Logistics of Bone Mineralization in the Chick Embryo Studied by 3D Cryo FIB‐SEM Imaging

Raguin, Émeline; Weinkamer, Richard; Schmitt, Clemens N. Z.; Curcuraci, Luca; Fratzl, Peter

doi:10.1002/advs.202301231

Cited by 13 publications

(5 citation statements)

References 83 publications

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“…In serial block face SEM (SBF-SEM) or Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), the top surface is imaged with repeated material removal from the top surface, and the resolution is limited by the slice thickness (∼30-100 nm). To ensure that biological samples are as close as possible to their native state, cryo-FIB-SEM has been developed [3][4][5][6][7] with a resolution as high as a few nanometers. However, depending on the imaging parameters, it takes 15-20 h to image a 10 mm thick sample with a slice Appl.…”

Section: Introductionmentioning

confidence: 99%

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Monte Carlo Simulation of Electron Interactions in an MeV-STEM for Thick Frozen Biological Sample Imaging

Wang,

Yang

2024

Applied Sciences

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A variety of volume electron microscopy techniques have been developed to visualize thick biological samples. However, the resolution is limited by the sliced section thickness (>30–60 nm). To preserve biological samples in a hydrated state, cryo-focused ion beam scanning electron microscopy has been developed, providing nm resolutions. However, this method is time-consuming, requiring 15–20 h to image a 10 μm thick sample with an 8 nm slice thickness. There is a pressing need for a method that allows the rapid and efficient study of thick biological samples while maintaining nanoscale resolution. The remarkable ability of mega-electron-volt (MeV) electrons to penetrate thick biological samples, even exceeding 10 μm in thickness, while maintaining nanoscale resolution, positions MeV-STEM as a suitable microscopy tool for such applications. Our research delves into understanding the interactions between MeV electrons and frozen biological specimens through Monte Carlo simulations. Single elastic scattering, plural elastic scattering, single inelastic scattering, and plural inelastic scattering events have been simulated. The electron trajectories, the beam profile, and the intensity change of electrons in each category have been investigated. Additionally, the effects of the detector collection angle and the focal position of the electron beam were investigated. As electrons penetrated deeper into the specimen, single and plural elastic scattered electrons diminished, and plural inelastic scattered electrons became dominant, and the beam profile became wider. Even after 10 μm of the specimen, 42% of the MeV electrons were collected within 10 mrad. This confirms that MeV-STEM can be employed to study thick biological samples.

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Section: Introductionmentioning

confidence: 99%

“…Sci. 2024, 14, 1888 2 of 11 thickness of 8 nm and an area of 3 × 2 mm 2 (8 nm pixel size) [7]. In addition, there are charging artifacts, curtaining artifacts, and linear artifacts [3,5].…”

Section: Introductionmentioning

confidence: 99%

Monte Carlo Simulation of Electron Interactions in an MeV-STEM for Thick Frozen Biological Sample Imaging

Wang,

Yang

2024

Applied Sciences

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“…In both SBF-SEM and FIB-SEM, the resolution is limited by the slice thickness (around 30-100 nm). Moreover, to ensure that biological samples are as close as possible to their native state, cryo-FIB-SEM has been developed [14][15][16][17][18]. Resolution in depth can be as high as a few nanometers.…”

Section: Introductionmentioning

confidence: 99%

“…However, depending on the imaging parameters, the imaging process often takes 15-25 s, and the milling process takes 7-36 s [15]. A total of 15-20 h is required to image a 10 µm thick sample with a slice thickness of 8 nm and an area of 3 × 2 µm 2 (8 nm pixel size) [18]. In addition, there are charging artifacts due to positively charged lipid deposits, curtaining artifacts due to density and content changes, and linear artifacts due to the milling process [14,16].…”

Section: Introductionmentioning

confidence: 99%

Towards Construction of a Novel Nanometer-Resolution MeV-STEM for Imaging Thick Frozen Biological Samples

