[3dem] [ccpem] cryo-EM
Marin van Heel
marin.vanheel at googlemail.com
Fri Aug 20 15:34:45 PDT 2021
Dear Teige and others
To understand the phase contrast principles (and many other basic
principles in Cryo-EM) you may want to look at my phase-contrast lecture
notes and other goodies at our single-particle website
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.singleparticles.org_methodology.html&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=J_-fFfmsY8jwoTP-j3WdgXQgr2qj5-wexg1J2iTNpK4&s=GzOyQ06zZDZKKp-we94_RAUiOGD9ENo11iqft4YzeP4&e= . Adding microns to the path
of the electrons in underfocus versus overfocus is not really relevant
since all that counts are the path (phase) differences, not their absolute
value.
I noticed that there is an updated version of this phase-contrast document
that apparently has not yet made it to the site. There is also one or
maybe more YouTube movies on the phase contrast issue. just search for it.
There certainly is an old one where I start in Portuguese but then switch
to English soon (so don't be shocked).
In terms of the linguistics issue that started off this discussion I prefer
"cryogenic electron microscopy" (abbreviated to cryo-EM). Of course, if
you prefer constructs like "super-resolution light cryo-microscopy", or
"atomic-force cryo-microscopy" or "electron phase-contrast
cryo-microscopy", be my guest.
Cheers,
Marin
On Fri, Aug 20, 2021 at 1:01 PM Teige Matthews-Palmer <teige.lg at gmail.com>
wrote:
> What *is *the property that gives rise to light and dark on the image?
>
> Different sums of electron detection events over a recording, mostly
> arising from the weak phase contrast phenomenon.
>
> If I’ve got this right a direct electron detector with a perfect detective
> quantum efficiency would count every incident electron and approximate its
> position on a grid of pixels.
>
> If the electron beam were supplying electrons all at exactly the same
> energy and direction, hitting all the detector pixels at a uniform rate,
> then you’d say areas picking up lower or higher rates of electrons indicate
> some obstruction from the sample.
>
> Given our thin cryoEM samples with low mass atoms and typical electron
> energies, most electrons simply pass straight through our samples as if it
> wasn’t there.
>
> A small number of electrons deposit some energy in the sample (thus lose
> some energy, damage the sample) and are scattered to high angles (an energy
> filter removes some of these from the image). These events would create
> amplitude contrast. However for the amount of radiation damage incurred,
> amplitude contrast of our samples is very little, discussed in R Henderson
> 1995 https://urldefense.proofpoint.com/v2/url?u=https-3A__pubmed.ncbi.nlm.nih.gov_7568675_&d=DwIFaQ&c=-35OiAkTchMrZOngvJPOeA&r=L7-zyQ-04fFCMRqzLIOnx7H0exGZHwIQe_wMPuY600I&m=J_-fFfmsY8jwoTP-j3WdgXQgr2qj5-wexg1J2iTNpK4&s=4bt2SsWVdhhZ9T0oaiagGS9PNnO8I__eEo4RtUOnIBk&e= (anyone got the PDF to
> share?)
>
> Even less electrons are scattered by the sample without depositing energy
> (inelastic scattering), but their path deflected and their phase altered by
> pi/2. As with diffraction from a lattice of scattering centres, the
> distances and distribution of scattering centres in our suspended particles
> also produces constructive interference of this scattering at angles
> relating to the distance between scattering centres.
> These scattered electron paths are longer than the transmitted ones (but
> only a tiny bit as the angles are small), before they refocus at the image
> plane, where the difference in their phase modifies the probability of an
> electron detection event.
> The amplitude of scattered is much less than transmitted, so the potential
> to modify the electron detection by way of this interference is low.
> The fundamental pi/2 phase difference has almost no effect on the
> detection of electrons on the detector.
> If the phases were exactly opposite i.e. shifted by pi, this 'gives the
> scattered electrons' the maximum ability to reduce detection.
