraction, SCBED) with a controllable distance (Fig. 3(a)) [5]. Each spot in the diffraction pattern contains two lobes related to the regions crossed by the two probes. Fig. 3(b) shows an example of SCBED pattern series where the left and right lobes are related to unstrained and increasingly strained regions respectively.

[1]  M Hÿtch et al, Nature 453 (2008), 1086–1089.

[2]  T Denneulin et al, Ultramicroscopy  160 (2016), 98–109.

[3]  T Denneulin and M Hÿtch, J. Phys. D: Appl. Phys. 49 (2016), 244003.

[4]  F Röder et al, Ultramicroscopy 161 (2016), 23–40.

[5]  F Houdellier et al, Ultramicroscopy 159, Part 1 (2015), 59–66.

Acknowledgments

This work was funded through the European Metrology Research Programme (EMRP) Project IND54 Nanostrain. The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union. This work has been supported by the French National Research Agency under the "Investissement d'Avenir" program reference No. ANR-10-EQPX-38-01. The authors acknowledge the "Conseil Regional Midi-Pyrénées" and the European FEDER for financial support within the CPER program.

Off-Axis Electron Holography permits the direct reconstruction of amplitude and phase of electron waves elastically scattered by an object (see, e.g., [1]). The technique employs the Möllenstedt biprism to mutually incline an object modulated wave and a plane reference wave to form an interference pattern at the detector plane. Limited coherence of the electron beam in presence of aberrations attenuates high spatial frequencies of the object exit wave spectrum, which is illustrated by the sideband envelope function for a non-corrected TEM in Fig. 1a.

In this work, we explore an extension of the conventional setup given by deliberately tilting the reference wave independent from the object wave. This allows the transfer of spatial frequencies beyond the conventional sideband information limit in Fig. 1a as predicted by a generalized transfer theory for Off-Axis Electron Holography [2]. This is because a reference wave tilted by q₀ compensates the aberration impact on the spatial frequency q₀ of the object wave spectrum. The resulting transfer envelope for a tilt of q_0x = -10/nm perpendicular to the post-specimen biprism is shown in Fig. 1b, where the contrast maximum of the total envelope (TCC) is located at q_0x. Thus, an off-axis hologram series with varying reference wave tilt allows in principle a linear synthesis of an effective coherent aperture with a radius reaching out beyond the conventional information limit. Furthermore, an object-independent measurement of aberrations as well as dark-field electron holography can be realized using this setup.

The experimental realization of an arbitrarily tilted reference wave is challenging and could be realized for the first time at the Hitachi HF3300C I2TEM at CEMES Toulouse for one direction [3]. We used an additional biprism placed in the illumination system. Three condenser lenses were adjusted to provide a demagnified image of the condenser biprism at the sample plane under parallel illumination (Fig. 2). The pre-specimen deflectors were adapted to maintain the incident wave vector of the object wave and to realize a tilt of the reference wave as a function of the condenser biprism voltage. Optimal condenser lens settings were found by means of paraxial ray tracing (Fig. 3) finally producing a mutual tilt of up to 20/nm at the object plane. We verified the kink-like phase modulation of the incident beam by means of holographic measurements. Contrast transfer theory including condenser aberrations and biprism instabilities was applied to explain detailed fringe contrast measurements. Finally, we have experimentally shown that dark-field holography [4] can be conducted with an object-independent reference.

[1]  H Lichte et al, Rep. Prog. Phys. 71 (2008) 016102

[2]  F Röder et al, Ultramic. 152 (2015) 63-74

[3]  F Röder et al, Ultramic. 161 (2016) 23–40

[4]  MJ Hÿtch et al, Nature 453 (2008), 1086–1089

Acknowledgments

We thank the Graduate Academy of the TU Dresden for the financial support. The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative-I3).

Correlative light and electron microscopy (CLEM) methods integrate light and electron microscopy on a single sample, literally bridging the gap between these two microscopy techniques. Most methods use fluorescent microscopy of thin or semi-thin sections to define a region-of-interest, which then is traced back in the EM to provide subcellular context information (e.g. membrane organization, non-labeled surroundings of the fluorescent structure). The most powerful application of CLEM is correlative live cell imaging-EM, by which dynamic information is inferred to structures seen in static EM pictures. By merging the strengths of the two techniques a novel and integrated type of image is created that combines parameters that cannot - or not easily - be obtained when using separate images of related events. However, because imaging requirements are intrinsically different between light and electron microscopy, creating conditions that are ideal for both modalities is a challenging process. Moreover, correlating fluorescent labeling to the cellular architecture seen in the EM is not always straightforward. The most pressing challenges in CLEM are currently therefore 1. development of sample preparation methods that are appropriate for both LM and EM imaging. 2. development of bi-modal probes that are visible in both LM and EM and 3. development of software that allows for rapid and accurate correlation of LM and EM images. 

The focus of our research is to develop new probes and CLEM pipelines that allow us to efficiently and with high accuracy define the three-dimensional (3D) ultrastructural context of fluorescently-tagged proteins previously localized in fixed or living cells. We apply our technologies to study the cellular pathways and mechanisms that control the cell’s digestive system – i.e. the endo-lysosomal system - in health and disease conditions. The main CLEM technology that we use in the lab is based on the use of immunogold labeling of ultrathin cryosections (the Tokuyasu technique) [1]. Most CLEM approaches, however, are restricted in their EM approach by the lack of 3D structural information. To overcome this limitation, we apply Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) as 3D-EM approach in a live cell-CLEM set up. To visualize endo-lysosomes in live cells we combine fluorescent tagged endo-lysosomal proteins (such as LAMP1-mGFP) with endocytic tracers (such as fluorescently labeled dextran). This approach enables live-cell tracking of specific endo-lysosomal compartments, after which the samples are fixed, stained and resin-embedded for FIB-SEM imaging. Figure 1 presents an example of Dextran-Alexa646 and/or LAMP1-mGFP labeled endo-lysosomal compartments in live cells (A) and in 3D-EM (D), providing the cellular context at ultrastructural resolution. In my presentation I will show various examples of both immunoEM and FIB.SEM-based CLEM approaches, which are designed to optimally image individual, membrane-bounded compartments. 

The combination of fluorescence light microscopy (FLM) and electron microscopy (EM) makes it possible to put molecular identity in its full ultrastructural context. With array tomography (AT) long ribbons of serial ultrathin sections are labelled via immunohistochemistry (IHC). After FLM imaging the same sections are processed for scanning electron microscopy (SEM) and re-analyzed. The resulting images are correlated and superimposed. Due to the serial nature of this approach it is possible to obtain large volumes of correlated multi-channel light and electron microscopic data. One drawback of this method is the large discrepancy of resolution between light and electron microscopy. To alleviate this we advanced AT for two super-resolution light microscopy techniques, Structured Illumination Microscopy (SIM) and direct Stochastic Optical Reconstruction Microscopy (dSTORM). We also devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. Together, these advances make it possible to map even small subcellular structures with high precision and confidence. We applied our super-resolution AT approach in C. elegans to address an important problem in connectomics research: the mapping of gap junctions at connectomes.

Cellular structure can be imaged at nanometer resolution using electron microscopy, but the biological molecules remain invisible in grayscale electron micrographs. With Correlative Light and Electron Microscopy (CLEM)[1], fluorescence recorded on a separate light microscope can be used to identify molecules in colour, but the resolution gap with electron microscope precludes accurate localization. The use of super-resolution fluorescence microscopy techniques combined with CLEM[2,3] holds great promise but is still one order of magnitude off in resolution compared to EM. In addition, accurate registration of the separate images may be challenging and correlation may require expert procedures to maintain, e.g., protein photo-switching under EM preparation conditions. Thus, while CLEM adds information represented in the colors of visible light, molecular identification and localization at the resolution of the electron microscope is still hampered. Therefore, we are exploring novel approaches to achieve color-EM using the electron beam to add information about the specimen to the electron micrographs.

Detection of electron-beam excited luminescence has been explored in the past. Cross-sections for visible luminescence (also called cathodoluminescence) are, however, small and most biological fluorescent labels are destroyed before sufficient signal has been collected to allow high-resolution localization [4]. Phosphorescent nanoparticles are considered as non-bleaching alternative, but particle sizes are not yet well-defined and bio-functionalization has in most cases still to be pursued[5]. 

Here, we explore detection of X-ray fluorescence to identify nanoparticle labels[6] at EM resolution in colour on tissue. We use Au and CdSe colloidal quantum dots, immuno-targeted to guanine quadruplexes, resp. insulin, as molecular labels in large-scale electron microscopy of pancreatic tissue. Energy dispersive detection of X-ray emission (EDX) during electron irradiation allows discrimination of both nanoparticles based on their elemental (Au vs Cd) composition. We will present electron microscopy images overlaid with false colour elemental maps displaying single nanoparticles. Separate resolution tests have shown that individual Au particles down to 3 nm in size can be detected using EDX, highlighting the potential for coloured identification of materials in biological electron microscopy, revealing both compositional and ultrastructural information at nanometer-scale resolution. 

[1] P. de Boer, J. P. Hoogenboom, B. N. G. Giepmans, Nature Methods 12, 503 (2015)

[2] Kopek, B.G., et al., Proc. Natl. Acad. Sci. U. S. A. 109, 6136 (2012)

[3] Johnson, E. et al., Scientific Reports 5 (2015)

[4] Niitsuma, J., Oikawa, H., Kimura, E., Ushiki, T. & Sekiguchi, T., J. Electron Microscopy 54, 325 (2005).

