From the genetic analysis, introduced by Keim & Worsham, we move on to the physical analysis of the attack materials as found. One major difference is that while testimony from the early days of the genetic analysis is included, this is not so for the physical analysis. The original analytical work done at USAMRIID and AFIP by Geisbert & Mullick among others is, for whatever reason, apparently not part of the NAS Committee Review.
One odd thing about Sandia is that prior to this, they had no experience with microbial samples - they mainly worked with semiconductors, nuclear reactor materials, etc. Why would the FBI ask a novice in this area to analyze samples that were mainly prepared elsewhere - and from what source? What happened to those samples between the letter and their arrival at Sandia, and why isn't that part of the NAS inquiry?
Dr. Michael also states in his testimony that there were no weaponized Ames samples to look at from Dugway, and that "no one was weaponizing Ames" - in marked contrast to reports that Dugway was doing precisely that as part of their biological threat assessment work in the late 1990s. It's logical that they would have used Ames, if the resultant powder was to be tested on animals for 'efficacy' (aka virulence), so they could compare it to previous benchmarks. Ames is the standard anthrax vaccine challenge strain, used to test vaccinated animals for immunity in drug trials. (It replaced the Vollum strain in the 1980s - Vollum ended up in Saddam Hussein's hands c. 1985)
For those and other reasons, the following testimony is less than convincing. The most obvious issue is failure to do adequate method validation with standards, particularly with ion beam sample preparation, which could easily have blown off a large percentage of the silica in the samples - especially near the surface.
An equally confounding issue is the sample preparation, which was largely done elsewhere - and why did they get the Leahy powder in the form of glassy clumps (see below)? They also had a "weaponized sample" for comparison - but the means of weaponization is not reported. You would think, for a new forensic method, they'd have a lot more standards - another oddity.
This also raises another question: what happened to all the material in the letters? Were archival samples preserved under lock and key in secure locations, or is it "all gone"?
The media analysis of this has been almost nonexistent. Analysis is out, kowtowing to the grand poo-bahs is in - like this remarkable quote from the New York Times editorial, written by an apparently anonymous author, Feb 27, 2010:
The F.B.I.’s conclusion rests in large part on pioneering laboratory techniques.... The National Academy of Sciences will complete a review of that lab work in coming months. But the techniques were devised with the aid of some of the country’s most sophisticated scientists, so they are presumably reliable.
Appeal to authority is a logical fallacy when it is the mainstay of your argument. Why? Here's a good discussion:
If there is a significant amount of legitimate dispute among the experts within a subject, then it will fallacious to make an Appeal to Authority using the disputing experts. This is because for almost any claim being made and "supported" by one expert there will be a counterclaim that is made and "supported" by another expert. In such cases an Appeal to Authority would tend to be futile. In such cases, the dispute has to be settled by consideration of the actual issues under dispute. Since either side in such a dispute can invoke experts, the dispute cannot be rationally settled by Appeals to Authority.
With that in mind, read on.
-begin transcript-
Chair: Just to remind you that this is an open session on the record, and we do have members of the public and the press with us, and I would like to remind them that the committee's charge is focused on the scientific methods and approaches used during the FBI's investigation and I would also like to remind everyone that they should not infer any opinions or preconceived ideas based on the questions or nature of the discussion coming from committee members, and we're very pleased to welcome our two outside speakers this morning.
Dr. Joseph Michaels from Sandia National Laboratories will be speaking first.
Testimony begins:
So today I'd like to tell you about some of the work we did in support of the FBI in this case. I'd like to acknowledge my colleague Paul -inaudible- he did much of the transmission electron microscopy work in our laboratory... what button do I push here to advance my slides?
Okay - I'll talk louder - very good. Just by way of introduction, what I'll be talking about - first of all, a few of the tools that we use for elemental microanalysis, in our laboratory, in this study. A few words on spectral imaging, because it's a new concept for a lot of people, and how we do it in the laboratory. Then I'll start talking about the attack materials, the Leahy and the New York Post materials with our scanning electron microscope, then I'll talk about Leahy, New York Post, and Daschle with STEM and time-of-flight SIMS, and then I'll answer the question of whether the powders are unique with respect to elemental signature.
- It's unclear what is meant by "unique with respect to elemental signature." The content of silicon is one issue - the presence of trace elements (tiny amounts of iron, tin, etc.) is another - and the isotopic composition of the material as a whole (carbon, oxygen and nitrogen isotopes in particular) is a third. No isotopic composition work was done, however - or at least, no such efforts were ever reported. Nevertheless, the best hope for a "unique fingerprint" is from the isotopic ratios - so why wasn't it done?
