Local thickness and composition analysis of TEM lamellae in the FIB

Lång, Christian; Hiscock, Matthew; Dawson, Michael J.; Hartfield, Cheryl

doi:10.1016/j.microrel.2014.07.043

Cited by 9 publications

(6 citation statements)

References 3 publications

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“…Quartz) could be used to increase the depth of focus approximately 2.2-fold (Supplementary Table 1). This is an important consideration to exploit super SIL imaging in correlative light and electron microscopy (CLEM), where cell lamella thickness is in the range of 50–300 nm 46 . A suitable technique for nanoscale resolution under cryogenic conditions, like cryo- super SIL STORM, can provide true complementarity between EM and fluorescence microscopy, crucial to realize the promise of CLEM in biology 47 .…”

Section: Resultsmentioning

confidence: 99%

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution

et al. 2019

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Super-resolution fluorescence microscopy plays a crucial role in our understanding of cell structure and function by reporting cellular ultrastructure with 20–30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions. This substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixation. Cryogenic conditions are, however, underutilised because of the lack of compatible high numerical aperture objectives. Here, using a low-cost super-hemispherical solid immersion lens ( super SIL) and a basic set-up we achieve 12 nm resolution under cryogenic conditions, to our knowledge the best yet attained in cells using simple set-ups and/or commercial systems. By also allowing multicolour imaging, and by paving the way to total-internal-reflection fluorescence imaging of mammalian cells under cryogenic conditions, s uper SIL microscopy opens a straightforward route to achieve unmatched resolution on bacterial and mammalian cell samples.

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Section: Resultsmentioning

confidence: 99%

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution

et al. 2019

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“…It is worth noting that the depth of focus of a superSIL microscope depends on the refractive index of the superSIL material, allowing a degree of depth "tuning". This is an important consideration to exploit superSIL imaging in CLEM, where lamella thickness is in the range of 50-300 nm [45]. If required, superSIL materials of lower refractive indexes (such as Quartz) can be used to increase the depth of focus approximately 2.2-fold (Table S1).…”

Section: Resultsmentioning

confidence: 99%

Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

Wang

Bateman

Zanetti-Domingues

et al. 2018

Preprint

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Super-resolution fluorescence microscopy achieves 20-30 nm resolution by using liquid-immersion objectives to optimize light collection and chemical sample fixation to minimize image blurring. It is known that fluorophore brightness increases substantially under cryogenic conditions and that cryofixation is far superior in preserving ultrastructure. However, cryogenic conditions have not been exploited to improve resolution or sample quality because liquid immersion media freezes at the objective, losing its optical properties. Here, simply by replacing the immersion fluid with a low-cost super-hemispherical solid immersion lens (superSIL), we effortlessly achieve <8 nm localisation precision and 12 nm resolution under cryogenic conditions in a low-cost, low-tech system. This is to our knowledge the best resolution yet attained in biological samples. Furthermore, we demonstrate multicolour imaging and show that the inexpensive setup outperforms 10-fold more costly superresolution microscopes. By also removing the barrier to total internal reflection fluorescence imaging of mammalian cells under cryogenic conditions, superSIL microscopy delivers a straightforward route to achieve unmatched nanoscale resolution on both bacterial and mammalian cell samples, which any laboratory can effortlessly and inexpensively implement.

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“…[1][2][3][4][5][6][7] Determination of sample thickness during the milling process might thus allow the milling process to be stopped at precisely the required endpoint and thereby improve both quality and throughput of sample preparation. In SEM and TEM instruments, information along the z dimension of electron transparent samples can be obtained by backscattered electron imaging, [8] energy dispersive X-ray spectroscopy (EDX) analysis, [8,9] TEM tomography, [10] zero-loss TEM imaging, [11] the calibration of secondary electron signal intensity ratio in the SEM image to electron energy loss spectroscopy (EELS) measurements, [12] or by comparison of a calibrated STEM image with a simulation of a known sample geometry, which is called quantitative STEM (qSTEM). [13,14] Most methods require independent calibration before each imaging session to account for specific imaging settings.…”

Section: Introductionmentioning

confidence: 99%

“…In SEM and TEM instruments, information along the z dimension of electron transparent samples can be obtained by backscattered electron imaging, [ 8 ] energy dispersive X‐ray spectroscopy (EDX) analysis, [ 8,9 ] TEM tomography, [ 10 ] zero‐loss TEM imaging, [ 11 ] the calibration of secondary electron signal intensity ratio in the SEM image to electron energy loss spectroscopy (EELS) measurements, [ 12 ] or by comparison of a calibrated STEM image with a simulation of a known sample geometry, which is called quantitative STEM (qSTEM). [ 13,14 ]…”

Section: Introductionmentioning

confidence: 99%

Robust Local Thickness Estimation of Sub‐Micrometer Specimen by 4D‐STEM

et al. 2023

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A quantitative four‐dimensional scanning transmission electron microscopy (4D‐STEM) imaging technique (q4STEM) for local thickness estimation across amorphous specimen such as obtained by focused ion beam (FIB)‐milling of lamellae for (cryo‐)TEM analysis is presented. This study is based on measuring spatially resolved diffraction patterns to obtain the angular distribution of electron scattering, or the ratio of integrated virtual dark and bright field STEM signals, and their quantitative evaluation using Monte Carlo simulations. The method is independent of signal intensity calibrations and only requires knowledge of the detector geometry, which is invariant for a given instrument. This study demonstrates that the method yields robust thickness estimates for sub‐micrometer amorphous specimen using both direct detection and light conversion 2D‐STEM detectors in a coincident FIB‐SEM and a conventional SEM. Due to its facile implementation and minimal dose reauirements, it is anticipated that this method will find applications for in situ thickness monitoring during lamella fabrication of beam‐sensitive materials.

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Local thickness and composition analysis of TEM lamellae in the FIB

Cited by 9 publications

References 3 publications

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution

Solid immersion microscopy images cells under cryogenic conditions with 12 nm resolution

Solid immersion microscopy readily and inexpensively enables 12 nm resolution on plunge-frozen cells

Robust Local Thickness Estimation of Sub‐Micrometer Specimen by 4D‐STEM

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