Imaging Emitted Covid 19 Samples with an Electron Microscope

  • #1

Summary:

Covid 19 has been around for many months. There is still a lack of clarity on the nature of virus emission from an infected person, in particular the size and nature of emitted particles. An important question is the distribution of particle sizes. This thread is for some brain storming to see if it would be feasible to somehow sample particles with Covid 19 virus from infected people.
After many months what comes out of the mouth and nose of someone infected with Covid 19 has not been directly observed.

A single virus particle of Covid 19 is about 120 nanometers in diameter. This means that to see the virus, visible light wavelengths of 400 nm to 700 nm are too long to produce images of individual virus particles. An electron microscope can image the virus as we have seen.

In addition, when someone coughs, sneezes, talks or simply breathes the virus particles are often known to be contained in water droplets of unpublished size distributions.

The issue of whether some of the water droplets are of aerosol size - and float in the air for hours or days - is very important and still unknown.

The water droplets evaporate in flight so the emitted sizes will decrease. But after a time the particles may have relatively less water and more virus and other left-over debris. I believe that these particles are called 'droplet nuclei'.

There must be a simple way to sample what is emitted by an infected person's mouth and nose.

For example, could an infected person exhale into a box of dry flowing nitrogen that contains a cryogenically cooled metal surface, so that the water droplets would land on the surface and quickly freeze. This sample would then be sealed in the box of dry nitrogen. Then metal surfaces with frozen water droplet samples would be available for examination in an electron microscope. Since the electron microscope operates at high vacuum, the water may have to be removed before the droplet nuclei, droplet areas, or virus particles themselves could be examined.

Why not?

Identifying the virus?

Any electron microscopists out there?

What might we see?

Another approach?

Any ideas?
 

Answers and Replies

  • #2
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We don't exhale pure water plus coronavirus particles. There is so much other stuff that you collect as well. That's why people do PCR tests. These have been done with objects around a person coughing/exhaling and so on, but the tests come with large uncertainties because no breath/cough/... is the same and different people lead to different results as well.
 
  • #3
The purpose would not be to identify viruses. The goal would be research to better understand what is emitted by people known to be infected with Covid 19. The particle size and viral content of the particles would be studied. There would be a new method and tool to quickly characterize the threat.

An electron microscope image can identify viruses as a Corona viruses by the image of the Corona. The size and shape of Corona viruses may be useful for further sorting. The interest would be to learn something about the droplets, or droplet nuclei in a new way after they are captured, for example, on cryogenically cooled plates. Or perhaps a probe of some design could be used to collect a sample emitted by the mouth or nose. The samples could also be collected elsewhere for other purposes?

What portion of virus bearing particles are in the aerosol size range? Would a square millimeter sample from a metal plate show something useful?
 
  • #4
Vanadium 50
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Some questions you might want to look up:
  • What is the typical size of a sample imaged by electron microscopy.
  • How much area do you plan to investigate - surely no less than 2 linear meters
  • How long does it take to run a sample?
From this, you will be able to calculate how long this will take. That will take you to your next question like
  • What is the value in getting this information decades or centuries hence?
 
  • #5
Those are issues after emitted Covid 19 virus images have first been observed in an electron microscope. There may be uncertainty of which Corona virus is being observed. It would seem that patients diagnosed with Covid 19 infections would probably produce different Corona virus images and concentrations than control groups.

Only a small number of samples would be needed to determine feasibility. An adequate sampling of the emitted breath that would be representative of the distribution of droplets and the contained virus. Imagine exhaling on a cool mirror and how water condensation appears. Pick a routine size for electron microscope samples and look at that size. Each water droplet with viruses that lands on a surface would leave an area with Covid 19, other Corona viruses, other viruses, bacteria, and other debris. All Corona viruses should stand out in the electron microscope images.

Why wouldn't a water droplet that landed on a surface and dried up show its contents with all its Corona viruses being obvious? We would also want to see other things in the emitted droplet. Are individual virus particles, ~ 120m nm dia., ever emitted or occur because of evaporation in flight? How much Covid 19 is in the form of evaporated 'droplet nuclei' that is aerosol size and stays suspended in air? Another method of sampling could be used for suspended aerosols such as filtering intended for an electron microscope examination.

It seems that aerosol or droplet issues are now determined by past studies and studies of how the virus infections are observed to transmit. Once the two most dominant transmission routes are reduced by 'personal distancing' or 'masks that reduce droplet emission' what are the other transmission routes? Why can't we image emitted droplets and droplet nuclei?
 