First of all, a little thing to mention on signature statistics. We really don't have much to say about variability within the bulk material. Our samples came - the powders that we got were on the orders of milligrams, so it's a small part of the entire amount that was mailed, so really within the bulk material I can't say much. Did we get a representative sample? I'm hoping we did.
More to the point, did they get samples of the powder in their native state, or had they been autoclaved first?
We can talk about variability between fields of view in our microscopes, and then we talk about the variability of signals from individual spores. So I'll be talking about these two variables, in general.
Just a quick review, if nobody has ever seen a scanning electron microscope or a transmission electron microscope here's our SEM in our laboratory, we use it as a test bed for various detector systems, this is the SEM, this is a new type of x-ray detector that we're developing, this is another x-ray detector over here, another x-ray detector over there, so we get lots of signals off this microscope. And over here is our STEM, it's a scanning transmission electron microscope, and I'll tell you a little bit about that in a minute. So, just by way of review again, a STEM or scanning transmission electron microscope, currently we're running about six nanometer image resolution on a modern piece of equipment. We can do microanalysis, that means we can determine elemental signatures down to about the one micron level, we can detect elements down to beryllium, we can do some diffraction work, and really the nice thing about an SEM is you can take whatever you want to look at, sprinkle it on a stub, and image it. The downside to the SEM is of course we can focus our probe to a nice small spot, but really when it interacts, these high energy electrons, with sample, gives us a resolution of about a micron or so in elemental.
Keep in mind that a high resolution image of an artifact produced during the sample prep process is not of much value.
The STEM, on the other hand, the scanning transmission electron microscope, it's a high resolution tool, we can image down to about 0.2 nanometeres with it, in terms of microanalysis we can expect to see about one-two nanometer spectral resolution - so that means if we have a feature that's one to two nanometers in size, we can analyze it. Again, the elements limited to atomic numbers greater than beryllium, difraction, and in this case we need some sample prep, so we need electron-transparent samples.
Generally in this study we're going to make use of the characteristic x-rays generated by the sample - by the electrons when they interact with the sample. So how did we approach this problem? One of the things we've been working on out at Sandia for many years is, how do we characterize materials. It's very important to the nuclear weapons complex, in terms of aging and reliability of the, uh, systems we build. And one of the areas we worked in is how do we comprehensively survey compositions and chemistries in large sample areas?
This is the same basic method used at the Armed Forces Institute of Pathology - and that study showed a very large silicon peak, which fits with the reported "cream or bone" color of the anthrax letter spores. (Anthrax spores are dark brown in their native state).
So, in the old days, I'll say, here's just a picture of some spores - if I wanted to do a compositional analysis, I put a beam here, well that's sort of interesting, I'd move my beam over here, and maybe that's another interesting spot, and then you put it here. So we're doing things very subjectively. We're putting the beam where we think interesting features appear. Now that computers have caught up, become faster, larger capacity - what we need are two-dimensional distributions of chemical phases. And that's why we came up with this concept of chemical component images. And what we do is we make a spectrum image of our sample, and the way we do that is we take our electron beam and we scan it pixel by pixel over our sample. In the old days we'd monitor one chemical signal at a time, in the modern days now we collect the entire x-ray spectrum from that sample from every pixel, so we end up with this nice data cube, where we have spatial dimensions in the x and y, and in depth we have our energy spectrum. So, that gives us an x-ray, a data cube. And so we can go back now and interrogate any pixel we like for any sort of elemental signatures within that data.
The bad thing about this is we end up with tens of millions of pieces of data out of this, and going through all this by hand, it becomes quite tedious. So we began working on statistical tools to analyze these data, these are talked about in these two patents here, notice they were filed June 1st of 2001, and then there's a number of papers where we use this to analyze these various materials of interest to us at Sandia.
So what we do is, here's our data cube, and we use this statistical tool, and what it comes up with in the end is the component images that make up that sample. So, it makes the associations for us, and just to give you an example here, this isn't from the case, this is just a test sample we did, we took a variety of materials like alumina, some iron-cobalt nanoparticles, and some other materials, mixed them all together, and said can we tell them apart?
So what you see here is, the red is the carbon support that we put it on, green is the alumina particles, blue is the iron cobalt, and the cyan is an outlier, it's one we didn't expect to see in there, but it's a calcium sulfur silicate type of material. So we can use this tool to scan the whole area, we don't have to subjectively put our beam here, there and everywhere to decide what we're going to see.