[5] Glenn, D.R. et al., Scientific Reports 2 (2012)

[6] A. Loukanov, N. Kamasawa, R. Danev, R. Shigemoto, K. Nagayama, Ultramicroscopy 110,366 (2010)

Lamin B1 (Lmnb1) is a major component of the nuclear lamina and together with other lamins (lamin A/C) plays a key role in the structure and function of the nucleus. Mutations in lamins and lamin-associated proteins have been indeed shown to cause various human diseases. There are indications that mammalian lamins A and B and drosophila Lamin Dm₀ are involved in nuclear envelope formation and nuclear morphology (Prüfert et al. J Cell Sci 117, 6105). To explore the specific role of Lamin B1 in defining nuclear morphology we applied electron microscopy, electron tomography and correlative light electron microscopy (CLEM) on different cell types in which murine lamin B1 (Lmnb1) was silenced or human lamin B1 (LMNB1) was over-expressed.
To investigate how Lmnb1 deficiency affects nuclei of post-mitotic neurons, we analyzed the nuclear morphology of primary mouse cortical neurons derived from Lmnb1-null (Lmnb1^Δ/Δ) embryos (Giacomini et al. Mol Biol Cell 27, 35). Nuclei of cultured Lmnb1^Δ/Δ neurons were smaller and rounder than those of Lmnb1^+/+ embryos (the area of Lmnb1^Δ/Δ nuclei was 43% less than that of Lmnb1^+/+). Moreover the Lmnb1^Δ/Δ nuclei presented a rounder shape than those of Lmnb1^+/+, as confirmed by increased circularity values (mean circularity was 0.62±0.16 n=28 and 0.9±0.87 n=39 for respectively Lmnb1^+/+ and Lmnb1^Δ/Δ nuclei). Taken together, these results indicate that optimal Lmnb1 levels are essential to maintain neuronal nuclear size and shape. Lmnb1 deficiency also affected the distribution and composition of nuclear pore complexes (NPCs) in mouse cortical neurons. Indeed in Lmnb1^Δ/Δ neurons, the NPCs were distributed irregularly, with some regions of the nuclear envelope hosting groups of NPCs located close to each other and other areas devoid of NPCs (Fig.1A-C). Electron tomography performed on serial semi-thin sections showed that in Lmnb1^Δ/Δ neurons the NPCs are distributed in parallel, closely packed rows (Fig.1D).
The effect of the LMNB1 over-expression on nuclear morphology was assessed in transfected HEK 293 and HeLa cells. HEK 293 cells transfected with a bicistronic plasmid containing LMNB1 and EGFP downstream of an IRES sequence were sorted by EGFP expression using fluorescence-activated cell sorting (FACS) and high pressure frozen and freeze substituted to maintain structural integrity as much as possible. To increase contrast at the nuclear membranes we set up a freeze substitution recipe with a pre-incubation step at -90°C in 2% hydrated acetone containing small percent of tannic acid and glutaraldehyde. The cell nuclei of HEK 293 over-expressing LMNB1 were lobulated, with highly folded nuclear membranes often associated with multi-membrane stacks and intra-nuclear membranes (Fig.2A). As in primary cortical neurons lacking Lmnb1, we observed clusters of NPCs at the nuclear membrane (Fig.2B). Immunolabeling performed on cryo-sections revealed the presence of LMNB1 on the membrane stacks associated with the nuclear envelope (Fig.2C). This result supports the idea that these membrane stacks are effectively multi-membrane-layered nuclear membranes. To better understand the fine localization of Lmnb1 we performed correlative light electron microscopy (CLEM) experiments on HEK 293 and HeLa cells overexpressing LMNB1 in fusion with EGFP and tdTomato downstream of an IRES sequence. The cells, grown on sapphire disks carbon coated with a finder grid pattern, were live imaged at the confocal laser scanning microscope and then high pressure frozen and freeze substituted (Fig.3). As for HEK 293, the cell nuclei of HeLa cells over-expressing LMNB1 were lobulated with highly folded nuclear membranes. The HeLa nuclei differed from HEK 293 nuclei for the absence of membrane stacks associated to the nuclear membranes and for the presence of peculiar morphologies (Fig.3A). Indeed our CLEM approach revealed the presence of highly folded intra-nuclear membranes delimiting large cytoplasmic regions (Fig.3B,D). This approach also revealed the presence of nuclei that branched apically in thin and long nuclear rods delimited by conventional nuclear membranes (Fig.3B,C). Altogether, our results indicate that proper levels of Lamin B1 are required to maintain structural integrity and morphology of the nuclear envelope.
Acknowledgement
We thank Roberta Ruffilli and all the EM Lab members for their support. Special thanks to Prof. Liberato Manna for supporting this research.

Our group uses electron cryo microscopy (cryoEM) / tomography (cryoET) and combines these with complementary techniques to understand biological processes mechanistically. The power of such an integrated structural biology approach to cell biology at the macromolecular level will be exemplified along the group’s analyses of crucial steps in the molecular interactions between viruses and their host cells . The main emphasis will be on understanding mechanisms of membrane modulation in vesicle formation at the inner nuclear envelope in cargo egress from the nucleus [1,2].

Although nucleo-cytoplasmic transport is typically mediated through nuclear pore complexes, herpesvirus capsids exit the nucleus via a unique vesicular pathway. Vesicular nucleo-cytoplasmic transport might also be a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins (pUL31 and pUL34), forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane- distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM. We further solved the crystal structure of the pseudorabies virus NEC. Fitting of the NEC crystal structure into the cryoEM-derived hexagonal lattice found in situ, provided details on the molecular basis of NEC coat formation and inner nuclear membrane remodeling. Altogether, this multimodal imaging study exemplified the power of an integrated structural cell biology approach to reveal novel mechanistic insights by combining in-situ data with information on the isolated macromolecules mediating the respective biological process of interest.

References:

[1] Hagen, C., Dent, K.C., Zeev-Ben-Mordehai, T., Grange, M., Bosse, J.B., Whittle, C., Klupp, B.G., Siebert, C.A., Vasishtan, D., Bäuerlein, F.J.B. Cheleski, J., Werner, S., Guttmann, P., Rehbein, S., Henzler, K., Demmerle, J., Adler, B., Koszinowski, U., Schermelleh, L., Schneider, G., Enquist, L.W., Plitzko, J.P., Mettenleiter, T.C., Grünewald, K. (2015) Structural Basis of Vesicle Formation at the Inner Nuclear Membrane. Cell 163: 1692–1701.

[2] Zeev-Ben-Mordehai, T., Weberruß, M., Lorenz, M., Cheleski, J., Hellberg, T., Whittle, C., El Omari, K., Vasishtan, D., Dent, K.C., Harlos, K., Franzke, K., Hagen, C., Klupp, B.G., Antonin, W., Mettenleiter, T.C., Grünewald, K. (2015) Crystal structure of the herpesvirus nuclear egress complex provides insights into inner nuclear membrane remodeling. Cell Reports 13: 2645–2652.

In correlative light and electron microscopy, the discerning power of fluorescence microscopy (FM) is combined with the ultra-high resolution of electron microscopy (EM). We fluorescently label protein complexes in cells, with the purpose of facilitating their identification in transmission electron tomograms. Our imaging strategy is to perform cryogenic selected volume tomography, continuously keeping the specimen in a vitreous state. To this end, we are developing a cryogenic workflow in collaboration with FEI, consisting of three microscopes: CorrSight light microscope (LM), Scios Dualbeam scanning electron microscope (SEM) with a focused ion beam (FIB) and Tecnai Arctica cryo–transmission electron microscope (TEM). The focus of my presentation is on the integration of the CorrSight light microscope into the workflow (1). This translates to performing cryogenic light microscopy and correlating the images with EM for guided ion beam milling and tomography (2).

The data we collected from cryogenic fluorescence microscopy of mammalian and bacterial cells on TEM grids shows great promise for complementing EM (Fig. 1). This is substantiated by alignment of LM and SEM images utilizing FEI MAPS software, allowing for fluorescence based navigation during SEM imaging (Fig. 2). My future efforts will focus on improving localization precision and correlation accuracy, to guide FIB-milling of sub-cellular structures in the SEM. FIB-milling is used to fabricate thin lamellae in cells, making them suitable for cryo-TEM. Our results indicate that tomograms can be recorded from lamellae using a 200kV cryo-TEM, but vitrification of the specimen is essential. The ultimate goal of our workflow is to obtain structural insight of the Type 7 Secretion System (T7SS), formed by the ESX-1 machinery. To this end, we will perform tomography of mycobacterial membranes inside professional antigen-presenting cells. The T7SS is an important part of the mycobacterial protein weaponry, as it mediates virulence and phagosomal escape (4, 5).

References

1. Arnold, J., J. Mahamid, A. de Marco, J.-J. Fernandez, T. Laugks, et al. 2016. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy. Biophysical Journal. 110: 860.

2. Mahamid, J., S. Pfeffer, M. Schaffer, E. Villa, R. Danev, et al. 2016. Visualizing the molecular sociology at the HeLa cell nuclear periphery. Science. 351: 969.

3. K.V. Korotkov, and W. Bitter. 2014. Take five — Type VII secretion systems of Mycobacteria. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1843: 1707.

4. Smith, S. 2004. Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Molecular Microbiology. 51: 359.

5. van der Wel, N., D. Hava, D. Houben, D. Fluitsma, M. van Zon, et al. 2007. M. tuberculosis and M. leprae Translocate from the Phagolysosome to the Cytosol in Myeloid Cells. Cell. 129: 1287.

The investigation of vitrified biological specimens (i.e. samples that are plunge or high pressure frozen) enables the visualization of cellular ultrastructure in a near native fully hydrated state, unadulterated by harmful prepara­tion methods. Here, we focus on two recent cryo imaging modalities and discuss their impact on cryo correlative workflows. First, we present confocal cryo fluorescence microscopy, utilizing a novel confocal detector scheme with improved signal to noise ratio (SNR) and resolution. Second, we show volume imaging of multicellular specimens by cryo focused ion beam scanning electron microscopy (FIB/SEM).

Confocal laser scanning microscopes (LSM) are renowned for their optical sectioning capability, a feature enabled by utilizing a pinhole that rejects out of focus light. Closing the pinhole improves lateral resolution, but also causes less light to reach the detector leading to reduced signal to noise ratios. In cryo fluorescence microscopy, the situation is aggravated by the fact that current­ly no immersion optics are available and consequently only numerical apertures below NA 1 are possible. Here we combined Airyscan, a novel detector module (available for ZEISS LSM 780, 800 and 880) together with a cryo correlative stage (Linkam CMS 196) for cryo fluorescent imaging of vitrified cells prepared on electron microscopy grids (Figure 1a). The Airyscan detection module allows the spatially resolved detection of fluorescence light otherwise rejected by the pinhole in a standard confocal system. We show that under cryo conditions even without immersion optics a significant increase in resolution and SNR can be obtained with Airyscan compared to standard confocal images (Figure 1 b&c).

In FIB/SEM tomography three-dimensional volumetric data from biological specimens is obtained by sequentially removing material with the ion beam and imaging the milled block faces by scanning with the electron beam. Only recently, it has been shown that this imaging method can be applied also to frozen hydrated specimens [1]. Cryo FIB/SEM tomography allows the mapping of large multicellular specimens in the near native state and is particularly suited to analyze samples that require remaining hydrated. We demonstrate this approach by imaging the cryo immobilized specimens (e.g. mouse optic nerve) (Figure 2).

Both methods by themselves promise significant advantages for biomedical research by being able to investigate biological specimens in the near native fully hydrated state. Yet correlating both imaging modalities, LSM and FIB/SEM of vitrified samples, has the potential to provide even deeper insights into biological context. In contrast to correlative workflows using resin embedded samples, cryo imaging workflows do not have to find the optimal balance between preserving cellular ultrastructure and maintaining the functional integrity of fluorophores. Moreover, major mechanisms leading to irreversible bleaching of fluorescent molecules are suppressed at cryo temperatures [2]. The correlation between cryo light and electron microscopy data will greatly benefit from an increase in resolution in fluorescence imaging. Cryo Airyscan is a first step in that direction and can deliver three-dimensional optical sectioning data that can be used to reliably target cellular structures in a FIB/SEM microscope, before the structural context is explored by cryo FIB/SEM tomography.

References:

(1) Schertel A et al., “Cryo FIB SEM: volume imaging of cellular ultrastructure in native frozen specimens.” J Struct Biol. 2013 Nov; 184(2):355 60. doi: 10.1016/j.jsb.2013.09.024.
(2) Kaufmann R, Hagen C, Grunewald K. Fluorescence cryo microscopy: current challenges and prospects. Current opinion in chemical biology. 2014; 20:86 91. DOI: 10.1016/j.cbpa.2014.05.007

Photoemission electron microscopy combined with x-ray magnetic circular dichroism (XMCD-PEEM) is a powerful synchrotron radiation technique that can be used to observe magnetic configurations of nano-objects with high spatial resolution. Combined with time resolution, using stroboscopic detection, the magnetisation dynamics of such objects can be studied with sub-nanosecond resolution.