  • #6
TeethWhitener
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https://www.pnas.org/content/117/22/11875
Ad Bax and crew at NIH have been looking at respiratory particle size distribution with laser light scattering.

EM is tough. If you're looking to determine how viral content correlates with droplet size, a better way might be to separate the particles based on size (maybe something along the lines of time-of-flight) and then run qPCR on the different sizes.
 
  • #7
https://www.pnas.org/content/117/22/11875
Ad Bax and crew at NIH have been looking at respiratory particle size distribution with laser light scattering.

EM is tough. If you're looking to determine how viral content correlates with droplet size, a better way might be to separate the particles based on size (maybe something along the lines of time-of-flight) and then run qPCR on the different sizes.
I'll search for the Ad Bax work at NIH.

I have a PurpleAir sensor for particulates that works on a low cost laser sensor. Highly recommend for learning about aerosols. I am still uncertain of the effects of fog. Search PurpleAir Map and click on a sensor to see the aerosols around you and the several measurements available.

Another issue that I am unclear about is viral load required for infection. By the infecting mechanism, it seems as if one Covid 19 particle would be enough. ? If you only get infected by one particle vs many particles at the same time, it would seem to give your immune system a considerable head start. ?

Why is EM tough? A particle lands on a surface, the area is dried, made safe, prepared and taken to an electron microscope. I assume that the Covid 19 EM images earlier this year, were biopsied, since I believe those were other body cells visible in the images. ?

Do qPCR tests give the number of Covid 19 virus particles or just a likely positive reading above a threshold?

What's a "super spreader"?
 
  • #8
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In addition to the problems mention about isolating virus. There is a huge practical problem of lab safety. This work would need to be done in a BSL3 lab with special aparatus to contain the infective particles. This would be very expensive compared to culturing virus for easier collection, isolation, and correlation with other tests, like DNA sequence. I doubt that you'll find people using these limited resources on this.

It isn't an unreasonable assumption that the virus that is collected on an NP swab is also what comes out when people sneeze.
 
  • #9
TeethWhitener
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Why is EM tough?
Have you done EM?
A particle lands on a surface, the area is dried, made safe, prepared and taken to an electron microscope. I assume that the Covid 19 EM images earlier this year, were biopsied, since I believe those were other body cells visible in the images. ?
There’s a lot of stuff going on here. First you wanted to do cryofixation, now you want to let the particle dry out? How are you going to measure particle size? How will wettability of the capture surface bias particle size measurements? How are you going to isolate the droplets you want to analyze from all the other microscopic droplets flying around in the air (for example, oil droplets from the EM’s vacuum pump)? I’m not saying it can’t be done, but it does seem like there are much easier ways to get the information you’re looking for.
Do qPCR tests give the number of Covid 19 virus particles or just a likely positive reading above a threshold?
The “q” stands for “quantitative.”
Another issue that I am unclear about is viral load required for infection.
As far as I’m aware, that’s still an open question. The SARS-CoV-2 virus targets a specific receptor in a specific cell type in the lung, so you would have to be extremely unlucky to be infected by a single virion. And yes, generally higher viral exposure correlates to a worse outcome, but it’s not linear.
 
  • #10
Electron microscopes exist in laboratories.

Covid 19 experiments exist in unsafe environments.

Small samples, for example, 2 mm x 2 mm metal squares, must have living virus deactivated where created and before being transported to the laboratory with the electron microscope.

I read that temperature of XX? for 30 minutes will kill the Covid 19 virus. (I considered buying a commercial box for my mail.) If a small metal box with the samples were heated, it would become sterile. These boxes could be heated once again on receipt. There is low cost medical equipment for sterilizing.

We have seen electron microscope pictures of Covid 19. The experts that produced those pictures probably have had a procedure for many years for handling their infectious samples.

Compare the above risks to what we are experiencing every day, partly because we don't have answers to some of the most basic questions. Aerosol vs large droplets? Why is it better to be outdoors rather than indoors? In my area the number of cases is now 1% of the population. Cases will double in less than a month.

So far, we have the electron microscope pictures that show us Covid 19.

Images have a pretty good record of showing us what is going on, even when we don't expect to see it.

Is there a technical reason why this won't work? Electron microscopes are there. A sampling technique that retains some of the emitted particle and Covid 19 information is needed?
 