Now, the following section points to a major flaw in their methodology:
A little bit about sample prep, for these tools. The blue were steps either performed at USAMRIID or NBFAC. The green are what we did at Sandia. We're not equipped to handle live anthracis, so everything that came to our lab had to be irradiated or fixed or killed in some manner. So, those steps were done at USAMRIID. For the scanning electron microscope we just take the dry powder, we access the sample, dust it onto a grid or a stub support in a disposable glove bag, and then we can image those directly, either using uncoated materials in the variable pressure SEM or doing some conductive coating if needed to be. We'll talk about the rest later.
"Irradiated or fixed or killed in some manner" is hardly definite. What precisely was done to each sample, and why didn't they compare the effects of autoclaving (which is what the Battelle scientists did with their samples on Oct 17 or 18, 2001) versus irradiation (which is what the USAMRIID scientists did - it's a far less destructive method). Indeed, one could wonder whether Battelle's autoclaving of the samples was a deliberate effort to destroy evidence...
So, what do weaponized materials look like, or materials treated for flow improvement? What we have on the - over here is an SEM image of, I think this is Bt, treated with silica nanoparticles, and you can see this fluffy sort of appearance you can sort of get an idea that there might be a spore there, and over here, and they're coated with this very fine coating of silica. When we look at that in our EDS, our energy dispersive spectrometer, you can see here that we see carbon, oxygen and a huge silicon peak, and just a little calcium associated with the spore bodies themselves. And really, when you coat these particles with silica, it's pretty obvious - in the x-ray spectrum. If we do a spectral image of that, so we scan at fairly low mag, you can see this is a 100 micron bar, these are clumps of spores, not individuals. You can see the various components that come up, you can see the silicon and oxygen, associated with the clumps of spores here, there are other things in here, you can see some magnesium phosphorous type material, and of course some calcium and phosphorous as well - so we see this distribution of elements, but we're not getting any idea what's spatially located inside individual spores- so just sort of an overall look at the data.
The above points towards another serious flaw - how was their "weaponized" material prepared? Was it prepared at Dugway, for example? Did they compare different kinds of weaponization methods, using surrogates? Did they examine what happened to such material after being autoclaved or carved up with their ion beam?
In terms of the attack materials, we had powders in my labs from both Leahy and the New York Post - this is what they looked like. Over here is the low-mag SEM of the New York Post material, and over here is the low-mag SEM of the Leahy material. And you notice in the New York Post are these fairly large chunks - this is a 100 micron bar, so these chunks are quite large - and in fact, they're very hard little pieces of material. In fact, we like to break them up to see more of the internal structure of these clumps. And you can take a pair of tweezers and clamp them on there and kind of click them, to make these particles break apart - so they're very hard, uh, little particles.
This flies in the face of every eyewitness report on the material - which dispersed "like a cloud of smoke" or "like steam from a teapot." How did it turn into glassy chunks? In the autoclave?
When we look at higher magnification, you can see little individual spore bodies, but again, we don't see that fluffy silica coating that we expect from a standard weaponized, uh, sort of material. Again, down here was Leahy material, it was smaller clumps, but we still saw clumps. This is a nice picture, this happened to show up on a book cover, if you've ever seen the book Microbial Forensics. And I was surprised when I bought the book and saw the picture I took in my lab on the cover three years ago. It was a little surprising.
What precisely is "standard weaponized material," again?
At any rate, at higher mag here, we can see the fact that those spore bodies here again are not covered with silica particles, they look as what you'd expect spores to look like - untreated spores. If we put our electron beam on here and do elemental analysis, what we see are some interesting things. First of all, here is the Leahy letter material, and we see carbon, oxygen and a very small silicon peak here. If we do quantitative analysis and again this may be termed semi-quantitative analysis, or just bad quantitative analysis, I put an error bar of plus or minus 50%, because of the nature of the material is not amenable to good quantitative analysis in the scanning electron microscope, as far as bulk characterization is concerned, we sort of get in the 1.2 to 2.3 weight percent range silicon. And if we looked in the New York Post we see the same sort of spectra, not very different from this one up here - and again, the silicon is 1.2 to 1.5 weight percent - not very much.
Nowhere in this does Sandia report what "untreated spores" look like. Appartently, they never even looked at any untreated dried spore material - another basic flaw in the methodology.
QUESTION: [inaudible] the range?
