Nejvíce citovaný článek - PubMed ID 30953993
Quantitative STEM imaging of electron beam induced mass loss of epoxy resin sections
Electron microscopy stands as a cornerstone in unraveling the intricate dynamics of viral infections, with its high-resolution capabilities offering invaluable insights into the interactions between viruses and the infected cells. Here, we present a comprehensive methodology designed to explore the three-dimensional interactions specifically between tick-borne encephalitis virus (TBEV) and host cells. This approach allows to study all stages of viral lifecycle, including replication, budding, maturation, and host cell defense mechanisms. The methodology encompasses a range of techniques, commencing with sample preparation using high-pressure freezing, followed by freeze substitution, epoxy embedding, and ultrathin sectioning. Subsequently, we employ electron tomography in conjunction with image processing and analysis techniques to unravel the intricate nuances of TBEV-host cell interactions.
- Klíčová slova
- Electron tomography, Tick-borne encephalitis virus, Transmission electron microscopy,
- MeSH
- buněčné linie MeSH
- interakce hostitele a patogenu * MeSH
- klíšťová encefalitida * virologie MeSH
- lidé MeSH
- mrazová substituce MeSH
- replikace viru MeSH
- tomografie elektronová * metody MeSH
- viry klíšťové encefalitidy * fyziologie ultrastruktura MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
We present a powerful method for the simultaneous detection of Au nanoparticles located on both sides of ultrathin sections. The method employs a high-resolution scanning electron microscope (HRSEM) operating in scanning transmission electron microscopy (STEM) mode in combination with the detection of backscattered electrons (BSE). The images are recorded simultaneously during STEM and BSE imaging at the precisely selected accelerating voltage. Under proper imaging conditions, the positions of Au nanoparticles on the top or bottom sides can be clearly differentiated, hence showing this method to be suitable for multiple immunolabelling using Au nanoparticles (NPs) as markers. The difference between the upper and lower Au NPs is so large that it is possible to apply common software tools (such as ImageJ) to enable their automatic differentiation. The effects of the section thickness, detector settings and accelerating voltage on the resulting image are shown. Our experimental results correspond to the results modelled in silico by Monte Carlo (MC) simulations.
A modern scanning electron microscope equipped with a pixelated detector of transmitted electrons can record a four-dimensional (4D) dataset containing a two-dimensional (2D) array of 2D nanobeam electron diffraction patterns; this is known as a four-dimensional scanning transmission electron microscopy (4D-STEM). In this work, we introduce a new version of our method called 4D-STEM/PNBD (powder nanobeam diffraction), which yields high-resolution powder diffractograms, whose quality is fully comparable to standard TEM/SAED (selected-area electron diffraction) patterns. Our method converts a complex 4D-STEM dataset measured on a nanocrystalline material to a single 2D powder electron diffractogram, which is easy to process with standard software. The original version of 4D-STEM/PNBD method, which suffered from low resolution, was improved in three important areas: (i) an optimized data collection protocol enables the experimental determination of the point spread function (PSF) of the primary electron beam, (ii) an improved data processing combines an entropy-based filtering of the whole dataset with a PSF-deconvolution of the individual 2D diffractograms and (iii) completely re-written software automates all calculations and requires just a minimal user input. The new method was applied to Au, TbF3 and TiO2 nanocrystals and the resolution of the 4D-STEM/PNBD diffractograms was even slightly better than that of TEM/SAED.
- Klíčová slova
- 4D-STEM, nanoparticle analysis, powder nanobeam electron diffraction,
- Publikační typ
- časopisecké články MeSH
We introduce a novel scanning electron microscopy (SEM) method which yields powder electron diffraction patterns. The only requirement is that the SEM microscope must be equipped with a pixelated detector of transmitted electrons. The pixelated detectors for SEM have been commercialized recently. They can be used routinely to collect a high number of electron diffraction patterns from individual nanocrystals and/or locations (this is called four-dimensional scanning transmission electron microscopy (4D-STEM), as we obtain two-dimensional (2D) information for each pixel of the 2D scanning array). Nevertheless, the individual 4D-STEM diffractograms are difficult to analyze due to the random orientation of nanocrystalline material. In our method, all individual diffractograms (showing randomly oriented diffraction spots from a few nanocrystals) are combined into one composite diffraction pattern (showing diffraction rings typical of polycrystalline/powder materials). The final powder diffraction pattern can be analyzed by means of standard programs for TEM/SAED (Selected-Area Electron Diffraction). We called our new method 4D-STEM/PNBD (Powder NanoBeam Diffraction) and applied it to three different systems: Au nano-islands (well diffracting nanocrystals with size ~20 nm), small TbF3 nanocrystals (size < 5 nm), and large NaYF4 nanocrystals (size > 100 nm). In all three cases, the STEM/PNBD results were comparable to those obtained from TEM/SAED. Therefore, the 4D-STEM/PNBD method enables fast and simple analysis of nanocrystalline materials, which opens quite new possibilities in the field of SEM.
- Klíčová slova
- 4D-STEM/PNBD, nanoparticle analysis, powder nanobeam electron diffraction,
- Publikační typ
- časopisecké články MeSH
The thickness of electron transparent samples can be measured in an electron microscope using several imaging techniques like electron energy loss spectroscopy (EELS) or quantitative scanning transmission electron microscopy (STEM). We extrapolate this method for using a back-scattered electron (BSE) detector in the scanning electron microscope (SEM). This brings the opportunity to measure the thickness not just of the electron transparent samples on TEM mesh grids, but, in addition, also the thickness of thin films on substrates. Nevertheless, the geometry of the microscope and the BSE detector poses a problem with precise calibration of the detector. We present a simple method which can be used for such a type of detector calibration that allows absolute (standardless) measurement of thickness, together with a proof of the method on test samples.
- Klíčová slova
- Monte Carlo simulation, SEM, back-scattered electrons, electron mirror, quantitative imaging, sample bias, standardless calibration, thin coating layers,
- Publikační typ
- časopisecké články MeSH
