this is one of his pages. There is also a presentation on Youtube. I have no idea how accurate that is either though. I think everyone is in the dark - how else can they be, if even the manufacturers don't seem to know what they made (which for me is a reason to believe they did not really make it but just followed orders to do this and that). And why would they all come out with similar 'solutions' if they all worked independently.
Why do EM images almost always show objects as regular circles?
The preparation for EM is an ultra thin slice through the analyzed substance. If the image shows nearly perfect circles, it would mean that all objects cut through in the slice a) were fully round (globular) and never in any other shape, b) formed an orderly formation, c) were all cut perfectly equatorially. Meeting all these three conditions together looks like a miracle even for one image, but there are hundreds of them.
Because those are ideal LNPs made under ideal conditions. Made by people who know how to handle the fluidic mixtures. PDI is at 0.1 or less. Very small amounts made.
Much different than the scaled up processes used by both Pfizer and Moderna. Their PDIs are over 0.1 sometimes close to 0.3 and look at the figure with the mRNA escaping. Lots of elliptical LNPs in that sample, which is the real vaccine.
EM images invariably show nice full-circle cross sections for various preparations. Considering the EM sample preparation procedure, such an outcome for various preparations in so many repeated takes is mathematically a miracle. The whole sample preparation is more like a violent blender-type full-scale destruction of whatever is taken there - while the resulting images are always nice and tidy. How come?
The slice cannot contain a 3D “ball” or “bubble” due to the sample’s thickness (or rather thinness). Ten perfectly round full circles in one image means that all these objects were perfectly aligned right at the moment of the cut - impossible with the sample preparation cycle. Moreover, they are often of roughly the same size - which would mean either full homogeneity of the sample (??), or another perfect alignment of these objects, not only along the x and y axes, but also vertically (all objects aligned with their largest round cross sections on the same plane). The latter would mean a miracle of all miracles. In one sample and one image, maybe, who knows, but even in your article there five such images.
Oh now I understand. Yes, mathematically impossible. And full homogeneity is rare in the scaled up processes but more likely in these specialized labs making small amounts.
They also dialyze the LNPs against a buffer and a filter. Would that make a difference?
In your article, right under “What is the structure of the current LNPs in the mRNA vaccines?”, there is a small image of ca. 100 nm circles (21 in full view, some on the edges of the image). Their diameters are almost identical. If this is a cross-section (a sample slice), all these 21 circles are most probably aligned along their equatorial circle of latitude. How would be possible?
Where are any cross-sections along circles of latitude other than the widest diameter? All smaller, from close to those visible, down to perfect circles of 1 nm. This would be a resulting image with all “3D balls” aligned at their widest diameter - what I called a formation. What about “3d balls” cut through at random angles and thus resulting in elliptical cross-sections? Not a single one of them here.
Maybe this could happen if you were to leave the sample aside for some time to “settle” - but with EM it is not possible due to the violent and chaotic process of sample preparation… including freezing these poor creatures to death - the process which in itself entails structure deformations. At this point, the sample is no longer a living organism (tissue), so it does not matter much, though…
Obviously, there is a lot of post-processing of EM images, which is another aspect - why aren’t we shown raw images? We could see and assess the actual differences between the source image material, along with its noise, and what “photoshop” EM masters can create from these raw files.
Quote "In the case of protein samples, vitrified specimens are made in the following way: a solution of protein (~ 2–5 µl) is applied to a copper or gold EM grid coated with a thin layer of carbon (typically between 10 – 20 nm in thickness) that contains several small holes. The majority of the protein solution is blotted away leaving a thin film containing the sample which is immediately plunged into either liquid ethane or liquid propane, maintained at temperatures around −180 °C. This step ensures rapid freezing of the sample in an amorphous or vitreous state where the aqueous phase does not form crystalline ice. The sample is then stored and imaged at temperatures ≤ −160°C which ensures that the ice remains in a vitreous state and prevents formation of crystalline forms that are populated at higher temperatures?
Yes, this is one of the stages. It involves rapid freezing to which I referred earlier.
Here we face another interesting issue. The protein sample must have been dehydrated. This process had to cause structural deformations - even though we are told otherwise, but there is no proof of retaining the sample physically intact. Besides, at this scale of dimensions we have no way of checking or measuring, so we only can “believe” what we imagine or hope.
We could, sort of, verify it if we had control samples on each stage of sample preparation - but this is done absolutely never, right?
My point is that we introduce a lot of unknown variables before obtaining the EM image. We then make images which we cannot really analyze for a number of reasons, where physical invisibility of the objects represented on the final images is only one.