That would be a relative error - yeah, it's not very good. And again, that's because when we do quantitative analysis, our first assumption is that the electron beam intercepts material that's all the same composition, and then we can do our correction factors. In this case, we are hitting void space, we're hitting the calcium rich spore center, we're hitting the silicon material, so it's really hard to do good quantitative analysis.
That's worth repeating - at this micro-nanoscale, their quantitative analysis is poor. Not only that, they're not used to looking at biological materials - this is a materials science lab that seems to spend most of its time characterizing metals, semiconductors, nuclear materials and the like.
QUESTION: I just want to make sure that you're not saying it's 1.2 to 1.5 plus or minus 50%
No, no - relative. Right, right. And again, both samples look very similar in this case, and they look very different from the previous material that I showed you that was weaponized.
QUESTION: Calcium, though, was really low? -[inaudible]- how much calcium was there?
Right there, I'm seeing about three to six weight percent - I don't think that's low, from what I've seen in the literature, actually, in terms of weight percentage. What's interesting is, if we change the electron voltage on our microscope, as we lower the voltage we become more and more surface sensitive, okay, because the electron beam doesn't penetrate as deeply. What you see over here is a result at 20 kV, 15 kV and 5 kV, and these are estimates of the range which the electron beam penetrates at those accelerating voltages, so this is like 3.3 microns, um, we see that as we go to lower voltages, so we're getting more and more surface sensitive, the silicon signal goes from up here and goes down to this black line. So that's a really preliminary indication that the silica and the silicon and oxygen, and I shouldn't say silica, because we don't know the stoichiometry of it, is not on the outside of the spores.
Again, their quantitative analysis is poor - and what is he saying? There's silica inside the spores? Another possibility is that when they sliced their samples up with the ion beam, they blew off the surface silicon - so when they turn up the analytical beam, they see the silicon that didn't get blown off. That's more evidence of artifacts.
Just looking through the literature, here's and EDX spectrum I dug up from this report, and you can see here, carbon, oxygen, a little silicon, phosphorous, and if you go back, very similar x-ray spectra. And again, this paper didn't indicate that they had added silicon anti-foam or things like that to the preparation, so the spectra are not inconsistent with what has been seen before.
Yeah, I actually dug that old paper up - and you know what? They speculate that silicon vacuum oils could have contaminated their preps. That's the best "prior evidence" they could come up with.
If we look at the Leahy material using the spectrum imaging approach, here's our SEM image that's ten micron bar, so again these are clumps of spores, we see that in the spore material, we see the calcium, sulfur, phosphorous, silicon and a few magnesium and sodium and oxygen, and they're all mixed together, so we don't have the resolution to separate out those different materials, different elements at this point, and again we see the support material.
So it is indicating that there is silicon in there, and oxygen, but at this point we don't know where. We have a hint that it might be on the inside.
That hint is spurious.
Just to summarize the SEM work, we know that silicon is present in both of these samples, the ones we have powders of, again microanalysis tells us it is there, but it's not quantitative. The low kV work shows us that it is probably on the inside of the spores, again, the spectral imaging really helps us out with some of these - with some of this information.
Repetition of an unsubstantiated claim... get used to it.
At this point, we knew we needed to go to a higher resolution tool, so this is where we started to apply the scanning transmission electron microscope, and this will tell us more about what's inside the spore bodies. And again now, we took a couple different approaches to specimen prep, again, these were done at USAMRIID or NBFAC later on, I think they're standard, sort of, preps.
You know, this is completely unprofessional - you have to compare different preps, and the specific details matter - a lot. "Standard sort of preps?" What exactly does that mean? Autoclaved first?
One thing is that microbiologists love to use these heavy metal stains so they can see things in bright field mode, we'd prefer not to have them, the uranium in uranyl acetate and the lead citrate, the lead in the lead citrate, cause all kinds of contamination peaks in our spectrum, so we prefer to leave that out. We eventually convinced them not to do that. And we still get plenty of image contrast in the STEM mode, the dark field stem mode.
The other approach we applies was sort of unique. We took the dry powder, we mounted it on a stub, and we took it to a tool called a focused ion beam, dual beam SEM, and made thin samples that way. And this is a tool I'll talk to you a little bit about later. It's interesting, this is some of the first work ever applied to a biological sample back in 2002, and unfortunately we couldn't publish it an now there's been lots of papers on the subject, so this is sort of old hat now. But this was a perfect way to make nice thin TEM samples of even single spores if you'd like to.
A quick look at Google Scholar reveals that artifact generation with the use of ion beams is a serious problem when dealing with biological samples.