  • #11
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  • #12
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Compare the above risks to what we are experiencing every day, partly because we don't have answers to some of the most basic questions. Aerosol vs large droplets? Why is it better to be outdoors rather than indoors? In my area the number of cases is now 1% of the population. Cases will double in less than a month.
The aerosol vs large droplet issue is less a matter of whether each of those types of particulates contain virus, and more a matter of whether the viruses in each of those types of particulate remain infectious. This isn’t a question EM can answer.

It’s better to be outdoors than indoors for the same reason it’s safer to drink from a lake with one drop of poison than a shot glass with one drop of poison.
 
  • #13
This publication discusses droplets and aerosols.
The coronavirus pandemic and aerosols: Does COVID-19 transmit via expiratory particles?
Sima Asadi, a Nicole Bouvier, b Anthony S. Wexler, c and William D. Ristenpart a

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7157964/

It does not seem as if what's emitted from an infected person's mouth is being observed in 2020.

------------------------------------------------------------------------------------

I'm watching some masks on TV being tested with a green laser. It shows airborne clouds of particles that pass through different masks. Some of the masks seem to create finer aerosols that float at the same heights for seconds. What are the masks doing to the virus threat? Do some masks process the droplets into smaller droplets, into aerosols? Do droplets stopped by various masks evaporate and release virus?

If there were images showing Covid 19 emitted in droplets emitted by these masks would that convince most people that they should have effective masks?

It's chaos.

So far I don't see why emitted samples of Covid 19 can't be observed in an electron microscope. Sampling? Let's focus on these technical issues in this thread.
 
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  • #14
Vanadium 50
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So far I don't see why emitted samples of Covid 19 can't be observed in an electron microscope.
You were told why in message #4, and reminded again in message #11.

i don't think you want a discussion. You just want us to agree with you.
 
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  • #15
TeethWhitener
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So far I don't see why emitted samples of Covid 19 can't be observed in an electron microscope. Sampling? Let's focus on these technical issues in this thread.
You seem to be pretty stuck on using EM. Why? Because you think you’ll be able to see the virus? What if you can’t? EM samples and images are often highly processed under ideal conditions to get really impactful pictures. It’s kind of like astronomy: None of those nebulae actually look like what you see on the cover of National Geographic.

So let’s say somehow you’re able to solve all of that and someone coughs on a TEM grid and all of the processing goes swimmingly and you see coronavirus in the images. Or do you? Is it a common cold coronavirus (up to 60% of common colds are caused by coronaviruses that are morphologically indistinguishable from SARS-CoV-2), or is it the real deal? Are those protrusions actually coronavirus spike proteins or is it some weird beam damage effect? Are those carbon blobs a lipid layer or pump oil? And the most important point: how on earth do you use EM to determine whether a virus is infectious?

I think it’s also important to point out that hundreds of EM groups are already working on the COVID problem. They’re mainly doing cryo-EM for protein structure determination.

I guess there are two points that you need to make much more clearly than you’ve made thus far: 1) what exactly is the question you’re trying to answer, and 2) why is EM a suitable tool to answer that question?
 
  • #16
You were told why in message #4, and reminded again in message #11.

i don't think you want a discussion. You just want us to agree with you.
Your post seemed to deal with problems viewing a large area with an electron microscope and the difficulties that would involve. You gave an example of "2 meters".

I don't have a specific experiment in mind but am more interested in the experimental capability to observe what we are dealing with - virus plus particle as emitted and anywhere elsewhere. An expert with experience in this kind of research would know how to sample using the minimal amount of measurements and to get useful results. I discuss examples to illustrate the kind of information sought and not as part of a proposal.

The publication in post #13 mentioned some breathing where water particles of about 1 micrometer were emitted. (Picture a person exhaling on a cool mirror.) For an application like that I don't see why one 100 x 100 um sample area might not provide information that would show us the virus particles in one micrometer droplets. ? A swab could be placed along side the sample for the electron microscope.

It seems that imaging a droplet after collection is feasible and may have been done by experts but sampling to answer various questions is the issue. Sample collection - epidemiologist? Sample preparation - electron microscopist. These are not my fields. My experience is in optical and infrared applications, lasers, high speed imaging.

I'm trying to pin down problems sampling and imaging with an electron microscope and find some better ideas.
 