We then get a perfect image of perfect circles arranged in a perfect way.
How come? After freezing to -160C and then bombarding with high energy? How come no deformations are shown or reported? The sample is dead and solidified, which may be a reason, but if this is so a) can we replicate these processes at (visual) microscopic level to make sure that we do not go fantasy land, and b) how images of dead samples relate to live organisms - can we reliably enter any adjustments or corrections, and how?
Not dehydrated. "In cryo-EM, the specimen is maintained in its hydrated native state by vitrifying the water into an amorphous (glass-like) solid. The samples have to be kept below the devitrification temperature of water (∼−135 °C) throughout all handling steps, including TEM imaging."
My general impression from various sources on EM is that we approach the subject from a completely wrong angle. We have developed an amazing technology, no question about it. Now we are trying to push into its vacuum chamber whatever we can get hold, just to see “how it looks like when attacked by an electron beam”. It’s obvious that seeing a living tissue at such magnifications is a huge attraction. Like, “Let’s see how your fingernails looks like. And a hair.” So we go there.
But, wait, the sample cannot be alive. So we destroy the sample in a number of ways, which we nicely call “preparation”. Whatever we do in this area, it will always impact the sample material. Then, we add another step, or yet another, to counter these compounding impacts. First it was dehydration. We are told that water is the (by volume) basis of live, thus dehydration was a bad choice, AND we didn’t know how it really affects the sample’s biological structures. Ok, we replaced dehydration with (quote from the linked article, highlights are mine):
“…_Complete_ vitrification of thicker samples usually _requires additional measures._ The most widely used method is high-pressure freezing (HPF): after a few milliseconds during which _pressure is raised to ∼2000 bar,_ rapid cooling proceeds. In this manner, samples up to ∼100–300 μm thick can be vitrified. Subsequently, vitreous sections (∼25–100 nm thick) have to be cut with a cryo-ultramicrotome. _Sectioning vitrified samples is a very demanding process and not exempt of artifacts,_ such as _compression and crevasses_ that become particularly severe in sections thicker than ∼100 nm. Recently, focused-ion beam (FIB) milling has shown to be an effective alternative in thinning cryo-samples. The method consists in using a focused _beam of ions_ (usually Ga2+) _to erode the sample_ until it reaches the desired thickness. _The bulk of the material is lost in the process;_ however, the result is a thin slab of sample without sectioning artifacts.”
Now, pressurizing a human body at 2,000 bar would have some impact, wouldn’t it? The water may be still there, but is it water? What is happening to the molecular/atomic/functional/unknowable structure at this pressure? I guess, we don’t know - if we knew, the authors would be awarded 10 Nobels in bulk and the whole universe would know because it would revolutionize everything. No more dangerous transport of liquid gases, as an example.
In short, in EM we do whatever we can to obtain a sample with parameters acceptable to the EM technology. We don’t care about the integrity of the sample, and we don’t care if it retains anything of the original live material. We only need a 10 nm slice, the rest has become irrelevant, and to such extent that we apply a lot of mathematics (instead of observation) and image post-processing to arrive at the end result which would justify the whole process. We are seeing (visually, graphically) interesting images, but due to all interim steps they have no connection with the original alive structure which was there at the beginning.
Every step of this process has some impact on the sample, but we don’t know what is happening there because there can be no control samples. The technology excludes the possibility of having control samples. The only reasonable conclusion is that we have no idea what is happening to “live” organisms subjected to EM, we only may be sure that they are effectively deformed and finally destroyed in the process in a number of ways.
To balance this conclusion, we could have a look at samples from EM experiments from, say, 1980s, with the modern state-of-the-art EM systems. The comparison would be of immense scientific value, showing the progress in technology and in the ability to examine the natural processes. Would be - but won’t be, because EM samples cannot be re-examined. It’s all one-off imaging. The particle beam used to visualize the sample impacts and destroys it, in part in ways of which we are not aware. However, when repeatability is excluded, the science (in its original definition) is gone. Observation, we have got rid of it when we started the sample preparation procedure. EM appears to be art, not science…
PS. So far, I have not found the answer to my original question about putting image content in a 3D perfect formation.
You might like to read about using high energy electron beams to cool targets. You might also like to consider that Human IVF uses vitrification to preserve sperm and egg.
"What Does Vitrification Involve? With vitrification, the solution containing the oocyte or embryo protects the cells from freezing-related damage. The cooling process is so rapid that the water molecules in the cells do not have time to form destructive ice crystals and instantly solidify into a glass-like structure."