> The angles of scattering are very small, so although scattered paths are
> longer than transmitted paths, the difference is not enough to delay the
> phase anywhere close to pi by the time the paths refocus.
> However if we underfocus the lens (here is a weird part that I’d like
> explained) the distance before refocusing becomes microns longer or many
> thousands wavelengths longer, that extra length allowing greater phase
> delay of the scattered path upon their interaction - (however, how does a
> fixed camera length accommodate this - where does this interaction need to
> happen in relation to the detector and how thick is this zone? Explanations
> that the lens is focused on a point above the specimen doesn’t seem helpful
> for my bodged intuition).
>
> Thanks for coming to my phase contrast for beginners ted talk.
>
> Since the small detection probability differences that create our image
> contrast come from scattering, the different charges of bits of our samples
> ought to deflect electrons differently. But, isn’t the atomic nucleus tiny
> compared to the electron cloud? And, what is the meaningful difference
> between say an amino group and a carboxylic acid group: is it the different
> electric fields? Aren’t electrons deflected to a range of angles by passing
> through any electron cloud - and then rather the distances ‘between’ clouds
> determines the enhanced diffracting angles just like x-ray diffraction… I
> guess these sizes have become similar and the gradients of the field starts
> to matter?
> What does the coulomb potential field of an atom in a molecule look like
> to an electron?
>
> On 20 Aug 2021, at 14:44, Colin Palmer - STFC UKRI <
> colin.palmer at STFC.AC.UK> wrote:
>
> Dear Quyen,
>
> The reconstructed maps are an indication of the way the imaging radiation
> scatters in the sample, rather than a property of the radiation itself.
>
> So, in X-ray crystallography, we detect X-rays, but the X-rays are
> scattered by the electrons in the sample and so the maps are electron
> density maps.
>
> In EM, we detect electrons, but in the sample those electrons are
> scattered by charge interactions with both negatively charged electrons and
> positively charged atomic nuclei. Therefore the maps are (ideally) maps of
> the charge or electrical potential density in the sample. But the details
> are complicated, hence this discussion!
>
> Best wishes,
> Colin
>
>
> *From:* Collaborative Computational Project in Electron cryo-Microscopy <
> CCPEM at JISCMAIL.AC.UK> *On Behalf Of *Quyen Hoang
> *Sent:* 20 August 2021 14:31
> *To:* CCPEM at JISCMAIL.AC.UK
> *Subject:* Re: [ccpem] cryo-EM
>
> Interesting discussion.
> As a crystallographer trying to learn cryo-EM (or cryoEM), I have learned
> a lot from listening in on these discussions.
>
> As others have pointed out, are the images on the micrographs not the
> result of electrons impacting the detector?
> So, are the light and dark parts not represent electron density (or
> distribution) on the micrographs?
> And then would the resulting average/combined images not be a direct
> representation of electron density?
>
> In X-ray crystallography, the resulting maps represent the calculated
> electron distribution that might have given raise to the X-ray diffraction
> images.
> But it seems that the reconstructed map of cryo-EM (or non-cryo) is a
> direct distribution of electrons on the micrographs?
> If so, are they not electron-distribution/density maps?
>
> Cheers,
> Quyen
>
>
> On Aug 20, 2021, at 6:37 AM, Patrick Shaw Stewart <pshawstewart at GMAIL.COM>
> wrote:
>
>
>
> what do you call the observed quantity represented in an EM map or
> tomogram?
>
>
> What *is *the property that gives rise to light and dark on the image? I
> asked a famous cryoEM expert that question. Strangely, I didn't get a
> clear answer. Does anyone have one?
>
> Is the ability to create images related to the fact that the density of
> protein is greater than the density of water?