We will describe the study of domain wall (DW) dynamics and magnetic skyrmions in multilayers of composition Pt/Co/MOx (M=Al, Mg), characterised by a large perpendicular magnetic anisotropy (PMA) and anti-symmetric exchange interaction (Dzyaloshinskii-Moriya interaction, DMI). Such interaction favours non-collinear magnetic textures such as chiral Néel walls and magnetic skyrmions.

In systems with large DMI, such as Pt/Co/AlOx with ultrathin Co, domain walls acquire a Néel chiral structure and move at large speeds when driven by current pulses. Most microscopic studies of domain wall dynamics have been performed using quasi-static measurements, where the DW position and structure are imaged before and after the excitation pulse. In order to better understand the details of the interaction between current and DW magnetisation, we have observed DWs during the application of the current pulses, by synchronising the current pulses with the x-ray pulses, in stroboscopic mode. The measurements reveal that the DWs move without inertia, showing a practically vanishing mass. The negligible inertial effects can be explained taking into account  the narrow domain wall width induced by the large PMA, the large damping parameter and the stabilising effect of the longitudinal field associated to the Dzyaloshinskii-Moriya interaction.

Magnetic skyrmions are nanometer scale whirling spin configurations, predicted in the 80's but only recently observed with high resolution magnetic microscopies. Their small size, topological protection and the fact that they can be moved by very small current densities call for applications to novel memory and logic devices where skyrmions are the information carriers. Here we report on the experimental observation of magnetic skyrmions in Pt/Co/MgO sputtered ultrathin magnetic nanostructures, stable at room temperature without applied magnetic field. XMCD-PEEM measurements allowed us to observe such skyrmions and to demonstrate their chiral Néel structure. The skyrmion sizes are typically of the order of 120 nm. Our experimental observations are well reproduced by micromagnetic simulations and numerical modelling. This allows the identification of the physical mechanisms governing the size and stability of the skyrmions, which are keys for the design of devices based on their manipulation.

REFERENCES

J. Vogel at al. Phys. Rev. Lett. 108, 247202 (2012)

O.Boulle et al., Nature Nanotechn. 11, 449 (2016)

Energy loss magnetic chiral dichroism (EMCD), established in 2006 [1] celebrates its 10^th anniversary. EMCD is the TEM equivalent of the X-ray magnetic circular dichroism (XMCD) technique routinely applied on synchrotron beam lines for the study of magnetic moments. The EMCD signal is detected as an asymmetry in the energy filtered diffraction pattern, or alternatively as a slight difference in the fine structure of energy loss spectra for particular momentum transfers in the inelastic interaction (Fig. 1). The extremely high spatial resolution of modern TEMs makes EMCD interesting for spintronic and micromagnetic applications. In  the last decade, the technique has evolved into a reliable tool demonstrating nm-resolution, site selectivity and separation of spin and orbital moments via sum rules.

One of the consequences of EMCD is that the outgoing inelastically scattered probe electrons have topological charge. Structurally, they are identical with vortex electrons that can be routinely created in the electron microscope [2]. Such vortices are characterized by a spiraling wavefront and a phase singularity at the center, similar to optical vortices that were first described by Nye & Berry [3]. Owing to their short wavelength, these matter waves can be focused to atomic size. Another novel aspect is their magnetic moment, quantized in multiples of the Bohr magneton, independent of the electron spin. These features make electron vortices extremely attractive as a nanoscale probe for magnetic materials.

The discovery of vortex electron beams has spurred efforts to use them for EMCD because of their intrinsic chirality. It became soon clear that atom-sized vortices are needed to achieve this goal [4], as shown in Fig. 2. At the time of writing, the closest successful approach to such beams is the shaping of the incident wave front with a Cs corrector such that it matches the point group symmetry of the selected atomic column, revealing  EMCD signals with atomic resolution [5]. A promising alternative is the use of holographic vortex filters in the outgoing beam to detect spin polarized transitions [6], as sketched in Fig. 3. Other than in the standard EMCD geometry, this approach does not require a precise alignment of the crystal, and would thus allow the study of nanocrystalline and amorphous materials.

Acknowledgements: The financial support of the Austrian Science Fund (I543-N20, J3732-N27) and of the European research council, project ERC-StG-306447 is gratefully acknowledged.

[1] P. Schattschneider et al., Nature 441 (2006)  486

[2] J. Verbeeck et al.,Nature 467 (2010) 301

[3] J. F. Nye and M. V. Berry, Proc.Roy. Soc. A 336 (1974) 165

[4] P. Schattschneider et al., Ultramicroscopy 136 (2014) 81

[5] J. Rusz et al., Microscopy and Microanalysis 21 (2015) 499

[6] T. Schachinger et al., submitted; also this conference

Key to the advancement of magnetic materials is the exploitation of emergent magnetic behavior arising from nanoscale confinement.   A comprehensive understanding of this behavior requires a technique capable of resolving it and mapping its relationship to parallel nanoscale property domains such as local structural imperfections, chemical environment, and interfacial proximity.  The relatively young electron absorption spectroscopy technique known as Electron Magnetic Circular Dichroism (EMCD) [1] promises to meet these criteria, and it has already demonstrated qualitative [2] and quantitative [3, 4] results for parallel electron probe illumination on sample regions in the sub 100 nanometer range.  While this already represents a spatial resolution superior to what can be achieved with x-rays [5], the use of parallel electron illumination means that high spatial resolution can only be achieved through the use of energy filtered diffraction patterns [3, 6] or energy filtered TEM imaging techniques [7].

In this work, we describe our progress in the development of an experimental methodology utilizing convergent electron probes for the purposes of acquiring EMCD datasets.  We name this method STEM-EMCD (see figure 1) and argue that it has two significant advantages over parallel beam illumination schemes.  First, the use of a convergent probe allows for the acquisition of arbitrarily large datasets, extending the effective acquisition time on a region of interest well beyond what is feasible for a single-shot experiment using parallel illumination.  This substantially improves the signal to noise ratio of EMCD spectral pairs, as shown in figure 1, and can be employed in such a way to mitigate significant beam damage.  Second, the diameter of the electron probe can be reduced to one nanometer or less.  If appropriate multivariate statistical methods are used to exploit spectral redundancy in the datacubes, it becomes possible to accurately approximate the raw data using a significantly reduced parameter space, dramatically suppressing the influence of statistical noise.  We demonstrate that this allows for sum rules to be applied on individual spectral pairs, enabling real-space maps of magnetic transitions to be generated with a spatial resolution approaching that of the beam diameter, as shown in figure 2.  We conclude with a discussion of some of the challenges still faced by the STEM-EMCD method as well as its prospects for directly addressing some of the most elusive questions in contemporary research in nanomagnetism.

[1]          Schattschneider, P. et al.: Detection of magnetic circular dichroism using a transmission electron microscope. Nature 441 (2006) 486.

[2]          Schattschneider, P. et al.: Magnetic circular dichroism in EELS: Towards 10 nm resolution. Ultramicroscopy 108 (2008) 433.

[3]          Lidbaum, H. et al.: Quantitative Magnetic Information from Reciprocal Space Maps in Transmission Electron Microscopy. Phys. Rev. Lett. 102 (2009) 037201.

[4]          Wang, Z.Q. et al.: Quantitative experimental determination of site-specific magnetic structures by transmitted electrons. Nat. Commun. 4 (2013) 1395.

[5]          Fischer, P.: Frontiers in imaging magnetism with polarized x-rays. Condens. Matter Phys. 2 (2015) 82.

[6]          Loukya, B. et al.: Electron magnetic chiral dichroism in CrO₂ thin films using monochromatic probe illumination in a transmission electron microscope. J. Magn. Magn. Mater. 324 (2012) 3754.

[7]          Lidbaum, H. et al.: Reciprocal and real space maps for EMCD experiments. Ultramicroscopy 110 (2010) 1380.

[8]          Thersleff, T. et al.: Quantitative analysis of magnetic spin and orbital moments from an oxidized iron (1 1 0) surface using electron magnetic circular dichroism. Sci. Rep. 5 (2015) 13012. 

Near-ambient pressure X-ray photoemission spectroscopy (NAP-XPS) [1] is a modification of XPS so that surfaces can be studied in presence of gases and not only in vacuum. This is necessary when you want to access chemical and electronic properties of catalytic surfaces in realistic reaction conditions (variable P, T). We have focused on the study of bimetallic catalytic surfaces and their chemical/electronic surface properties during the oxidation of carbon monoxide. Indeed, the low temperature catalytic oxidation of carbon monoxide to carbon dioxide is an important reaction used in several areas and in particular for removing the CO traces from the H₂ combustible in fuel cells. Pt and Au are well known catalysts for the CO oxidation reaction, however on both metals the oxygen dissociation remains the rate determining step. Associating Pt or Au with a second metal, that facilitates the oxygen activation while keeping free sites for CO adsorption on the surface, appears as an effective way to improve the catalytic performances of these two metals. The surfaces Pd₇₀Au₃₀(110) and Pt₃Sn(111) have been chosen on the basis of the performance of real catalysts (bimetallic nanoparticles supported on oxides).

The NAP-XPS experiments were performed at ALS beamlines 9.3.2 & 11.0.2 (Berkeley, USA) and, more recently, at SOLEIL beamline TEMPO (St Aubin, France). NAP-XPS allows a fine characterization of electronic properties of catalytic surfaces including reactants, products and inert spectator species. In addition, NAP-XPS can also detect the gas phase species near the surface and can therefore monitor the reaction rates, at least qualitatively. The inherent variable photon energy of synchrotron sources enables depth profiling measurements which can be used to explore the segregation of species into the subsurface region under gas environments.

In the case of Pd₇₀Au₃₀(110) we have a very rich Au-surface (~ 85% of Au as determined by LEISS) due to Au segregation to the surface at thermodynamic equilibrium. By environmental STM [2] we could notice that the surface reorganizes at atomic level and eventually roughens just after adsorption of the reactive gases both under oxygen and CO. This is followed by a reversal of segregation where Pd migrates to the surface as it is determined by varying the photon kinetic energy in NAP-XPS. For Pt₃Sn(111) we studied the two terminations (2x2) and (√3x√3)R30° obtained at different annealing temperatures. For both surfaces we have identified the presence of bridge and top sites during adsorption of CO. Oxygen adsorption studied by PM-IRRAS shows a higher interaction of oxygen with Pt₃Sn(111)-(√3×√3)R30° than with Pt₃Sn(111)-(2x2). By NAP-XPS we observe, in both surfaces, the partial segregation of Sn to the surface with increasing temperature in the presence of oxygen and under reaction conditions [3]. Finally we studied the evolution of the surfaces by NAP-XPSunder reaction conditions. A general result was observed for the studied bimetallic surfaces: the activity of the surface is higher in presence of chemisorbed oxygen (that we can compare to a“loose”oxide) but it rapidly decreaseswhen stoichiometric oxides are formed on the surface at higher temperatures (Figures 1 & 2).

