[3dem] amplitude contrast valuesHi,

Philip Köck Philip.Koeck at ki.se
Wed Aug 6 03:17:42 PDT 2014

Hi Marin, David and other 3dem readers.

For me this is quite an interesting topic so I'll give you my views hoping for some more feedback.

The absorption potential can only have one sign since the opposite sign would describe amplification and this is not possible anywhere
in a normal specimen. The amplitude of the electron wave can't increase even locally due to interaction with the specimen unless the specimen
were to contribute electrons with the correct energy and phase to the incoming wave (like a laser, but with electrons).

The electrostatic potential however is positive on the whole but can be negative in the vicinity of partial negative charges.

Therefore assuming a proportionality seems like physical nonsense to me from the beginning.

One could argue that the assumption is still reasonably good in the absence of negative charges, but then I don't see why it is
made in such a general way for cryo specimens of, for example, proteins.
As I see it, areas of negative electrostatic potential are quite common in fully hydrated proteins.

All in all I find it very strange that this assumption is made without any further discussion by almost every researcher and every
software package in the field.

All the best,


Från: 3dem-bounces at ncmir.ucsd.edu [mailto:3dem-bounces at ncmir.ucsd.edu] För Marin van Heel
Skickat: den 25 juli 2014 13:44
Till: David Gene Morgan; 3dem at ncmir.ucsd.edu
Ämne: Re: [3dem] amplitude contrast valuesHi,

Dear David

The amplitude and phase information are two independent properties of the sample which are transferred through a linear optical system by the Amplitude Transfer Function (ATF) and the Phase Contrast Transfer Function (PhCTF), respectively. That is standard Fourier Optics. Merging of the amplitude and phase information transfer into a single hybrid contrast transfer function necessarily implies assuming a proportionality between the phase and amplitude information of the sample over all spatial frequencies. Assuming such a proportionality is certainly incorrect in general and the experimental evidence supporting such assumptions fail to convince me, even in the cases where one is only looking at low-resolution negative-stain images. The practical solution is simple: ignore the fiddle-factor "Q" altogether by setting it to zero and first take out all irrelevant and disturbing low-resolution amplitudes by high-pass filtering the images prior to the CTF determination. Reference: M. van Heel: On the imaging of relatively strong objects in partially coherent illumination in optics and electron optics, Optik 47 (1978) 389-408) and paper in preparation.

Hope this helps


On 21/07/2014 14:02, David Gene Morgan wrote:

    I'm trying to do some automated defocus determination (initially trying Niko's ctffind3 program) and would like some input on appropriate amplitude contrast values for various types of samples.  I know that ice embedded images usually are said to have an amplitude contrast of 0.07 to 0.1.  I also know that with protein/nucleic acid complexes in ice, amplitude contrast may need to be set a bit higher (say to as much as 0.15 for some data I worked on awhile ago).

    But I don't have any sort of feeling for appropriate values of amp;itude contrast for different sorts of negative stains (either cryo negative stain or standard negative stain).  It should be "high," but I don't know how high might be appropriate.

    If anyone has either some practical or theoretical insight into this, I'd appreciate some suggestions.  Also, does anyone know about an appropriate amplitude contrast for a standard Au/Pd waffle grid?

    Thanks in advance for all your help.


    Prof Dr Ir Marin van Heel

    Professor of Cryo-EM Data Processing

    Leiden University
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    Emeritus Professor of Structural Biology

    Imperial College London
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