>
> Thx, Patrick
>
>
> On Thu, Aug 19, 2021 at 11:14 AM Gerard Kleywegt <gerard at ebi.ac.uk> wrote:
>
> Hi all,
>
> By hyphenating "cryo" and "electron" you are turning it into a
> compound noun modifier (note the difference between "the amino acid is" an
>
> "the amino-acid sequence is" - in the latter case "amino-acid" is a
> compound modifier of the noun "sequence", hence hyphenated). Thus,
> in "cryo-electron microscopy" the "cryo-electron" modifies the noun
> "microscopy", which is not what you intend.
>
> I am in the Henderson School of Nomenclature, so cryo-EM but electron
> cryo-microscopy. Cryogenic electron microscopy (not hyphenated!) is also
> fine it seems to me.
>
> Now that I have everybody's attention - maybe we can sort the following
> issue out once and for all: what do you call the observed quantity
> represented in an EM map or tomogram? People often say "electron density"
> which it is not and which I try to avoid. Electric potential?
> Electrostatic potential? Something else entirely?
>
> --Gerard
>
>
>
> On Thu, 19 Aug 2021, Ben Engel wrote:
>
> > This debate again? :)
> > Think of it as cryo-(electron microscopy). Cryo is modifying the
> compound noun "electron microscopy". It's amazing to me how people insist
> that
> > this means cold electrons. We use compound nouns all the time.
> >
> > If I told you it was cryo-cat food, would you think the cat is frozen or
> the food? Would you really reorder the words to cat cryofood? If the
> > hyphen bothers you, just call it cryo cat food. Or if you must, even
> cryogenic cat food. But personally, I have no issues hyphenating a compound
> > noun. So cryo-electron microscopy for me.
> >
> > Ben :)
> >
> > On Thu, Aug 19, 2021 at 9:11 AM Reinhard Rachel <
> Reinhard.Rachel at biologie.uni-regensburg.de> wrote:
> > > Goodmorning, a linguistic question. We all write about cryo-EM,
> but what is
> > > its correct full name, cryo-electron microscopy or cryogenic
> electron
> > > microscopy? The first one seems most used, but isn't the second
> one more
> > > correct?
> > >
> >
> > my understanding, and my preference:
> >
> > electron cryo-microscopy.
> > (this was not so much my idea, but some day, I listened to Richard
> H, and he
> > had an argument about "cryoEM" etc. His argument was clear).
> > This would result in ECM - and this abbreviation is used also in
> other parts
> > of our life, I know.
> > We have this problem.
> >
> > "cryo electron microscopy " would suggest that the electron is in
> a cryo
> > state, which is not the case
> > it is the microscope, which is - partly, in the area of the sample
> - is in a
> > cryo-state, where the sample and some other parts (which act as
> > anticontaminants ) are cooled down to below 100K, using a cryogen.
> >
> > my two cents.
> > Reinhard
> >
> >
> > --
> > Prof. Dr. Reinhard Rachel
> > University of Regensburg
> > Centre for EM / Anatomy
> > Faculty of Biology & Preclin. Med.
> > Universitaetsstrasse 31
> > D-93053 Regensburg - Germany
> > tel +49 941 943 -2837, -1666
> > mail reinhard.rachel at biologie.uni-regensburg.de
> > office: VKL 3.1.29
> > member of the IFSM board
> >
> > Next microscopy conferences:
> > - next European EM conf.: EMC2024 in Kopenhagen, August 2024
> > - MC2021 virtual in Vienna, 22-26 Aug 2021(D-A-CH + MCM conference)
> > - IMC20 in Busan, South Korea: Sept, 2023
> > - next virtual Microbiol. conferences: VAAM March 2022 Düsseldorf
> >
> >
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> Best wishes,
>
> --Gerard
>
> ---
> Gerard J. Kleywegt, EMBL-EBI, Hinxton, UK
> Head of Molecular and Cellular Structure
> gerard at ebi.ac.uk pdbe.org emdb-empiar.org
> PA: Roisin Dunlop pdbe_admin at ebi.ac.uk
>
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