References
[1] D.F. Ogletree et al., Rev. Sci. Instr. 73 (2002) 3872; M. Salmeron, R. Schlögl, Surf. Sci. Rep. 63 (2008) 169

[2] F.J. Cadete Santos Aires, C. Deranlot, Proc. EUREM12, Vol.III, P. Ciampor, L. Frank, P. Tománek, R. Kolarik, Brno 2000, I263; Y. Jugnet et al., Surf. Sci. 521 (2002) L639; M.A. Languille et al., Catal. Today 260 (2016) 39.

[3] Y. Jugnet et al., J. Phys. Chem. Lett. 12 (2012) 3707.

Propelled by rapid advances in synthesis, characterization, and computational modeling, materials science is fast progressing toward a “materials-by-design” paradigm. While we can envision a very large number of materials combinations, we are unable to synthesize them in practice because existing characterization and modeling approaches fail to capture the inherent complexities of such systems. This shortcoming is amplified by the fundamental disconnect between highly local and volume-averaged structure-property models resulting from electron microscopy and X-ray diffraction investigations, respectively. Here we show how complementary analysis techniques can reveal previously overlooked local compositional and valence fluctuations around secondary phases in an oxide thin film, yielding powerful new insight into low-pressure thin film synthesis. We explore the model complex oxide, La2MnNiO6 (LMNO), which possesses a hierarchy of structural, chemical, and magnetic ordering across multiple length scales. This material shows great promise for next-generation spintronics and thermoelectrics, but its implementation is hindered by a poor understanding of the underlying structure that governs its macroscale magnetic performance. Using aberration-corrected scanning transmission electron microscopy (STEM) and energy-dispersive X-ray spectroscopy (STEM-EDS), we confirm the onset of cation ordering upon annealing; however, three-dimensional composition mapping using atom probe tomography (APT) reveals a fine distribution of NiO secondary phases, as shown in Figure 1. Electron energy loss spectroscopy (STEM-EELS) mapping of the chemical environment surrounding these particles shows significant fine structure changes, indicating a reduction in Mn valence, as shown in Figure 2. We consider these results in light of ab initio calculations, which show that the NiO phase and a transition region with reduced Mn valence is an inevitable result of low-oxygen pressure growth processes that are commonly used in fabrication of complex oxide heterojunctions. We argue that kinetic limitations on the reincorporation of NiO nuclei “locks” them into the film structure during synthesis; the resulting nanoscale network disrupts the long-range ferromagnetic ordering of the matrix, degrading macroscale magnetic properties. This array of experimental and theoretical techniques allows us to better understand the relationship between structure and magnetic properties, illustrating the need for a correlative, multidimensional approach to thin film characterization.

Atomic-scale investigation of interface phenomena in two-dimensionally Sr-doped La₂CuO₄ and La₂CuO₄/ La_2-xSr_xNiO₄ superlattices

Y. Wang, Y. E. Suyolcu, W. Sigle, U. Salzberger, F. Baiutti, G. Gregori, G. Cristiani, G. Logvenov, J. Maier, and P.A. van Aken

Max Planck Institute for Solid State Research, Heisenbergstr. 1, 70569 Stuttgart, Germany

Physics phenomena at interfaces of complex oxide heterostructures have stimulated intense research activity due to the occurrence of a broad range of electric and magnetic functionalities that do not pertain to the constituting single phases alone. Interface effects have been proven to be a powerful tool for improving or even inducing novel functionalities [1, 2]. In the case of interface superconductivity, the interatomic structure relaxation and charge transfer play a key role [3].

In this work, we combine atomic-resolved quantitative STEM imaging with analytical STEM-EELS/EDX analysis to investigate the interface effects in La₂CuO₄ (LCO)-based hetero-structures, i.e. superlattices of Sr two-dimensionally (2D) doped LCO, and LCO/La_2-xSr_xNiO₄ (LSNO) hetero-structures, both exhibiting T_c up to 40 K, despite its non-superconducting constituents. HAADF images of the structure are displayed in Fig.1 confirming high-quality epitaxy. A detailed study of the cation distribution and concentration at the doped interfaces was performed by EDXS and EELS analyses. In the case of Sr-2D doped LCO superlattices, the analysis shows that Sr cations undergo an asymmetric redistribution, as a result of the MBE growth process. Oxygen K-edge fine structure analysis reveals that the hole concentration profile on the downward side of the nominal SrO layer is clearly decoupled from the Sr-profile indicating an accumulation of positive charges compensating the negatively charged SrO planes (Fig.2).

These results were supplemented by quantitative analysis of atomic-resolved high-angle annular dark-field (HAADF) and annular bright-field (ABF) images, which were simultaneously acquired. From these images the local lattice and copper-apical-oxygen distortions at the interfaces were evaluated. The relation of these results with the observed T_c will be discussed.

References:

[1] A.Gozar et al., Nature 455 (2008), p.782.

[2] F.Baiutti et al., Nat. Commun. 6 (2015), p. 8586.

[3] Y.Wang et al., ACS Appl. Mater. Interfaces (2016) DOI :10.1021/acsami.5b12813

[4] The research leading to these results has received funding from the European Union Seventh Framework Program under Grant Agreement 312483-ESTEEM2 (Integrated Infrastructure Initiative I3).

Development and innovations of modern high-performance materials rely crucially on the repertoire of characterization methods available. While individual techniques provide information on a specific aspect of the investigated material, a comprehensive understanding requires a correlative approach in which complementary information are needed from exactly the same Region of Interest (RoI). Therefore, correlative methodologies combining several techniques are indispensable in a wide range of scientific disciplines including materials science [1].

In contrast to the traditional EDX and EELS spectroscopies, SIMS is well-known for high-sensitivity (ppm), high dynamic range and more interestingly, isotope analysis for all the elements of the periodic table. This presentation will highlight the benefits of correlating SIMS imaging with complementary analysis using Electron Back Scattered Diffraction (EBSD) and Atomic Force Microscopy (AFM). As the SIMS images do not reveal crystallographic orientations of the grains in the microstructure, studies that aim to correlate structural defects such as twinning and grain boundary orientation in a multiphase material require that the strengths of SIMS are coupled with that of EBSD to probe the local crystallographic characteristics. As a first step, we correlate the SIMS with EBSD imaging to complement specific chemical characteristics seen in SIMS with the corresponding crystallographic characteristic captured in EBSD.

To further characterize the material and for correct interpretation, the exact topography of the RoI is required. We do topographic analysis using an AFM on the exact location where SIMS and EBSD analysis are carried out. In this way, a three-way correlative methodology is developed. As each of these analyses is done on separate instruments, specific methodological strategies are employed to overcome the challenges.

To illustrate this methodology, we selected the topic of hydrogen embrittlement in a TWinning Induced Plasticity (TWIP) steel [2]. As conventional techniques such as EDX are not capable of mapping hydrogen distribution, the SIMS approach brings a particularly strong advantage to this study. Prior to the SIMS analysis, the steel samples were electrochemically charged with deuterium to distinguish it from hydrogen naturally present in the sample. The SIMS images of H^- and D^- distributions are obtained from a Cameca NanoSIMS50 with a Cs⁺ primary ion beam. Then, EBSD and AFM analyses are carried out on the exact location to correlate SIMS chemical characteristics with crystallographic defects such as twins, grain boundaries and sample topography.

In this presentation, we will introduce and demonstrate this correlative paradigm for materials characterization. We will also present the scientific and technical challenges and discuss the strategies to go beyond the current state-of-the-art.

References:

T. Wirtz et al, Nanotechnology, Vol. 26, 434001, 2015.

L. Mosecker et al, Mat. Sci. Engr. A, 642, p71-85, 2015.

Acknowledgments: This work was co-funded by the Luxembourg National Research Fund (FNR) by the grant C13/MS/5951975.

Contrast in atomically-resolved EDX elemental mapping in real space or in reciprocal space channelling patterns, derived from characteristic X-ray emissions excited by a given probe, is generated by the variation in emission rate as the incident wave function is scanned over the target atoms in a crystal. This is achieved either by raster scanning a focused coherent probe in real space at a fixed orientation (conventional EDX mapping), or by a systematic scan in angle of a collimated beam, the entire EDX spectrum being collected for each pixel (ALCHEMI). Whilst the signal is essentially governed by the probability density of the probe wave function on each atom site, delocalization of the primary ionization event may lead to a reduction in contrast since the electron wave function is effectively sampled over a finite region.  The total ionization potential may be described in real space as a Lorentzian function of half-width b, and the event becomes more localized with an increase in X-ray energy. Thus coherent contrast provides a map in real or reciprocal space of the electron beam interaction with the ionization potential of a specific atomic species.

However incoherent scattering by an absorptive potential leads to a progressive increase in a separate incoherent background component, this being associated with an EDX spectrum which is representative of the overall specimen composition. Generally speaking, coherent contrast is generated within the top 10 – 30 nm, and this initial contrast is progressively undermined by the incoherent background contribution in the form of an absorptive potential in atomic resolved maps or thermal diffuse scattering Kikuchi band contrast in channelling patterns [1].

The relative detected proportion of coherent compared with incoherent signal is altered by X-ray absorption within the specimen. It is envisaged that strong absorption of soft X-rays (described by a small mean free path λ) will enhance coherent contrast in both lattice image and channelling patterns compared to that derived from more energetic X-rays. High energy X-rays should have better contrast in the coherent signal due to increased localization, but this begins to be dominated by an incoherent signal that builds up with thickness. Softer X-rays may have somewhat diminished coherent contrast due to greater intrinsic delocalization, but this is offset by a more strongly attenuated incoherent signal with increasing thickness.

X-ray channelling patterns near the orientation were obtained from GaAs using 300 keV electrons together with a custom-built acquisition script run on an FEI Titan TEM. Fig. 1 shows PCA noise-reduced patterns compared with simulations in which both delocalization and absorption are taken into account. Close inspection reveals greater overall dynamic contrast occurs for softer X-rays. Experimental K/L ratio maps are shown in Fig. 2 (tilt 26^o and take off angle 21^o), being consistent with the calculation where the central As K/L contrast ratio starts to diminish with increasing thickness due to X-ray absorption (λ_K = 16 μm, λ_L = 0.15 μm, (b_K= 0.024 Å, b_L= 0.13 Å) .  A similar but smaller effect occurs for the mapped Ga K/L ratio (λ_K = 42 μm, λ_L = 1.2 μm,  b_K= 0.027 Å, b_L= 0.15 Å) since Ga L-shell X-rays are less strongly absorbed than the As L. Without X-ray absorption, a central enhancement remains for all thicknesses, and the harder X-rays retain more contrast.  Fig. 3 shows the separate coherent and incoherent components together with the total contribution. Note the incoherent contrast is similar for all excitations.

