Development and application of STEM for the biological sciences

Smart acquisition EELS

A DEC VAXstation 3200 color graphics computer workstation has been interfaced to a Vacuum Generators HB501A Scanning Transmission Electron Microscope (STEM) for on-line image and spectrum acquisition. Electron Energy Loss Spectra (EELS) or Auger spectra may be recorded (serially) using pulse or analog modes (using the voltage to frequency converters supplied by VG). High resolution bright field (BF) / annular dark field (ADF) image pairs may be recorded simultaneously as well as simultaneous pairs of Auger / secondary images. A resolution of 2Å (or better) has been achieved with an experimental pole piece (Cs = 1.2mm. and 0.7mm.). The image quality obtained with this system is greatly improved over the Polaroid system supplied with the microscope.A schematic of the overall system is shown in FIG 1. To avoid possible ground loop noise the entire computer system has been optically isolated from the STEM. There are four 16 bit digital to analog converters (DAC) for controlling the x,y position, the EELS energy and the Auger energy. The output of four detectors is connected back to the computer for pulse counting using a Qbus board manufactured by Codar Technologies. This board has three AMD 9513 counter IC's (with five 16 bit counters each) to count the electron or VFC pulses. The EELS spectra are acquired using two 32 bit counters (16 bit counters concatenated) and may function in a constant dwell time or constant count (constant signal to noise ratio) mode. The Auger and BF/ADF image counters work in a constant dwell time 16 bit mode. The average CPU overhead has been measured at 50 microsec per point in polled mode (images) and 500 microsec in interrupt driven mode (spectra).

Imaging with high-angle scattered electrons and secondary electrons in the STEM

Contrast and resolution of scanning transmission electron microscope imaging modes

Scanning Transmission Electron Tomography of Blood Platelets in Thick Sections

At present transmission electron energy loss spectrum (EELS) analysis is only carried out in dedicated research instruments such as transmission electron microscopes (TEMs) or scanning transmission electron microscopes. This article presents a new design of second-order geometric aberration corrected EELS spectrometer attachment using split plates, which enables conventional scanning electron microscopes (SEMs) to provide TEM-like EELS spectra. Correction to a third-order dominant geometric aberration pattern has been achieved, which indicates that most of the second-order geometric aberration component is eliminated. This second-order aberration corrected spectrometer attachment design can enable SEMs to provide transmission EELS spectrums.

Three-Dimensional Visualization of Whole Synapses by Stem Tomography

Strategy for optimizing experimental settings for studying low atomic number colloidal assemblies using liquid phase scanning transmission electron microscopy

Theory of the spatial resolution of (scanning) transmission electron microscopy in liquid water or ice layers

Compressed sensing electron tomography of needle-shaped biological specimens – Potential for improved reconstruction fidelity with reduced dose

Scanning transmission electron microscopy (STEM) has been widely utilized with annular detectors such as annular dark field (ADF) and annular bright field (ABF).Recently, segmented and pixelated detectors have allowed for the detection of detailed electron scattering intensity in the diffraction plane.Utilizing this richer information, low-dose STEM imaging capabilities have been discussed [1,2].Optimum-bright field (OBF) STEM, a non-iterative imaging technique developed recently, uses a segmented or pixelated detector for low-dose observation [3].OBF images are reconstructed from multiple images recorded simultaneously with a segmented/pixelated detector (Fig. 1).The reconstruction process ensures that the reconstructed image achieves the maximum signal-to-noise ratio (SNR) under the weak phase object approximation (WPOA).As the reconstruction process is noniterative, OBF images can be displayed synchronously with the probe scan.OBF STEM is starting to be used as a dose-efficient imaging technique for beam-sensitive materials.Recently, we demonstrated low-dose atomic resolution observations of FAU-type zeolite using OBF STEM, successfully visualizing all atomic sites [4].Figure 2 compares the OBF image with integrated differential phase contrast (iDPC), annular bright field (ABF), and bright field (BF) imaging techniques.The OBF image displays the highest SNR, clearly visualizing both oxygen and silicon sites.Additionally, since the noise in the OBF image can be considered as white noise, its interpretation is straightforward.In contrast, noise in the iDPC image is intensified in the low-frequency region, leading to reduced image contrast.Although OBF STEM is a promising technique for low-dose observations, adjusting observation conditions such as aberrations and crystal orientations for beam-sensitive specimens remains challenging.However, OBF reconstruction can account for residual aberrations and crystal misorientation.We developed a method to estimate actual observation conditions, including residual aberrations, crystal misorientation, and specimen thickness, using data recorded by a pixelated detector.Under the conditions of a thick weak phase object approximation (tWPOA) [5,6], we formulated a mathematical expression that defines the relationship between the 4D dataset and observation condition parameters.We found that it is possible to estimate these parameters separately by fitting the dataset to the corresponding mathematical expressions.We tested this method on a low-dose observation of FAU-type zeolite, with the dataset intentionally recorded under suboptimal observation conditions.The OBF reconstructed image assuming ideal observation conditions appeared opaque due to residual aberrations and crystal misorientation.In contrast, the OBF image that took into account the estimated observation condition parameters accurately visualized all atomic sites of FAU-type zeolite.This method significantly enhances the efficiency of low-dose observations.Moreover, if real-time estimation becomes feasible, the dataset could be recorded under nearly ideal conditions by adjusting the electron microscope based on the estimated parameters [7].

The scanning transmission electron microscope (STEM) was a rediscovery or reinvention whose conception was based on techniques and processes in nuclear physics, and it was lamentably ignorant of any precedent. This chapter is concerns the principles of operation of the STEM, paying particular attention to the limits of performance, it is helpful first to understand the principles behind the formation of images and spectra in the STEM. Axial bright-field imaging is performed in the STEM by placing a small detector on the optic axis beyond the sample. The most commonly used imaging mode in STEM is that using an annular dark-field detector. Spectroscopy is an important application in STEM, with the common ones being energy-dispersive x-ray and electron energy loss spectroscopy. The last few years have seen dramatic advances in the technology of spherical aberration correction for TEM, and commercial correctors are available for both STEM and conventional TEM instruments.

Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography

Investigating the atomic scale structure and chemistry of grain boundaries in high-Tc superconductors

Structures of large T antigen at the origin of SV40 DNA replication by atomic force microscopy