Yang,

Wang,

Maxson

et al. 2024

Photonics

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Driven by life-science applications, a mega-electron-volt Scanning Transmission Electron Microscope (MeV-STEM) has been proposed here to image thick frozen biological samples as a conventional Transmission Electron Microscope (TEM) may not be suitable to image samples thicker than 300–500 nm and various volume electron microscopy (EM) techniques either suffering from low resolution, or low speed. The high penetration of inelastic scattering signals of MeV electrons could make the MeV-STEM an appropriate microscope for biological samples as thick as 10 μm or more with a nanoscale resolution, considering the effect of electron energy, beam broadening, and low-dose limit on resolution. The best resolution is inversely related to the sample thickness and changes from 6 nm to 24 nm when the sample thickness increases from 1 μm to 10 μm. To achieve such a resolution in STEM, the imaging electrons must be focused on the specimen with a nm size and an mrad semi-convergence angle. This requires an electron beam emittance of a few picometers, which is ~1000 times smaller than the presently achieved nm emittance, in conjunction with less than 10−4 energy spread and 1 nA current. We numerically simulated two different approaches that are potentially applicable to build a compact MeV-STEM instrument: (1) DC (Direct Current) gun, aperture, superconducting radio-frequency (SRF) cavities, and STEM column; (2) SRF gun, aperture, SRF cavities, and STEM column. Beam dynamic simulations show promising results, which meet the needs of an MeV-STEM, a few-picometer emittance, less than 10−4 energy spread, and 0.1–1 nA current from both options. Also, we designed a compact STEM column based on permanent quadrupole quintuplet, not only to demagnify the beam size from 1 μm at the source point to 2 nm at the specimen but also to provide the freedom of changing the magnifications at the specimen and a scanning system to raster the electron beam across the sample with a step size of 2 nm and the repetition rate of 1 MHz. This makes it possible to build a compact MeV-STEM and use it to study thick, large-volume samples in cell biology.

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“…The use of three-dimensional visualization techniques is crucial in this regard [9][10][11][12][13]. Among these, the X-Ray microtomography (microCT) technique is gaining popularity for researching both healthy embryogenesis and developmental abnormalities [14][15][16].…”

Section: Introductionmentioning

confidence: 99%

High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis

Rzhepakovsky,

Piskov,

Avanesyan

et al. 2023

Applied Sciences

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X-ray contrast techniques were tested on the chick embryos in early periods of embryogenesis. For contrast stain, reagents with radiopacity in various concentrations were used: silver proteinate, eosin, Lugol’s solution (I2KI), phosphomolybdic acid and phosphotungstic acid under heating at 25 °C and 40 °C and exposure for 24 and 48 h. The use of silver proteinate, eosin and I2KI in various concentrations in the contrast of the chick embryo in the early period of embryogenesis did not make it possible to obtain microtomographic results that provide reliable microstructural analysis. The most optimal and effective method of embryo staining at the HH22–HH34 embryonic stages reliably determined the staining of 1% phosphotungstic acid at 40 °C heating and exposure for 24 h. Taking into account the size of the chick embryos and their structures at the HH22–HH34 embryonic stages, the features of the development, location of organs, and the minimum permissible parameters of microtomography for obtaining high-quality and reliable results were determined by the isometric spatial resolution of 8.87 μm, X-ray voltage 50 kV, X-ray current 500 μA, and the use of filters started from Al 0.5 mm. Microtomographic results were obtained, characterized by the appearance of the chick embryo at the HH22–HH34 embryonic stages, and they visualized the locations and structures of the chick embryo organs and provided calculation of their volume and X-ray density. The results of the work open up significant prospects for using the chick embryo at the early embryonic period of embryogenesis as an alternative model for screening teratogenicity.

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Logistics of Bone Mineralization in the Chick Embryo Studied by 3D Cryo FIB‐SEM Imaging

Cited by 13 publications

References 83 publications

Monte Carlo Simulation of Electron Interactions in an MeV-STEM for Thick Frozen Biological Sample Imaging

Monte Carlo Simulation of Electron Interactions in an MeV-STEM for Thick Frozen Biological Sample Imaging

Towards Construction of a Novel Nanometer-Resolution MeV-STEM for Imaging Thick Frozen Biological Samples

High-Performance Microcomputing Tomography of Chick Embryo in the Early Stages of Embryogenesis

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