In conclusion, experimental observations are consistent with a model that predicts an increase in contrast from soft X-rays compared with more energetic X-rays, thus demonstrating that X-ray absorption may play a greater role than delocalization in determining overall contrast. It is anticipated that X-ray absorption, enhanced by grazing angle specimen-detector geometry, may be useful in enhancing the coherent/incoherent scattering for soft X-rays. Calculations have shown analogous effects occur in atomically-resolved X-ray STEM imaging [1].

[1] C.J. Rossouw, ‘Channelling and atomic resolution STEM using X-ray emissions with absorption’, Microsc. Microanal. 21 (2015), 1077 - 1078.

Direct electron detectors have played a key role in the recent increase in the power of single particle electron cryomicroscopy (cryo-EM). In this talk, I will summarise the background to these recent developments, give a practical guide to their optimal use, and discuss future directions.

Over the last decade, great progress has been made in the instrumentation of transmission electron microscopy (TEM) through the introduction of aberration correctors, electron energy monochromators, and a wide variety of TEM specimen holders designed for in-situ applications. With these new capabilities, a wealth of advanced experimental data can be easily generated by advanced TEM systems.  However, the development of a revolutionary image capture device has been lacking; this effectively makes imaging detectors the main bottleneck when striving to achieve the full potential performance of an advanced TEM.

Traditional image detectors use scintillator and optical transfer path (fiber-coupling or lens) to convert high energy electrons to photons that are subsequently transferred to the imaging sensor to form an image.  One of the disadvantages of this indirect detection approach is the loss of image resolution and sensitivity during the electron-photon conversion and the photon transfer as additional noise sources that significantly degrade the signal-to-noise ratio (SNR) of the image detector.

Very recently direct detection imaging devices have been successfully developed based on technology advancement made in CMOS (complementary metal oxide semiconductor) design and manufacturing, high speed data architectures, vastly increased memory densities and speed, etc. The elimination of scintillator and subsequent optical transfer path has significantly improved the detective quantum efficiency (DQE) – a critical measure of SNR for resolution and sensitivity of an imaging device. The state-of-the-art direct detection imaging device further boosts the DQE and image quality under extremely low beam conditions by electron counting at high speed (e.g. 400 fps @ 4k x 4k resolution) to eliminate the sensor readout noise and minimize the electron scattering noise. Figure 1 shows the comparison of DQE measurement at 300 kV for K2 direct detection cameras operated in electron counting (solid blue) and linear (dotted blue) mode, and scintillator/fiber optical coupled cameras (purple). It is clear that electron counting has significantly restored the DQE performance of direct detection cameras for all frequencies.

This new generation of direct detection imaging device has revolutionized the field of cryo-electron microscopy (cryoEM) in structural biology and is starting to impact many applications in electron microscopy of materials science, for example, in-situ microscopy, 4D STEM, imaging beam sensitive materials, quantitative measurement of radiation damage or quantitative electron microscopy, etc. Direct detection and electron counting are poised to advance electron microscopy into a new era.

In scanning transmission electron microscopy (STEM), one can obtain a variety of STEM images such as bright-field (BF) and annular dark-field (ADF) STEM images by changing the shape of the scintillator. However, the intensity distribution of convergent beam electron diffraction (CBED) patterns at the detector plane is yet to be fully utilized. Meanwhile, direct electron detectors with fast frame rate have recently been commercialized and used in electron microscopy. Such detectors, when used for recording CBED pattern images for each STEM probe position, are called pixelated STEM detectors. With the obtained 4-dimensional (4D) dataset, any shape of STEM detector can be synthesized in a post processing by a free selection of the integration area. Therefore, we can synthesize variety of STEM images such as differential phase contrast (DPC) and annular bright field (ABF) images, if we once record the image signal with a pixelated detector.

The 4D dataset can also be used for the advanced image processing techniques such as ptychography, which has been shown to provide high efficiency for reconstructing the phase image of an object [1,2]. Using ptychography, not only the phase contrast can be enhanced but also the effect of lens aberrations to the image such as defocus can be corrected by the post processing using the information collected by a pixelated detector.

Experiments were performed using an aberration corrected microscope (JEOL JEM-ARM200F) equipped with a pixelated detector (pnDetector pnCCD), the fast direct electron detector, which can record images at a speed of 1,000 fps in a full frame mode (264 x 264 pixels). Binning or windowing can increase the speed. The camera was placed below the ADF detector to enable simultaneous recording.

Figure 1 shows STEM images of a monolayer graphene obtained at 80 kV. Fig. 1a shows an ADF image obtained with the probe current of approximately 0.2 pA and the dwell time for a pixel of 0.5 ms. Because of a combination of low dose and residual uncorrected aberrations, the lattice contrast of graphene is almost buried in the noise originated from 50 Hz commercial frequency. Fig. 1b shows a reconstructed phase image using ptychography. The image contrast is significantly improved compared to the simultaneous ADF image, but the contrast transfer of the image (see the Fourier transform displayed in the inset) is anisotropic, resulting in uncertain positions of carbon atoms. This is because there remains large two-fold astigmatism and defocus in the image shown in Fig. 1(b), because we could not adjust those by observation of the ADF image due to the weak image signal. Fig. 1c is a phase image in which the aberrations are corrected through post processing using the same 4D dataset by applying correction functions in the spatial frequency domain. The image is no longer anisotropic and the carbon atomic positions can be unambiguously determined. Although the same amount of electron dose is used to form the ADF and the corrected phase images, the result clearly shows the benefit of ptychographic phase reconstruction in improving image signal to noise and being able to correct aberrations through post processing.

Figure 2 shows the ptychographic phase maps of the 4D dataset at a certain spatial frequency. With an aberration-free electron probe, they would be flat phase on the two sidebands, and the phase difference between the bands be π. In Fig. 2a, the map of the original dataset, there is a phase gradient inside each sideband because the aberrations were present in the electron probe. Fig. 2b is the correction function that compensates for the aberration seen in Fig. 2a. Fig. 2c is the corrected phase map and corresponding to the image in Fig. 1c. Here, only defocus and two-fold astigmatism are corrected but corrections of other higher order aberrations are in principle possible.

Figure 3 shows through focus images created in the same way as above. As we know the full information on the electron wave at the condenser aperture plane, we can induce any aberrations such as defocus.

References

[1] PD Nellist et al., Nature, 374 (1995) p. 630.

[2] TJ Pennycook et al., Ultramicroscopy, 151 (2015) p. 160.

Acknowledgement

PDN and HY acknowledge funding from the EPSRC through grant number EP/M010708/1.

Typically, the Nyquist frequency determines the sampling rate required to resolve a specific feature in a dataset. In practice this requires oversampling by twice the maximum relevant frequency. As such this provides a lower limit for the electron dose necessary to acquire the data experimentally. However, by applying the compressive sensing (CS) principles of sparsity and incoherence, this limit can be overcome and therefore a substantial reduction of the total electron dose can be achieved. CS theory then allows a recovery of the essential image information from randomly undersampled images. The theory of CS has been successfully applied in a number of areas, e.g. astronomy, MRI scanning, electron tomography [1], and most recently first experimental designs for STEM have been reported [2,3]. Despite these developments, CS acquisition schemes able to recover quantifiable information from atomically resolved images of beam-sensitive materials, time-changing processes and spectroscopic datasets have so far not been established  for STEM.
Here we demonstrate such a CS-based STEM acquisition implementation and benchmark its performance at atomic resolution using a variety of materials, including complex oxide ceramics with an inhomogeneous distribution of point defects as illustrated in fig. 1, but also highly beam-sensitive catalysts with atomic resolution [4]. The implementation relies on custom-built hardware and software which controls an electrostatic beam shutter to blank the electron beam during all but a few randomly chosen pixels through a regular image (or indeed spectrum image) acquisition. The total electron dose used to form a whole dataset can thus be tailored according to the electron damage threshold of the sample under investigation whilst maintaining all other acquisition parameters identical to regular data acquisition, allowing a seamless transition from ‘fully sampled’ to ultra-low-dose conditions. It will be shown that datasets acquired in this fashion down to a few % of the total incoming dose (see fig. 2) can be reconstructed to yield a truthful atomic resolution representation of the sample, using a variety of reconstruction algorithms [5,6]. We show that this implementation of CS can also be extended to 2D spectroscopy mapping and simultaneous HAADF image acquisition.. These results open the door to practical ultra-low dose high-resolution imaging and spectroscopy in the STEM for very beam sensitive samples, where the level of sampling can simply be chosen depending on the damage threshold of the material being investigated [7].
References
[1] Z. Saghi et al., Nano Letters 11 (2011), 4666
[2] Q.M. Ramasse et al., 15th Frontiers of Electron Microscopy in Materials Sciences (FEMS); 2015, September 13-18; Tahoe City, California, p76;
[3] A. Béché et al. , Appl. Phys. Lett. 108 (2016)
[4] D. Mücke-Herzberg et al. submitted
[5] A. Stevens et al. , Microscopy 63 (2014), 41-5;
[6] J. Ma. et al. submitted
[7] SuperSTEM is the UK EPSRC National Facility for Aberration-Corrected STEM, supported by the Engineering and Physical Science Research Council. PNNL, a multiprogram national laboratory, is operated by Battelle for the U.S. Department of Energy under Contract No. DE-AC05-76RLO1830. PAM, ZS and RL acknowledge funding under ERC Advanced Grant 291522-3DIMAGE.

Transmission electron microscopy (TEM) is a very powerful technique to investigate materials down to their atomic components. Its versatility allows quantifying samples from their shape to the nature of their constituents and surroundings. However, the strong interaction of the electron beam with matter potentially induces damages in samples under investigation, especially for those composed of soft matter such as zeolites, metal organic frameworks (MOFs) and most life science samples. Current workarounds involve the reduction of the beam intensity, such as in the so-called low dose imaging, or increase of the detector performances as provided e.g. by direct electron detectors.

Recently, improvement in signal processing lead to the development of compressed sensing [1, 2], a numerical algorithm based on the assumption that real life images are sparse in some particular well-chosen basis. Images can then be expressed with much less components than what required by the Nyquist sampling theorem. By extension, not all pixels in a given image are necessary and only a random selection of them is sufficient to retrieve the original image with fidelity. By definition, compressed sensing is a very dose efficient technique as only parts of the sample need to be exposed to the electron beam to reconstruct a faithful image. So far, only theoretical implementations of compressed sensing were investigated in the TEM community, focused mostly on the case of tomographic reconstructions [3].

Very recently, we demonstrated the first physical implementation of compressed sensing in a Scanning TEM (STEM) based on the use of a solenoid as a fast beam blanker [4]. The solenoid is placed in the condenser plane of a STEM in a specially designed condenser aperture holder with feedthrough electrical contacts. By synchronizing the STEM signal with the current source driving the solenoid, we successfully acquired compressed images by shifting the beam away from the region of interest on blanked pixels. The case of both medium scale imaging (Fig.1) and high resolution imaging (Fig. 2) was investigated and reconstructed using the SPGL1 algorithm [5] with a signal compression of 80%. The present setup, although still quite far from low dose imaging conditions, has lead to successful imaging of a cross grating and a SrTiO₃ sample. Latest results revealed that a checkerboard pattern, the most demanding pattern for the fast beam blanker, could be acquired with a dwell time of 40 µs and a beam deflection of 225 nm, as shown in Fig. 3.