Vache volante! I’m reminded of the scene in Monte Python’s Holy Grail, when the insulting French soldiers hurled all manor of garbage at King Arthur, including a cow. Yes, hurling junk is and has been a rather offensive act. It’s just us cowards that don’t want to get pricked; yeh, right?
I have a maybe stupid question: the cationic lipids would only be cleared in acidic milieu, r8? So the loss of transfecting ability wouldn't appear even if they would be disintegrated?
Or did I got it wrong and you can claim by now: "Hey! You aren't sick by now than don't worry cause the vial did not work and didn't hurt you?"
Not a stupid question at all. I've been thinking about what happens to these ionizable lipids? The PEG lipids do not enter the cell, so it is only cholesterol, the helper lipid and ALC-0315 or SM102
But first let's zoom out.
After the protein covered nanoparticle is engulfed into a an endosome, it has to break down inside the endosome, then the endosome has to break apart to release the mRNA and lipids and any other gunk in the LNPs into the cytosome. If this isn't done within 6 hours, then the mRNA doesn't escape and those endosomes turn into lysosomes.
And that's when things get interesting. Imagine all those lysosomes full of those lipids, mRNA and what not just sitting there. What does the cell do with this?
1. lysosomes are the cells recyclying centres...they get rid of bad proteins and the like. And if they can't the cellular stress and dysfunction... so similar to lysosomal storage diseases. Which are nasty and have overlap with vaccine injuries. So all that signalling occurring?
Oswald Ripening
Nice
Outstanding Canning
Outstanding
I have been following Dr Bines for a while. https://drbine.substack.com/p/die-plorre-ist-wohl-innen-flussig?utm_source=profile&utm_medium=reader2
this is one of his pages. There is also a presentation on Youtube. I have no idea how accurate that is either though. I think everyone is in the dark - how else can they be, if even the manufacturers don't seem to know what they made (which for me is a reason to believe they did not really make it but just followed orders to do this and that). And why would they all come out with similar 'solutions' if they all worked independently.
Dr Bine (Sabine) is a lady.
https://twitter.com/Sabisteb
aw I did not know that! thanks for the correction
Absolute insanity.
Thank you for staying on top of this.
Why do EM images almost always show objects as regular circles?
The preparation for EM is an ultra thin slice through the analyzed substance. If the image shows nearly perfect circles, it would mean that all objects cut through in the slice a) were fully round (globular) and never in any other shape, b) formed an orderly formation, c) were all cut perfectly equatorially. Meeting all these three conditions together looks like a miracle even for one image, but there are hundreds of them.
Because those are ideal LNPs made under ideal conditions. Made by people who know how to handle the fluidic mixtures. PDI is at 0.1 or less. Very small amounts made.
Much different than the scaled up processes used by both Pfizer and Moderna. Their PDIs are over 0.1 sometimes close to 0.3 and look at the figure with the mRNA escaping. Lots of elliptical LNPs in that sample, which is the real vaccine.
EM images invariably show nice full-circle cross sections for various preparations. Considering the EM sample preparation procedure, such an outcome for various preparations in so many repeated takes is mathematically a miracle. The whole sample preparation is more like a violent blender-type full-scale destruction of whatever is taken there - while the resulting images are always nice and tidy. How come?
The slice cannot contain a 3D “ball” or “bubble” due to the sample’s thickness (or rather thinness). Ten perfectly round full circles in one image means that all these objects were perfectly aligned right at the moment of the cut - impossible with the sample preparation cycle. Moreover, they are often of roughly the same size - which would mean either full homogeneity of the sample (??), or another perfect alignment of these objects, not only along the x and y axes, but also vertically (all objects aligned with their largest round cross sections on the same plane). The latter would mean a miracle of all miracles. In one sample and one image, maybe, who knows, but even in your article there five such images.
Oh now I understand. Yes, mathematically impossible. And full homogeneity is rare in the scaled up processes but more likely in these specialized labs making small amounts.
They also dialyze the LNPs against a buffer and a filter. Would that make a difference?
Just trying to understand.
In your article, right under “What is the structure of the current LNPs in the mRNA vaccines?”, there is a small image of ca. 100 nm circles (21 in full view, some on the edges of the image). Their diameters are almost identical. If this is a cross-section (a sample slice), all these 21 circles are most probably aligned along their equatorial circle of latitude. How would be possible?
Where are any cross-sections along circles of latitude other than the widest diameter? All smaller, from close to those visible, down to perfect circles of 1 nm. This would be a resulting image with all “3D balls” aligned at their widest diameter - what I called a formation. What about “3d balls” cut through at random angles and thus resulting in elliptical cross-sections? Not a single one of them here.
