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The higher conductivity facilitates scanning electron microscope (SEM) imaging, by reducing sample charging under the electron beam.
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Low-energy electron flooding (20 eV) was used to reduce sample charging.
The data stack was imaged using backscattered electron detection at 2.5 kV, on a 2048 × 2048 array with 25 μs dwell time at a chamber pressure of 0.56 mbar in a water vapour atmosphere to earth and reduce sample charging.
Samples were gold-palladium coated using a Denton Vacuum Desk II gold-cathode sputter to reduce sample charging and analyzed using a ZEISS 1540 XB field emission gun – scanning electron microscope (FEG-SEM) at 3.0 kV.
The gold substrates were grounded to eliminate sample charging.
Search parameters for reduced samples against IPI human were as follows: (i) one missed cleavage Asp-N; (ii) tolerance 5 p.p.m. (precursor), 0.5 Da (fragment ions); (iii) charge state 2+, 3+, 4+; and (iv) oxidation cysteine, methionine (variable).
The low vacuum pressure in the sample chamber was reduced until charging levels on the sample surface were reduced to the level at which electron imaging of the sample surface was possible.
This carbon sandwich method can reduce the charging effect as well [ 48, 59].
To compensate for sample charging, all spectra were charge corrected against the C 1s adventitious carbon peak at 284.8 eV.
The binding energy was calibrated by taking the C1s peak (284.6 eV) as a reference with the application of electron shower gun to reduce charging effect of the samples.
Each sample had been placed onto a carbon membrane, and an Au sputter coating was applied to reduce charging effects.
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