By further improving the experimental setup, we expect reducing the acquisition dwell time to values within the µs range. Such improvements will offer possibilities for realizing improved images and tomography experiments of electron beam sensitive materials.

[1] Candes E. J. et al., Communications on Pure and Applied Mathematics, 59 (2006) 1207-1223.

[2] Donoho, D., IEEE Transactions on Information Theory 52 (2006) 1289-1306.

[3] Saghi Z. et al., Advanced Structural and Chemical Imaging 1 (2015) 1-10.

[4] Béché A., Goris B., Freitag B. and Verbeeck J., APL 108 (2016) 093103.

[5] Van den Berg E. and Friedlander M. P., SIAM Journal on Scientific Computing, 31 (2008) 890-912.

Aknowledgments: A.B and J.V. acknowledge funding from the ERC Grant No. 278510 VORTEX and under a contract for an Integrated Infrastructure Initiative No. 312483 ESTEEM2. B.G. acknowledges the FWO for a postdoctoral research grant.

Spectro-microscopy techniques like electron energy-loss spectroscopy (EELS) and cathodoluminescence (CL) are routinely used nowadays for optical characterization of nanostructures with wonderful spatial resolution. With advanced monochromators, the energy resolution in EELS can be as good as 10 meV [1]. Nevertheless, with monochromators, there is a trade-off between the signal intensity and the energy resolution. Conversely, the spatial resolution in common optical microscopes is diffraction limited, though the spectral resolution is very often down to the sub meV regime. Hence, it is a high time to look for a technique, which can combine the spectacular spectral resolution of optical probes and spatial resolution of the electron probe. The possibility of integrating these two probes was first anticipated by Prof. Archie Howie in 1999 [2], and a few years later, a compact theoretical formalism of the technique named electron energy-gain spectroscopy (EEGS) was proposed [3].

In EEGS, electrons pick up energy from external illumination, which is very often the coherent output of a finely tuned laser. Although the electron and photon do not couple linearly in free space due to energy-momentum mismatch, the presence of a nanostructure can break the mismatch by providing the extra momentum through induced light fields, like the evanescent plasmonic field of the nanostructure. Employing synchronized femtosecond pulses of electrons and very intense pulse of photons, electron energy gain (EEG) has been demonstrated, a technique commonly called as photon-induced near-field electron microscopy (PINEM) [4]. It has shown that electrons can absorb or emit photons and the resulting spectra consist of peaks positioned at energies integral multiples of the laser photon energy on both sides of the zero loss peak. Multiphoton absorption/emission is a non-linear phenomenon, occurring at very high laser peak intensity (~ GWcm^-2), but this can also be triggered at comparatively lower laser intensity, if the laser wavelength is tuned at particle plasmon wavelength [5]. The multiphoton emission and absorption probabilities depend only on the temporal ratio of the electron and photon pulse and the delay between them. The laser can be described as a coherent photon state, which excites a coherent plasmon state and the electron can absorb photon resulting in gain. And the reverse process is a stimulated photon emission by the electron (SEELS) in which the electron loses one photon energy. For large number of incident photons, these two probabilities are the same [5]. For a moderate laser intensity the EEGS or SEELS probability is few ten times larger than the EELS probability [3,5].

In our lab, we are developing an EEGS setup in one of the existing STEM (VG HB 501). The goal is to do spectroscopy by varying the laser energy and detecting the energy gain of electrons. We use a paraboloid mirror (used in CL) to focus the laser output on the sample surface, which can be used over a wide wavelength range without any change of optics. In a normal EEG set up, a pulsed laser is used as excitation source and a pulsed gun is used to detect only those electrons which goes through energy gain or loss in interaction with the laser pulse. In this work we are not using a pulsed gun. A pulsed gun is expensive to make, pulse duration is not easily tunable. A high brightness and thus high spatial resolution is not achieved easily with pulsed gun. We will use a Q-switched pulsed dye laser with tunable wavelength (570-900 nm) and typical pulse duration of approximately 30 ns.

The instrumental details and some preliminary results will be discussed. We expect that geometries like nanostars (Figure 1(b)) which can confine a huge amount of field might be a promising candidate that can yield much stronger EEGS signals at moderate laser intensity.

References:

[1] O. L. Krivanek et al. Nature. 2014, 514, 209.

[2] A. Howie, Inst. Phys. Conf. Ser. 1999, 161, 311.

[3] F. J.  García de Abajo, and M. Kociak, New J. Phys. 2008, 10, 073035.

[4] B. Barwick et al.  Nature 2009, 462, 902.

[5] A. A. Garcia, and F. J.  García de Abajo, New J. Phys. 2013, 15, 103021.

Following a recent suggestion [1] that interaction-free measurements are possible with electrons, we have analyzed the possibilities to use this concept for imaging of biological specimen with reduced damage. We have also made preliminary designs for an atomic resolution interaction-free electron microscope, or “quantum electron microscope” [2], for one example see figure 1.

The idea of interaction-free measurements follows Elitzur and Vaidman [3], who proposed that an opaque object may be observed by detecting a photon that did not interact with that object. This concept uses a Mach-Zehnder interferometer in which one branch of the particle wave passes through the specimen (“specimen beam”) while the other part follows another path (“reference beam”).  For a substantial reduction of the interaction, the amplitude of the wave passing through the specimen must be small and the interaction must be repeated many times. To implement this concept in an electron microscope would require a number of unique components not found in conventional transmission electron microscopes. These components include a coherent electron beam-splitter or two-state-coupler, and a resonator structure to allow each electron to interrogate the specimen multiple times. A two-state-coupler has the function of moving the electron wave slowly between the reference beam and the specimen beam, as in a Rabi-oscillation. We have come up with four different choices for this: a thin crystal, a grating mirror, a standing light wave and an electro-dynamical pseudopotential.

Figures 2 shows a typical example of the theoretical output of a measurement, where for each pixel there is signal in the reference beam, the specimen beam and the inelastic channel, possibly detected in an energy loss measurement. Figure 3 shows the advantage of interaction free measurement in the detection of the presence of a dark pixel as compared to dark field microscopy. In the analysis of the image contrast that interaction-free microscopy can create, our tentative conclusion is that transparent specimen can be detected, but different transparencies cannot be distinguished [4]. Other modes of operation of a microscope with multiple interactions with the specimen, however, may enable this.

This research is funded by the Gordon and Betty Moore Foundation.

1] Putnam, W.; Yanik, M. Phys. Rev. A 2009, 80, 040902.

2] Kruit, P.; R. G. Hobbs, C-S. Kim, Y. Yang, V. R. Manfrinato, J. Hammer, S. Thomas, P. Weber, B. Klopfer, C. Kohstall, T. Juffmann, M. A. Kasevich, P.Hommelhoff, K. K. Berggren. Ultramicroscopy (2016), doi:10.1016/j.ultramic.2016.03.004.

3] Elitzur, A. C.; Vaidman, L. Found. Phys. 1993, 23, 987–997.

4] Thomas, S.; Kohstall, C.; Kruit, P.; Hommelhoff, P. Phys. Rev. A 2014, 90, 053840.

Electron diffraction, and its use to study the nanoscale crystallography of materials, has had something of a renaissance in recent years. The ease with which electron diffraction patterns can now be acquired with modern instrumentation, coupled with developments in electron optical techniques, faster detectors and sensitive cameras have all contributed to microscopists looking again at what novel information electron diffraction can provide. In this presentation, we will focus on scanning electron diffraction (SED), whereby electron diffraction patterns are acquired at each real space pixel, so that following a raster scan across a region of interest, a rich 4D data set is obtained that contains a wealth of information about the phases present, their orientation, defective regions and strain. Automation of pattern acquisition and sensitive recording devices also enable very fast data collection and open new avenues for recording unique crystallographic data from highly beam-sensitive materials [1]. SED can also be applied whilst precessing the beam [2] and this combination, known, in general, as scanning precession electron diffraction (SPED), which may lead to more accurate measurements of local crystallography through access to higher order reflections and greater sensitivity to in-plane rotations, strain, etc.

Figure 1 shows an example of SED analysis, with and without precession, considering the effects of strain brought about by the addition of Sb in a GaAs nanowire, whose wire axis is parallel to the axis in the zinc blende structure ([0001] in wurtzite). Fig 1 (a) shows a series of ‘virtual dark field’ (VDF) images created by plotting the intensities of particular reflections, from an unprecessed pattern series, as a function of real space position. The contrast seen in the VDF resembles the contrast seen in conventional DF images showing changes in intensity brought about through planar bending in the doped region. In Fig 1(b) the strain components are plotted, derived from measuring the distortions of precessed diffraction patterns relative to a reference (undistorted) region well away from the Sb region. The magnitude and direction of the strain is as expected from finite element modelling.

In reality, any 2D crystallographic map such as those shown in Figure 1, is really a projection of a 3D structure. As such, in order to be sensitive to changes in strain and orientation in 3 orthogonal directions, it becomes necessary to tilt the sample and ultimately to record a full tilt series of crystallographic information so that, using tomographic methods, a complete 3D reconstruction of the local crystallography is made possible [3]. Recently, we have combined SPED and tomography to reconstruct both real space morphology and orientation maps in three dimensions, to interrogate the local crystallography of sub-volumes of material and to determine, for example in the case of a Ni-base superalloy, a novel 3D orientation relationship between a matrix phase and an embedded carbide particle, see Figure 2. To extend this to map strain, as illustrated in Fig 1 (b), into 3D is considerably more challenging as such ‘tensor tomography’ demands a new approach to recover, in general, six strain components at every voxel. We will discuss progress in this regard and show how a model-fitting approach may be the best route forward [4].

[1] E.F. Rauch and L. Dupuy, Arch. Metall. Mater. 50, 87–99 (2005).

[2] P.A. Midgley and R. Vincent, Ultramicroscopy, 53, 271–282 (1994).

[3] A.S. Eggeman, R. Krakow and P.A. Midgley, Nature Commun. 6, 7267 (2015).

[4] The authors acknowledge the many people who have contributed to this work including R. Leary. R. Krakow, J. Barnard, J.M. Thomas, A. van Helvoort, Z. Saghi, M. Benning. C. Schoenlieb, J. Ma, O. Messe, C. Rae, A. Knowles, H. Stone, A. Ferrari, S. Hodge, U. Sassi, D. de Fazio. 

The authors acknowledge funding from the Royal Society and from the ERC grant 291522-3DIMAGE. DJ acknowledges an an Associateship from the Cambridge NanoDTC.

The properties of nanoscaled materials can be changed by applying strain and as such there is an interest in the accurate measurement of deformation with nm-scale resolution. This was until recently considered as a difficult problem. However, the last ten years has seen a great deal of development in techniques that can be used to measure deformation with the required resolution [1,2]. Today there are many different approaches which can be used to recover valuable information about the deformation. Each of these techniques has strengths and weaknesses and requires different set ups in the electron microscope [2]. In this presentation we will present dark field electron holography, nanobeam electron diffraction (NBED), precession diffraction (NPED) and the geometrical phase analysis (GPA) of TEM and high angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) images. We will then discuss which technique is most suitable for different types of materials problems and benchmark their performance with respect to the accuracy of the measurements, precision and spatial resolution.