Maybe this could happen if you were to leave the sample aside for some time to “settle” - but with EM it is not possible due to the violent and chaotic process of sample preparation… including freezing these poor creatures to death - the process which in itself entails structure deformations. At this point, the sample is no longer a living organism (tissue), so it does not matter much, though…
Obviously, there is a lot of post-processing of EM images, which is another aspect - why aren’t we shown raw images? We could see and assess the actual differences between the source image material, along with its noise, and what “photoshop” EM masters can create from these raw files.
Definitely beyond my ability to answer, sorry. I did find a write up with raw cryoEM images but these are all LNPs with blebs.
https://www.criver.com/eureka/revolutionizing-lipid-nanoparticle-analysis-machine-learning
Thank you for your commitment and willingness to do into the subject. Your work here is very informative, I like it a lot. Have a great weekend.
Dan
Quote "In the case of protein samples, vitrified specimens are made in the following way: a solution of protein (~ 2–5 µl) is applied to a copper or gold EM grid coated with a thin layer of carbon (typically between 10 – 20 nm in thickness) that contains several small holes. The majority of the protein solution is blotted away leaving a thin film containing the sample which is immediately plunged into either liquid ethane or liquid propane, maintained at temperatures around −180 °C. This step ensures rapid freezing of the sample in an amorphous or vitreous state where the aqueous phase does not form crystalline ice. The sample is then stored and imaged at temperatures ≤ −160°C which ensures that the ice remains in a vitreous state and prevents formation of crystalline forms that are populated at higher temperatures?
Yes, this is one of the stages. It involves rapid freezing to which I referred earlier.
Here we face another interesting issue. The protein sample must have been dehydrated. This process had to cause structural deformations - even though we are told otherwise, but there is no proof of retaining the sample physically intact. Besides, at this scale of dimensions we have no way of checking or measuring, so we only can “believe” what we imagine or hope.
We could, sort of, verify it if we had control samples on each stage of sample preparation - but this is done absolutely never, right?
My point is that we introduce a lot of unknown variables before obtaining the EM image. We then make images which we cannot really analyze for a number of reasons, where physical invisibility of the objects represented on the final images is only one.
We then get a perfect image of perfect circles arranged in a perfect way.
How come? After freezing to -160C and then bombarding with high energy? How come no deformations are shown or reported? The sample is dead and solidified, which may be a reason, but if this is so a) can we replicate these processes at (visual) microscopic level to make sure that we do not go fantasy land, and b) how images of dead samples relate to live organisms - can we reliably enter any adjustments or corrections, and how?
Not dehydrated. "In cryo-EM, the specimen is maintained in its hydrated native state by vitrifying the water into an amorphous (glass-like) solid. The samples have to be kept below the devitrification temperature of water (∼−135 °C) throughout all handling steps, including TEM imaging."
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7110493/
Geoff, thank you for the link and contribution.
My general impression from various sources on EM is that we approach the subject from a completely wrong angle. We have developed an amazing technology, no question about it. Now we are trying to push into its vacuum chamber whatever we can get hold, just to see “how it looks like when attacked by an electron beam”. It’s obvious that seeing a living tissue at such magnifications is a huge attraction. Like, “Let’s see how your fingernails looks like. And a hair.” So we go there.
But, wait, the sample cannot be alive. So we destroy the sample in a number of ways, which we nicely call “preparation”. Whatever we do in this area, it will always impact the sample material. Then, we add another step, or yet another, to counter these compounding impacts. First it was dehydration. We are told that water is the (by volume) basis of live, thus dehydration was a bad choice, AND we didn’t know how it really affects the sample’s biological structures. Ok, we replaced dehydration with (quote from the linked article, highlights are mine):
“…_Complete_ vitrification of thicker samples usually _requires additional measures._ The most widely used method is high-pressure freezing (HPF): after a few milliseconds during which _pressure is raised to ∼2000 bar,_ rapid cooling proceeds. In this manner, samples up to ∼100–300 μm thick can be vitrified. Subsequently, vitreous sections (∼25–100 nm thick) have to be cut with a cryo-ultramicrotome. _Sectioning vitrified samples is a very demanding process and not exempt of artifacts,_ such as _compression and crevasses_ that become particularly severe in sections thicker than ∼100 nm. Recently, focused-ion beam (FIB) milling has shown to be an effective alternative in thinning cryo-samples. The method consists in using a focused _beam of ions_ (usually Ga2+) _to erode the sample_ until it reaches the desired thickness. _The bulk of the material is lost in the process;_ however, the result is a thin slab of sample without sectioning artifacts.”
