Publication

Towards near-atomic resolution imaging of microsecond nanoscale dynamics

Gabriele Bongiovanni
2023
EPFL thesis
Abstract

Time-resolved electron microscopy has made significant progress in recent years, with some groups now working on instruments that offer attosecond temporal resolution. While much of the research in the field revolves around the improvement of temporal resolution, atomic-resolution imaging of nanoscale dynamics has remained elusive.This thesis describes the development of two methods for time-resolved electron microscopy which afford near-atomic spatial resolution. The first consists of a modification of a commercial transmission electron microscope to generate bright and intense microsecond electron pulses, which are then used to image fast and irreversible nanoscale dynamics with atomic resolution (Chapters 2 and 3). The second is a novel approach to time-resolved cryo-electron microscopy which boosts the temporal resolution to the microsecond timescale (Chapters 4 and 5).Chapter 2 describes the irradiation of a Schottky emitter with microsecond laser pulses. The temperature of the filament rises to extreme values for brief periods of time, causing a significant increase in emission current. Even though the temperatures reached by the filament tip during laser irradiation are well beyond the maximum value recommended by the manufacturer, we show that the brief and localized heating provided by the focused laser pulse provides a way to extract large currents without damaging the filament.An electrostatic deflector, placed below the accelerator, chops the laser-boosted electron beam into microsecond pulses, as described in Chapter 3. We show that a 5 µs pulse generated with this method is brighter than the continuous electron beam and can be used to capture an atomic-resolution image of a gold nanoparticle in a single shot. Two possible applications of these pulses are then discussed. Drift-corrected imaging, especially in the presence of large amounts of drift, is significantly improved when bright electron pulses are used instead of the continuous beam. In addition, these pulses can be employed to capture irreversible dynamics occurring on the microsecond timescale with atomic spatial resolution. Chapter 4 provides details of a novel method for microsecond time-resolved cryo-electron microscopy. The high temporal resolution is achieved by irradiating a cryo specimen with a laser beam, causing it to locally melt. The embedded biomolecules can undergo dynamics in liquid until the laser is switched off, at which point the sample revitrifies within a few microseconds trapping particles in their intermediate configurations. The chapter shows that it is possible to obtain a near-atomic resolution reconstruction from revitrified sample areas, and the result looks identical to a map obtained from conventional sample areas. In addition, the projection angles are more uniformly distributed after revitrification.Chapter 5 shows that it is not necessary to modify a transmission electron microscope to perform melting and revitrification experiments on a cryo sample. The chapter introduces a simplified setup, requiring an optical microscope, that allows performing such experiments and verify their outcome on the fly. We present the advantages and disadvantages of this new setup, in the hope it will encourage the adoption of our method by other research groups and boost the development of microsecond time-resolved cryo-electron microscopy.

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Related concepts (32)
Transmission electron microscopy
Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.
Transmission electron cryomicroscopy
Transmission electron cryomicroscopy (CryoTEM), commonly known as cryo-EM, is a form of cryogenic electron microscopy, more specifically a type of transmission electron microscopy (TEM) where the sample is studied at cryogenic temperatures (generally liquid-nitrogen temperatures). Cryo-EM is gaining popularity in structural biology. The utility of transmission electron cryomicroscopy stems from the fact that it allows the observation of specimens that have not been stained or fixed in any way, showing them in their native environment.
Cryogenic electron microscopy
Cryogenic electron microscopy (cryo-EM) is a cryomicroscopy technique applied on samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid ethane or a mixture of liquid ethane and propane. While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution.
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