Figure 1(a) shows a HAADF STEM image of a 100-nm-thick Si test specimen containing 10-nm-thick SiGe layers with different Ge concentrations. As the specimen has been grown epitaxially we expect no deformation to be measured in the ex direction. However, due to the expanded lattice parameter of the SiGe layers relative to the Si reference, tensile deformation is expected in the ez direction. Figure 1 shows deformation maps that have been acquired by (b) GPA of HAADF images (c) dark holography and (d) precession diffraction. These are compared to finite element simulations that are shown in Figure 1(e). These results reveal that all of the different techniques provide accurate measurements of the deformation. Figure 2(a) shows a HAADF STEM image of a SiGe test device structure with a gate length of 35 nm. Finite element simulations showing the expected deformation in the struture is shown in Figures 2(b) and (c). Deformation maps are shown in Figure 2(d) and (e) for precession diffraction, (f) and (g) for dark holography and (h) and (i) for GPA of HAADF STEM images [4]. Again, accurate measurements of the deformation are made, but the precision and spatial resolution depends on the experimental technique that has been used. As well as presenting state of the art results from a range of strained materials we will highlight improvements that are required for all of the different techniques in order to optimise their performance and provide the best possible measurements of deformation.

Acknowledgements : These experiments have been performed on the platform nanocharacterisation (PFNC) at Minatec. The work has been funded by the ERC Starting Grant 306365 « Holoview ».

References
[1] M. Hytch et al. Ultramicroscopy 74, 131–146 (1998)
[2] M. Hytch et al. Nature 453, 1086-1089 (2008)
[3] D. Cooper et al. Micron 80, 145-165 (2016)
[4] D. Cooper et al. Nano letters 15, 5289-5294 (2015)

Orientation and phase are routinely determined with the automated ACOM tool developed for Transmission Electron Microscopes [1]. With this attachment, the beam is scanned over the area of interest and all the diffraction patterns are collected and kept in memory for further off-line analysis. The present work concerns a novel approach that makes use of the memorized data to compute a structural image of the sample through a straightforward post-processing algorithm that consists in weighting the similarities between the neighbor diffraction patterns [2].

The successive diffraction patterns acquired within a given crystal are anticipated to be nearly identical, while an abrupt change is expected when the beam is crossing a grain or a phase boundary. Plotting the value of a correlation coefficient that compares the intensities of every pixels of the neighbor diffraction patterns produces a contrasted picture in which all structural features that modify the local diffracting conditions are highlighted.

Fig. 1 gives a typical example where grain boundaries for a polycrystalline sample are retrieved. Of particular interest is the fact that the grain boundary contrasts are directly related to the boundary inclination. Indeed, the sharp changes in diffraction patterns expected for boundaries parallel to the electron beam are associated to a strong contrast. A weak and extended contrast indicates qualitatively a gradual modification of the diffracting signal as expected for inclined boundaries. Quantitative evaluation needs different processing [3].

The correlation coefficient is sensitive to any structural component that modifies the diffracting conditions. This is valid for dislocations, too. Two of them appear in the upper grain in figure 1.

Moreover, the correlation coefficient is less sensitive to non-visibility conditions. This is because it is constructed on the difference between the intensities of all reflections including the faint ones. In particular, if the sample is in a so-called two beam condition, the main reflection g remains unchanged when the beam is crossing a dislocation line whose Burgers vector is normal to g. This reflection will be dominant in the bright field image and the dislocation will not be visible. Being constant, g will not contribute to the correlation coefficient. By contrast faint reflections that always exist in the diffraction pattern - even in two beam conditions - will be affected by the distortion around the defect line. Figure 2 compares the virtual bright-field image and the correlation coefficient map for a deformed ferritic steel sample. The thin foil is slightly bended (less than 2°, mainly from left to right) so that the contrast conditions are not homogeneous in the micrograph and part of the structural information is missing. The correlation coefficient is less sensitive to the exact illumination conditions and consequently additional dislocations appear in the map.

[1] E.F. Rauch, M. Véron, Mater. Charact. 98 (2014) 1–9.

[2] Á.K. Kiss, E.F. Rauch, J.L. Lábár, Ultramicroscopy 163 (2016) 31–37.

[3] Á K. Kiss E F. Rauch B Pécz J Szívós and J L. Lábár, Microsc Microanal 21(2015) 422–435

An electron vortex beam is propagating electron which carries an orbital angular momentum (OAM) [1]. Because an electron has an electric charge and a mass, electron vortex beam is considered to have a magnetic moment and a moment of force, with which imaging of magnetic materials and manipulation of nano-sized objects are expected [2,3].

Electron vortex beams are generated using amplitude holograms such as binary masks of forked gratings [2] and spiral zone plates [4], and using phase holograms of forked gratings as their diffracted waves when the holograms are illuminated by a plane wave [2]. Recently, spiral phase plates with a smooth and continuous variation of the thickness of Si₃N₄ membranes were successfully prepared by nano-fabrication techniques, which were not realized in the first report by Uchida and Tonomura [1].

In the present study, the forked grating masks are used as a selected area aperture for electron diffractive imaging, which imposes a real space constraint. Diffractive imaging is lens less imaging for reconstructing the wave field from a diffraction pattern. Diffractive imaging requires a constraint in real space, or “support region”, which corresponds to a beam illuminated area of the specimen. The support region has so far been restricted by a narrow beam itself or a single circular hole inserted at the first image plane of microscopes. In the present study, the support region is restricted by a forked grating. Forked gratings provide not only a transmitted peak but also discrete Bragg peaks which contain rich information of the object located in the support region, and thus one can expect more reliable phase retrieval than the case using a hole mask.

The patterns of the forked gratings with a 5 μm diameter were designed by computer hologram whose Burgers vector of b = 1. The forked gratings were fabricated from 1μm thick PtPd films deposited on 50nm thick Si₃N₄ membranes by using a focused ion beam (FIB) instrument. The forked gratings were introduced into a position of selected-are aperture of a transmission electron microscope. Diffraction patterns were taken by a Gatan imaging fileter with a 16 bit 2k x 2k CCD camera.

Figures 1(a) and 1(b) show a TEM image of the forked grating and its diffraction pattern, respectively. Figures 1(c), 1(d), 1(e) and 1(f) show retrieved amplitudes of image and diffraction pattern, and retrieved phases of image and diffraction patterns, respectively. The retrieved diffraction amplitude (Fig. 1(d)) shows a good agreement with the experimental amplitude (Fig.1(b)). A flat phase in the retrieved phase of the image (Fig.1(e)), which is consistent to the plane wave incidence, is well reproduced. A deformation of the wavefront of incident electron beam by a quadruple magnetic field produced by a stigmator of the intermediate lens of the microscope is also successfully visualized by the present method.

A wavefront deformed by a specimen is also reconstructed. We used Au nano-plates as test specimens. Figures 2(a) and 2(b) show an experimental TEM image and diffraction pattern of a Au nano-plate. Figures 2(c), 2(d), 2(e) and 2(f) show retrieved amplitudes of the image and diffraction pattern, and retrieved phase of the image and diffraction pattern, respectively. The retrieved diffraction amplitude (Fig. 2(d)) shows a good agreement with the experimental amplitude (Fig. 2(b)). The thickness of the Au nano-plate is obtained from the phase map (Fig. 2(e)), which is consistent to the thickness determined by electron holography. We discuss how the phase retrieval using a forked grating mask is effective by comparing with the cases using a regular grating and a single circular hole.

A method for ab-initio structure factor retrieval from large-angle rocking-beam electron diffraction (LARBED)[1] data of thin crystals is described and tested.
This method determines crystal structure factors and specimen thickness from the intensities of the diffraction spots alone, solving a nonlinear least-squares problem. No additional information, such as atomicity or information about chemical composition, have been made use of.

In addition to a demonstration of the method on 120 keV experimental data of SrTiO₃, where 456 structure factors have been retrieved, we analyze the dependence of the success of the reconstruction on specimen thickness and tilt range by applying this algorithm to simulated data. Our numerical experiments show that the dynamical inversion by gradient optimization works best if the beam tilt range is large and the specimen not too thick. At specimen thicknesses which allow for moderate multiple scattering, the large tilt amplitude effectively removes local minima in this global optimization problem, making ab-initio structure factor retrieval possible.

In addition, a dynamic parallelism framework  based on the Compute Unified Device Architecture (CUDA) is introduced, reducing the runtime of the optimization routine by two orders of magnitude when compared to running on a CPU.

The authors acknowledge funding by the Carl Zeiss Foundation as well as the German Research Foundation (Grant no. KO 2911/7-1 and SFB 951).

[1] Koch, C.T., 2011. Aberration-compensated large-angle rocking-beam electron diffraction. Ultramicroscopy, 111, 828–840. doi:10.1016/j.ultramic.2010.12.014

Scanning TEM (STEM) in combination with a small convergence angle (nano-beam electron diffraction, NBED) can be used, e.g., to determine the local strain state of a sample by measuring the distance between two non-overlapping Bragg discs in the diffraction pattern [1]. Automated disc-detection algorithms [2] reveal a high precision which allows for measuring small shifts of diffracted discs relative to the undiffracted disc to determine local strain with a spatial resolution below 1 nm. Simulation of NBED patterns is an important tool to optimize the precision of an experimental measurement as well as to predict the resolution of measured strain profiles at interfaces [3]. In this context, it is important to optimize such simulations to describe experimental measurements as accurately as possible. The present study shows by a comparison of experiment and simulation that for both types of simulations, the absorptive-potential and the frozen-lattice method, deviations to experiment occur if additional effects, such as electron noise or the influence of the modulation-transfer function (MTF) of the CCD camera, are not taken into account.

Fig 1(a) shows the undiffracted 000 disc of an experimental tilt series in pure silicon which was compared with simulations (Fig 1(b) shows the raw absorptive-potential simulations). The selected experimental disc in Fig 2(a) shows smooth edges in contrast to the raw simulation (b) with much sharper edges. Fig 2(c) shows the simulated disc after adding background and applying MTF and noise. It is shown that results for 000-disc detection of the modified simulation agree sufficiently well with experiment for all tilts giving a reliable tool to investigate the effect of sample conditions on NBED-disc detection.

With these optimized simulations, we separately investigated three effects on the 000-disc detection procedure: the effect of (1) scanning into a strained layer (without composition change), (2) scanning into a layer of different material (but without changes in the crystal structure), and (3) the effect of varying specimen thickness.

The latter effect occurs in experiment, e.g., in case of selective layer etching. A GaAs supercell was set up with [001] electron beam direction with two plateaus on the top and on the bottom surface as shown in the schematic illustration in Fig. 3(a). Different NBED disc-detection algorithms [2] were used to detect the deviation of the 000-disc position from the initial center for a scan along the [100] direction across the plateaus. Fig. 3(b) shows the deviations in μrad where each line stands for a certain detection algorithm (Selective Edge Detection [2] and Radial Gradient Maximization [2], both with and without radius fitting) - in all cases the slopes on the surface lead to a measured deviation of the disc position of up to 30-40 μrad. This reveals a consequence for experimental measurements: On the one hand, if non-homogeneous surface topology is an unwanted factor, it can lead to artifacts in disc-position determination. On the other hand, it also might offer the possibility for precise measurements of topology or sample orientation.