Now, pressurizing a human body at 2,000 bar would have some impact, wouldn’t it? The water may be still there, but is it water? What is happening to the molecular/atomic/functional/unknowable structure at this pressure? I guess, we don’t know - if we knew, the authors would be awarded 10 Nobels in bulk and the whole universe would know because it would revolutionize everything. No more dangerous transport of liquid gases, as an example.
In short, in EM we do whatever we can to obtain a sample with parameters acceptable to the EM technology. We don’t care about the integrity of the sample, and we don’t care if it retains anything of the original live material. We only need a 10 nm slice, the rest has become irrelevant, and to such extent that we apply a lot of mathematics (instead of observation) and image post-processing to arrive at the end result which would justify the whole process. We are seeing (visually, graphically) interesting images, but due to all interim steps they have no connection with the original alive structure which was there at the beginning.
Every step of this process has some impact on the sample, but we don’t know what is happening there because there can be no control samples. The technology excludes the possibility of having control samples. The only reasonable conclusion is that we have no idea what is happening to “live” organisms subjected to EM, we only may be sure that they are effectively deformed and finally destroyed in the process in a number of ways.
To balance this conclusion, we could have a look at samples from EM experiments from, say, 1980s, with the modern state-of-the-art EM systems. The comparison would be of immense scientific value, showing the progress in technology and in the ability to examine the natural processes. Would be - but won’t be, because EM samples cannot be re-examined. It’s all one-off imaging. The particle beam used to visualize the sample impacts and destroys it, in part in ways of which we are not aware. However, when repeatability is excluded, the science (in its original definition) is gone. Observation, we have got rid of it when we started the sample preparation procedure. EM appears to be art, not science…
PS. So far, I have not found the answer to my original question about putting image content in a 3D perfect formation.
You might like to read about using high energy electron beams to cool targets. You might also like to consider that Human IVF uses vitrification to preserve sperm and egg.
"What Does Vitrification Involve? With vitrification, the solution containing the oocyte or embryo protects the cells from freezing-related damage. The cooling process is so rapid that the water molecules in the cells do not have time to form destructive ice crystals and instantly solidify into a glass-like structure."
I first studied vitrification in 1974.
https://en.wikipedia.org/wiki/Electron_cooling
Thank you for directing me there, I will read more. Regards, Dan.
Vache volante! I’m reminded of the scene in Monte Python’s Holy Grail, when the insulting French soldiers hurled all manor of garbage at King Arthur, including a cow. Yes, hurling junk is and has been a rather offensive act. It’s just us cowards that don’t want to get pricked; yeh, right?
Nice paper on Doxil cryo-electron tomography
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2712644/
Indeed. Thank you very much. Fascinating
I have a maybe stupid question: the cationic lipids would only be cleared in acidic milieu, r8? So the loss of transfecting ability wouldn't appear even if they would be disintegrated?
Or did I got it wrong and you can claim by now: "Hey! You aren't sick by now than don't worry cause the vial did not work and didn't hurt you?"
And off topic:
https://rumble.com/v4nuq95-the-good-the-bad-the-transfected..html
Would be proud if you find the time to watch my talk with Kevin McCairn.
Not a stupid question at all. I've been thinking about what happens to these ionizable lipids? The PEG lipids do not enter the cell, so it is only cholesterol, the helper lipid and ALC-0315 or SM102
But first let's zoom out.
After the protein covered nanoparticle is engulfed into a an endosome, it has to break down inside the endosome, then the endosome has to break apart to release the mRNA and lipids and any other gunk in the LNPs into the cytosome. If this isn't done within 6 hours, then the mRNA doesn't escape and those endosomes turn into lysosomes.
And that's when things get interesting. Imagine all those lysosomes full of those lipids, mRNA and what not just sitting there. What does the cell do with this?
1. lysosomes are the cells recyclying centres...they get rid of bad proteins and the like. And if they can't the cellular stress and dysfunction... so similar to lysosomal storage diseases. Which are nasty and have overlap with vaccine injuries. So all that signalling occurring?
2. And SARSCoV2 itself, interferes with this system...https://onlinelibrary.wiley.com/doi/10.1002/jmv.29200
3. so what does the vax spike protein do? Or the mRNA? or those lipids?
4. and is this why fasting helps so much with both the virus and the vax? https://pubmed.ncbi.nlm.nih.gov/37883971/
Just stuff to get you going. And yes this takes a long time to show up.
Maybe you covered this in your talk? I promise I will listen to it. Good work Narf
I would think the ionized part is only solving in acidic milieu and this is the part which scares me really to death.
A very interesting paper:
https://pubmed.ncbi.nlm.nih.gov/33901441/
Correct.
Exactly as designed.