  • #17
You seem to be pretty stuck on using EM. Why? Because you think you’ll be able to see the virus? What if you can’t? EM samples and images are often highly processed under ideal conditions to get really impactful pictures. It’s kind of like astronomy: None of those nebulae actually look like what you see on the cover of National Geographic.
The electron microscopy seems to have the only potential to put the virus together with its surrounding particle (or what's left of it after collection). An EM expert would know about image processing issues.

I lived near Johns Hopkins University and used to attend some of the lectures at the Hubble lab that processes the images. I played tennis with one of the guys that processed the pictures. I was going to ask him how the images came about, but never did.

So let’s say somehow you’re able to solve all of that and someone coughs on a TEM grid and all of the processing goes swimmingly and you see coronavirus in the images. Or do you? Is it a common cold coronavirus (up to 60% of common colds are caused by coronaviruses that are morphologically indistinguishable from SARS-CoV-2), or is it the real deal? Are those protrusions actually coronavirus spike proteins or is it some weird beam damage effect? Are those carbon blobs a lipid layer or pump oil? And the most important point: how on earth do you use EM to determine whether a virus is infectious?
It is always good to consider that you have gotten perfect pretend data and ask, so what?

I did learn that some Covid 19 images, I believe the ones that showed the corona, may have been from transmission electron microscopes. Another issue to resolve.....

Look at the example of the 1 micron particles in breath in post #13, where a qPCR swab might be placed next to the sample for the EM. The swab would identify viruses in the cloud.

I think it’s also important to point out that hundreds of EM groups are already working on the COVID problem. They’re mainly doing cryo-EM for protein structure determination.
With hundreds of EM groups, which ones are sampling in front of the mouths of infected Covid 19 patients? It seems as if this would have been done. But I have assumed that no groups have because of publications like the one in post #12. Do you know of one EM group that has sampled infected Covid 19 patients?

I guess there are two points that you need to make much more clearly than you’ve made thus far: 1) what exactly is the question you’re trying to answer, and 2) why is EM a suitable tool to answer that question?
1) Can electron microscope images be applied to Covid 19 problems by providing information both on the virus and its surrounding matter and droplet? 2) electron microscopes have produced images of viruses and - until we learn otherwise - there is currently no show-stopper with sampling.

Looks reasonable enough to try it.
 
  • #18
TeethWhitener
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1) Can electron microscope images be applied to Covid 19 problems by providing information both on the virus and its surrounding matter and droplet?
This just sounds like a solution in search of a problem. This happens occasionally in science where you have invested in a really fancy or expensive tool and you have to go hunting for applications. However, I’ve found (especially when pitching to funding agencies) that a much more effective approach is to first isolate a question of primary importance (something like “what is the smallest particle that will be airborne and still yield infectious virus after x minutes?”) and then figure out which tools are best to answer the problem.

So asking “can EM help?” is jumping the gun a little bit (unless you’re an EM operator). What needs to be asked first is “what do we need to know that we don’t know already?” Then, based on the inevitably myriad answers to that question, one (edit: or more than one) of them might be answerable most efficaciously by applying EM. I don’t really think droplet size or whatever (I’m still not sure what you’re trying to answer) is it.
 
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  • #19
There are many Covid 19 problems and this seems a feasible solution to try.

Here is a quote from the publication in post #12 about breathing and 1 micron particles. This is an example of a very important problem that seems workable.

"Their experimental work involved artificially generated and aged aerosols using a nebulizer and maintaining it suspended in the air with a Goldberg drum. But if pre- or asymptomatic infected individuals do not sneeze or cough, how do they generate aerosols? In fact long ago it was established that ordinary breathing and speech both emit large quantities of aerosol particles (Duguid 1946; Papineni and Rosenthal 1997). These expiratory particles are typically about 1 micron in diameter, and thus invisible to the naked eye; most people unfamiliar with aerosols are completely unaware that they exist. The particles are sufficiently large, however, to carry viruses such as SARS-CoV-2, and they are also in the correct size range to be readily inhaled deep into the respiratory tract of a susceptible individual (Heyder et al. 1986). Recent work on influenza (another viral respiratory disease) has established that viable virus can indeed be emitted from an infected individual by breathing or speaking, without coughing or sneezing (Yan et al. 2018)."

The capability has been demonstrated by electron microscopes but collecting samples needs work.
 
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  • #20
TeethWhitener
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There are many Covid 19 problems and this seems a feasible solution to try.
Already things have gone awry: you still have yet to clearly name a problem where “this” is clearly a feasible solution.