[1] F Uesugi et al., Ultramicroscopy 111(2011), p. 995-998

[2] K Müller et al. Microsc. Microanal. 18 (2012), p. 995-1009

[3] C Mahr et al., Ultramicroscopy 158 (2015), p. 38-48

[4] This work was supported by the German Research Foundation (DFG) under contract number RO 2057/11-1 and MU 3660/1-1.

Localisation microscopy is a powerful tool for imaging structures at a lengthscale of tens of nm, but its utility for live cell imaging is limited by the time it takes to acquire the data needed for a super-resolution image. The acquisition time can be cut by more than two orders of magnitude by using advanced algorithms which can analyse dense data, trading off acquisition and processing time.

Modelling the entire localisation microscopy dataset using a Hidden Markov Model allows localisation information to be extracted from extremely dense datasets. This Bayesian analysis of blinking and bleaching (3B) is able to image dynamic processes in live cells at a timescale of a few seconds, though it is very computationally intensive, requiring at least several hours of analysis. We demonstrate the performance of 3B on various live cell systems, including cardiac myocytes and podosomes, showing a resolution of tens of nm with acquisition times down to a second.

While analysing higher density images can improve the speed at which the data required for a super-resolution reconstruction is acquired, there are still limits to the speed which can be achieved. Unlike in conventional fluorescence microscopy, these limits are not just set by the properties of the microscope, but are determined by the structure of the sample itself. This is because the local sample structure affects how quickly the information necessary to create an image of a certain resolution can be transmitted through the optical system. The theoretical limits will be discussed, and the effect on live cell experiments demonstrated.

Time-lapse sub-diffraction imaging of cells labeled with genetically-encoded labels continues remains a challenging task. We have focused our efforts on developing techniques that work well under challenging conditions, combined with the semi-rational engineering of "smart" fluorescent proteins. In this presentation I will discuss our work in developing live cell sub-diffraction imaging by combining superresolution optical fluctuation imaging (SOFI) with new photochromic probes based on the enhanced green fluorescent protein, EGFP. In particular, I will discuss our recent progress in enhancing the temporal resolution of SOFI imaging while also providing reliability estimates for the obtained images [1], followed by our work on engineering and understanding improved fluorescent proteins with properties specifically tailored to sub-diffraction imaging. In particular I will discuss our recent rsGreens [2], which display robust optical performance while also expressing well in E. coli and HeLa cells. Since our rsGreens are based on EGFP, they should be widely useful in existing experiments. In the last part I will highlight ongoing work on how these developments can be applied to live cell interaction sensing.

[1] Vandenberg et al, Biomed Opt Expr 2016
[2] Duwé et al, ACS Nano 2015

Total Internal Reflection Fluorescence (TIRF) microscopy is becoming a widespread technique to study cellular processes occurring near the contact region with the glass substrate [1]. The characteristics of TIRF microscopy are directly related to the singular properties of evanescent waves, such as the exponential decay of the electric field along the z direction. This providing a selective excitation of fluorescent molecules close to the interface. Determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process [2]. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters. The first one is obviously the distance z, which separates the dye molecules from the surface, as the emitted fluorescence signal is mainly governed by the exponential decay of the evanescent wave. But TIRF images contrast is also affected by unknown parameters such as the local concentration of dyes, their dipole moment orientation and consequences on their related angular emission pattern (which influences the detection efficiency η_d), and also their absorption cross section (σ_abs) and their fluorescence lifetime (τ). Moreover, these last three parameters (η_d, σ_abs, τ) can be strongly altered as a function of z near the surface [3].

To get around this problem, we propose two strategies allowing us to map the membrane-substrate separation distance with a nanometric resolution (typically 10 nm). The first one is dedicated to study the adhesion of Giant Unilamellar Vesicles (GUVs), which are often used as a biomimetic system to reproduce cells spreading. This approach, called normalized TIRF, is based on dual observation, which combine epi-fluorescence microscopy and TIRF microscopy: TIRF images are normalized by epi-fluorescence ones [4, 5]. Figure 1 shows an example of a negatively charged GUV completely spread on a thin layer of a positively charged polyelectrolyte (PDDA) recovering the coverslip. The second technique is devoted to explore the adhesion of living cells. This last is called variable-angle TIRF (vaTIRF) microscopy. vaTIRF is an old technique introduced in the middle of 80s and quickly forgot due to the high complexity of the first experimental setup used. We propose an improved straightforward version of vaTIRF microscopy adapted to modern TIRF setup. This technique involves the recording of a stack of several TIRF images, by gradually increasing the incident angle θ on the sample. We developed a comprehensive theory to extract the membrane/substrate separation distance from fluorescently labeled cell membranes [6], as illustrated in figure 2 for a MDA-MB-231 cell in adhesion on fibronectin. Finally, we demonstrate that these two techniques (nTIRF and vaTIRF) are useful to quantify the adhesion of vesicles and cells from weak to strong membrane-surface interactions, achieved on various functionalized substrates with polymers or proteins, such as collagen and fibronectin.

[1] D. Axelrod, Total Internal Reflection Fluorescence Microscopy, Meth. in Cell Biol. 89, 169-221 (2008).

[2] E. Sackmann and A-S. Smith, Physics of cell adhesion: some lessons from cell-mimetic systems, Soft Matter 10, 1644-1659 (2014).

[3] J. Mertz, Radiative absorption, fluorescence, and scattering of a classical dipole near a lossless interface: a unified description, J. Opt. Soc. Am. B 17, 1906-1913 (2000).

[4] M. Cardoso Dos Santos, R. Déturche, C. Vézy and R. Jaffiol, Axial nanoscale localization by normalized total internal reflection fluorescence microscopy, Opt. Lett. 39, 869-872 (2014).

[5] M. Cardoso Dos Santos, C. Vézy and R. Jaffiol, Nanoscale characterization of vesicle adhesion by normalized total internal reflection fluorescence microscopy, accepted in BBA Biomembranes (2016).

[6] M. Cardoso Dos Santos, R. Déturche, C. Vézy and R. Jaffiol, Nanoscale topography of cells in adhesion revealed by variable-angle total internal reflection fluorescence microscopy, submitted in Biophys. J. (2016).

Under blue light irradiation green FPs can act as light-induced electron donors in photochemical reactions with various electron acceptors. These reactions are accompanied by green-to-red photoconversion (oxidative redding) which is among the processes significantly contributing to the photostability of many GFP family proteins. We suppose that inhibition of redding will have similar consequences in super-resolution microscopy (PALM) as it does in epifluorescent microscopy, namely, enhanced photostability and average fluorophore brightness. Understanding of the primary mechanisms of electron transfer (ET) occurring in excited state allows to control the photostability. In our recent work [1] we consider two mechanistic hypotheses:

1) ET proceeds via direct ET to a surface-docked oxidant (tunnel mechanism)

2) Photoinduced ET proceeds via a hopping mechanism (in which the first step is ET from the chromophore to some intermediate acceptor, and the second step is ET from this acceptor to an outside oxidant molecule)

Hopping mechanism appears to be the most likely process according to the quantum-mechanical calculations. One of the approaches to reduce ET via hopping mechanism is an elimination of the potential internal electron acceptors, located in the side chain of fluorescent protein. We determined such critical residues in the polypeptide chain of PAGFP using quantum-chemical simulations, and changed them to amino acid that is not able to be an efficient electron acceptor, namely, leucine. According to both epifluorescent and TIRF-experiments’ data, this substitution leads to the highly improved photostability in comparison with the original PAGFP. It has also been shown that in TIRF-experiments PAGFP Y145L had significantly lower blinking rate than PAGFP (see Fig.1). This property could be potentially used in SPT (single particle tracking) technique, where transitions to the dark states impose serious constraints and appear to be a drawback.

[1]. Bogdanov et al., JACS, 2016, in press

Abstract

Chromatin presents a unique challenge in structural biology.  Its fundamental units, DNA and the nucleosome, are well-characterised at the Ångstrom level.¹  Yet, the higher-order organisation of the chromatin ensemble, particularly during interphase, remains largely to be determined.  Single molecule localisation microscopy (SMLM) appears uniquely positioned to answer many remaining questions about chromatin structure – it is possible to determine structures to accuracies of better than 20 nm,² and analysis of spatial point data has been used by some groups to determine the scaling behaviour and fractal dimension of chromatin.³  This is of particular interest when considering nuclear molecular transport and gene activation: it has been suggested that cells use the very structure of chromatin to regulate transcription and expression.⁴

Whilst imaging has been able to shed light on the general organisation of chromatin and other fibrous structures in the cell, there is a lack of methods to fully characterise their folding and looping behaviour.  To date, most SMLM analysis has focussed on clustering-based algorithms such as Ripley’s K function or more recently Bayesian clustering.⁵  It is clear, however, that different approaches are needed to tackle the analysis of elongated or fibrous structures within cells, and in three dimensions.

Here, we demonstrate the power of 3D pair correlation analysis combined with careful labelling of regions of interest in chromatin, showing that the fractal dimension of chromatin can be determined across the entire nucleus for structures 30 nm in size upwards.  This provides a platform for analysis complementary to imaging, describing differential folding behaviour across the nucleus, yielding results comparable to Hi-C and other recent methods.  In addition to applying spatial statistics to probe chromatin structure on the nanoscale, we incorporate photon statistics into the analysis.  This allows us to draw conclusions about chromatin structure with greater confidence by adding a weighting to reduce the influence of data points localised with lower precision than others.

Understanding how chromatin fibres fold at all stages in the cell cycle is crucial to the future development of drugs and novel treatments.  More and more, drugs target specific loci yet in many cases lack the ability to be directed to the target of interest with great accuracy.⁶  We hope that by understanding the organisation of different regions of the genome, drugs can be designed to be delivered to their targets in ‘smarter’ ways.

References

1.  Flors, C. & Earnshaw, W. C. Super-resolution fluorescence microscopy as a tool to study the nanoscale organization of chromosomes. Curr. Opin. Chem. Biol. 15, 838–44 (2011).

2.  Deschout, H. et al. Precisely and accurately localizing single emitters in fluorescence microscopy. Nat. Methods 11, 253–66 (2014).

3.  Récamier, V. et al. Single cell correlation fractal dimension of chromatin. Nucleus 5, 75–84 (2014).

4.  Bancaud, A. et al. Molecular crowding affects diffusion and binding of nuclear proteins in heterochromatin and reveals the fractal organization of chromatin. EMBO J. 28, 3785–3798 (2009).

5.  Rubin-Delanchy, P. et al. Bayesian cluster identification in single-molecule localization microscopy data. Nat. Methods (2015). doi:10.1038/nmeth.3612

6.  Rajendran, L., Knölker, H.-J. & Simons, K. Subcellular targeting strategies for drug design and delivery. Nat. Rev. Drug Discov. 9, 29–42 (2010).