Benchmarking the ideal sample thickness in cryo-EM

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

Download Full-text

Benchmarking ideal sample thickness in cryo-EM using MicroED

10.1101/2021.07.02.450941 ◽

2021 ◽

Author(s):

Michael W. Martynowycz ◽

Max T. B. Clabbers ◽

Johan Unge ◽

Johan Hattne ◽

Tamir Gonen

Keyword(s):

Free Path ◽

Focused Ion Beam ◽

Ion Beam ◽

Mean Free Path ◽

Sample Thickness ◽

Proteinase K ◽

Specimen Thickness ◽

Quality Of Data ◽

Inelastic Mean Free Path

The relationship between sample thickness and quality of data obtained by microcrystal electron diffraction (MicroED) is investigated. Several EM grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion-beam scanning electron microscope (FIB/SEM) where the crystals were then systematically thinned into lamellae between 95 nm and 1650 nm thick. MicroED data were collected at either 120, 200, or 300 kV accelerating voltages. Lamellae thicknesses were converted to multiples of the calculated inelastic mean free path (MFP) of electrons at each accelerating voltage to allow the results to be compared on a common scale. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined from crystalline lamellae only up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages but the quality was insufficient to yield structures. No diffraction data were observed from lamellae thicker than four times the calculated inelastic mean free path. The quality of the determined structures and crystallographic statistics were similar for all lamellae up to 2x the inelastic mean free path in thickness, but quickly deteriorated at greater thicknesses. This study provides a benchmark with respect to the ideal limit for biological specimen thickness with implications for all cryo-EM methods.

Download Full-text

Determination of Mean Free Path for Inelastic Scattering in Poly(2-Vinyl Pyridine) by Low-Loss Spectrum Imaging

Microscopy and Microanalysis ◽

10.1017/s1431927600033614 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 224-225

Author(s):

A. Aitouchen ◽

T. Chou ◽

M. Libera ◽

M. Misra

Keyword(s):

Free Path ◽

Free Fall ◽

Mean Free Path ◽

Specimen Thickness ◽

Thickness Determination ◽

Vinyl Pyridine ◽

Integrated Intensity ◽

Inelastic Mean Free Path ◽

Electron Energy Loss

The common experimental method to determine the total inelastic mean free path i by electron energy-loss spectroscopy (EELS) is by the relation : t/λi= ln(It/IO) [1] where t is the specimen thickness, It, is the total integrated intensity, and Io is the intensity of the zero-loss peak. The accuracy of this measurement depends on the thickness determination. Model geometries like cubes, wedges, and spheres enable accurate thickness determination from transmission images.Spherical polymers with diameters of order 10-200nm can be made from a number of high-Tg polymers by solvent atomization. This research studied atomized spheres of poly(2-vinyl pyridine) [PVP]. A solution of 0.1% PVP in THF was nebulized. After solvent evaporation during free fall within the chamber atmosphere, solid spherical polymer particles with a range of diameters were collected on holey-carbon TEM grids at the bottom of the atomization chamber.

Download Full-text

Determination of Inelastic Mean Free Path by Electron Energy-Loss Spectroscopy in TEM: A Model Study Using Si and Ge

MRS Proceedings ◽

10.1557/proc-0982-kk07-13 ◽

2006 ◽

Vol 982 ◽

Author(s):

Chongmin Wang ◽

Bret D. Cannon

Keyword(s):

Free Path ◽

Mean Free Path ◽

Thickness Measurement ◽

Least Square ◽

Specimen Thickness ◽

Low Loss ◽

Diffraction Profile ◽

Key Factor ◽

Inelastic Mean Free Path

ABSTRACTAlthough the inelastic mean free path for Si and Ge have been measured previously, reported experimental values for silicon range from 121 nm to 160 nm for 200 keV and a large collection angle. A key factor responsible for this uncertainty is the lack of an accurate measurement of the specimen thickness at the point at which the EELS spectra are obtained. In this research, we have evaluated a systematic methodology for determination of the specimen thickness. In the thickness measurement based on converging beam electron diffraction, CBED, instead of the classic “trial and error” straight-line-fitting method to either the maxima or minima, a non-linear least square fitting of the theoretical diffraction profile to the energy filtered two-beam CBED is used. The low-loss EELS spectrum is also obtained from the same location. The inelastic mean free path was determined using the measured thickness and EELS data. Furthermore, attempt is also made to obtain the dielectric function from the low-loss spectrum. The established method will be extended to other materials and the results will be compared with numerical simulations.

Download Full-text

Measurement of the Inelastic Mean Free Path by EELS Analyses of Submicron Spheres

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100133965 ◽

1990 ◽

Vol 48 (2) ◽

pp. 74-75

Author(s):

Laura A. Bonney

Keyword(s):

Energy Loss ◽

Free Path ◽

Analytical Electron Microscopy ◽

Mean Free Path ◽

Thickness Measurement ◽

Crystal Defects ◽

Sample Thickness ◽

Area Of Interest ◽

Image Position ◽

Inelastic Mean Free Path

Accurate measurement of sample thickness is important for analytical electron microscopy (AEM) but is often difficult and tedious. Unlike other thickness measurement methods, with electron energy loss spectroscopy (EELS) thickness may be measured in both amorphous and crystalline specimens and at the same location and orientation at which other data is collected in the electron microscope. Thickness values may be obtained from convergent-beam electron diffraction (CBED) data only if the sample is crystalline with large grains of uniform thickness. Sample thickness may be measured from crystal defects projected through the entire foil, but such defects are not always conveniently located in the area of interest. The distance between contamination spots on the upper and lower surfaces of the specimen may be measured, but this is not considered accurate and contamination is not desirable in microanalysis.Sample thickness t may be determined with EELS by the relation:(1)where It is the total intensity in the EEL spectrum, Iz is the intensity in the zero loss peak, and λ is the inelastic mean free path for energy loss of an incident electron in the sample.

Download Full-text

Local thickness determination by Electron Energy Loss Spectroscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100172450 ◽

1994 ◽

Vol 52 ◽

pp. 944-945

Author(s):

Suichu Luo ◽

John R. Dunlap ◽

Richard W. Williams ◽

David C. Joy

Keyword(s):

Energy Loss ◽

Analytical Electron Microscopy ◽

Mean Free Path ◽

Single Scattering ◽

Specimen Thickness ◽

Three Dimension ◽

Angular Correction ◽

Electron Intensity ◽

Image Position ◽

Inelastic Mean Free Path

In analytical electron microscopy, it is often important to know the local thickness of a sample. The conventional method used for measuring specimen thickness by EELS is:where t is the specimen thickness, λi is the total inelastic mean free path, IT is the total intensity in an EEL spectrum, and I0 is the zero loss peak intensity. This is rigorouslycorrect only if the electrons are collected over all scattering angles and all energy losses. However, in most experiments only a fraction of the scattered electrons are collected due to a limited collection semi-angle. To overcome this problem we present a method based on three-dimension Poisson statistics, which takes into account both the inelastic and elastic mixed angular correction.The three-dimension Poisson formula is given by:where I is the unscattered electron intensity; t is the sample thickness; λi and λe are the inelastic and elastic scattering mean free paths; Si (θ) and Se(θ) are normalized single inelastic and elastic angular scattering distributions respectively ; F(E) is the single scattering normalized energy loss distribution; D(E,θ) is the plural scattering distribution,

Download Full-text