The issue here is that you asked for focus on the technical issues, and every time we bring one up, your response is “well, that’s something the EM experts will be able to handle,” in an utter vacuum of evidence. I’ve already given you at least one problem that the EM experts will never be able to solve:
And the most important point: how on earth do you use EM to determine whether a virus is infectious?
Here’s the reason: even if you put infectious particles into an EM, by the time you’ve blasted them with 100 keV electrons, they will no longer be infectious. So you can never use EM to look at an infectious particle.

I’m trying to give you the benefit of the doubt, but I get the sneaking suspicion @Vanadium 50 was right all along (sorry, V50, I guess I’m still in curmudgeon denial).
i don't think you want a discussion. You just want us to agree with you.
 
  • #21
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Here’s the reason: even if you put infectious particles into an EM, by the time you’ve blasted them with 100 keV electrons, they will no longer be infectious. So you can never use EM to look at an infectious particle.
................................................
I never mentioned that the EM examination would provide any information on how infectious Covid 19 from patients was. With an additional sampling process, other measurements could be made for how long the emitted virus remains infectious.

After the virus is collected at a site with infected Covid 19 patients the samples would be "deactivated" as discussed in post #10. The virus would be sterilized after collection. See post #10. (I was replying to Post #8 but neglected to use 'reply').

There are publications on how long the virus remains infectious airborne and also on surfaces. The aerosols were generated in a lab, not patients. I believe that a prominent early publication established that the virus was still infectious at 24 hours airborne. A formite examination of one of the cruise ship cabins indicated that the virus was still infectious at 17 days. I have not heard of airborne measurements hours after emission from patients.

I am 77 and have severe asthma so I have spent some time looking for certain information.

If you want an application to evaluate for feasibility, use the application of post #19. Assume simple breathing, 1 micrometer water droplets, a successful sampling technique, sterilized samples with dead virus available for preparation at an EM facility and a team of specialists. This would bear on the question of whether asymptomatic people are emitting 1 micrometer water droplets that are not controlled by the recommended masks. Showing the pictures should be convincing evidence.

I am hoping that an epidemiologist or an electron microscopist might make some comments.
 
  • #22
Some questions you might want to look up:
  • What is the typical size of a sample imaged by electron microscopy.
An EM image size was listed as from 6 mm across to 12 um across varied by zooming. Using low magnification, a scientist would find images of virus and other material (that was originally in a water droplet) and zoom in to examine it.

How much area do you plan to investigate - surely no less than 2 linear meters
Let's assume an application as described in posts #19 and #21, breathing and emitting about 1 micrometer diameter water droplets with virus. Those droplets are collected, dried and sterilized. The residues exist on a metal surface, say, 2 x 2 mm. The plate would be scanned by a scientist to locate residues and each residue would be examined under high magnification. I'd say that 100 images of residues with virus counts could be examined in about 1 day. This would be done for research on Covid 19 in the breath of infected people.

  • How long does it take to run a sample?
'Running a sample' sounds like a developed test for a virus measurement.

The purpose here is to find a tool to characterize the virus droplets and use it briefly for research - targeted at the many unanswered questions.

From this, you will be able to calculate how long this will take. That will take you to your next question like
  • What is the value in getting this information decades or centuries hence?
 
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  • #23
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Here’s the reason: even if you put infectious particles into an EM, by the time you’ve blasted them with 100 keV electrons, they will no longer be infectious. So you can never use EM to look at an infectious particle.
............................................................
Do you mean the "low vacuum or wet conditions in variable or environmental SEM"? I was not aware of that type of SEM.

"Specimens are observed in high vacuum in conventional SEM, or in low vacuum or wet conditions in variable pressure or environmental SEM, and at a wide range of cryogenic or elevated temperatures with specialized instruments.[1]" Wikipedia , Scanning Electron Microscope
 
  • #24
" There is still a lack of clarity on the nature of virus emission from an infected person, in particular the size and nature of emitted particles. "

Reactive, not adhering to the fundamental root-cause analysis principle. Understanding the consequences of problems is not proactive. Each and All resources should be expended on solving the problem at the nature of the problem, i.e., how to kill the virus without damaging the host. Have more resources than can be effectively used? Then find another problem where you start at the base. Should you develop an experiment where you determine what will happen to the virus after 100 generations of dogs have contracted it? Of course not. Everything and everyone possible should be focused on the base of the problem. Far too much time spent on unnecessary research in thousands of areas.
 

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