So, back to our weaponized Bt surrogate - here's a bright-field TEM images of just the spores sprinkled on a grid. Okay, so this is our first approach, to sprinkle them on a grid and take a look here's the spore body, here's all this silica nanoparticles all around it - so that's pretty obvious, if we then go to a uh, thin section, and ultramicrotome thin section, you can see the spores here and then the silica particles all around the outside of the spore body.
If we do some spectrum imaging of that, here's a spore, here's some of that silica nanoparticles, and if we do the spectrum imaging approach, we now see that the calcium is in the spore body, we see the green phase, uh, as indicated on this spectrum over here, is the silicon and the oxygen, and we see some other things, some calcium, phosphates and things like that in the actual ah, preparation. And this has been published in this paper here.
If we go back now and look at the weaponized material, here's our image, and now here's a component image showing you where this component is, silicon and oxygen, we see that all this fluffy material around the outside of the spores, is located on the outside of the spores itselves. Okay, so that's the traditional weaponization approach - everything is on the outside of the spore.
Again, the method of sample prep is not mentioned - and the only powders Sandia got were chunky and glassy - which indicates that these were the autoclaved samples, not the powders in their native state. Not only that, they didn't get ANY of the Daschle material - only as pre-prepped and mounted samples. Who did that work?
If we now start to look at the attack materials, using the same technique, now this is an ultramicrotome section, this one happened to be fixed and stained, so we do see peaks from lead and uranium in it, so here's an image showing all the stain elements, you have your lead, your uranium, your osmium, this is the actual STEM annular dark-field image, and this is the image we worked from mostly because it gave us enough contrast to see what we are looking at, and we didn't need to do bright field in this case.
They based their analysis on a single image of a pre-prepped slide? What?
When we looked at this, we can see here's the silicon and the oxygen component, if you look closely it's this material here, and that appears to be located in or on the spore coat, not on the outside of the exosporium. And down here is a colorized image showing the different materials shown here, the green is the silica and oxygen, the red is some of the stain elements in the exosporium, and of course the spore body itself.
QUESTION: Can you go back to the weaponized spores? Can you see if it's the exosporium?
Well, if you look the exosporium is out here, and here is the silica - okay?
If we look at the Leahy letter material, a little bit higher magnification, this is again a fixed and stained section, here we see the silicon component, and out here we see the exosporium with the red is the stain, it's got lead, uranium, carbon a bunch of other elements in it, osmium - and down here underneath the exosporium we see the green layer, and that again is the silicon and oxygen associated with the spore coat.
So, they see silicon and oxygen associated with the spore coat, and this is the Leahy material - which had been autoclaved. And the quantitative analysis is poor, as well...
QUESTION: Were the preparations for the surrogate Bt silica and these Leahy samples - were they prepared exactly the same way?
I believe so.
QUESTION: Irradiated then fixed?
Yes. And in fact I didn't say this but we did get samples of the Bt irradiated and not irradiated to start with, compared the two images and really saw no difference from the irradiation.
How did "I believe so" turn into "Yes?"
QUESTION: Joseph, I was trying to understand that sample prep - did they take powdered material and then suspend it and redry it? It looks like there were dehydration steps.
I believe that's what they did - and you'd have to ask somebody from -
Now, he's changed his story again - irradiated? I believe so. Suspended and re-dried? I believe so. You get the sense that this guy has never worked with biological materials in his life before this.
QUESTION: Formaldehyde fixing? They never looked directly at dry samples straight out of the envelope?
Well, we did, but they had to be irradiated first - and I'll show you - that's why we had to do the focused ion beam work - so we're on to two different specimen techniques to compare the data from. So, I'll show you that in a moment.
QUESTION: But these have been in suspension and then re-dried?
I believe that's correct.
Well, that's a good way to reduce the surface silicon content, I imagine... I find this discussion surreal.
Just to bring that up right now, this is the focused ion beam tool, I don't know how much you are familiar with these tools - what we do in the focused ion beam tool is we combine the scanning electron microscope with an ion column, and the ion column is there because we can focus it to a fine spot, and we can micromachine materials with it. So we can actually watch what we are doing with the scanning electron microscope, while we remove nano-scale amounts of material from the sample. It's quite a great tool - it started off twenty years ago in the semiconductor industry and now they make these small dual beams that fit in your laboratory, and if you have $1.8 million dollars you can have one. Sounds not like a lot of money when you say it fast.
Anyway, in specimen prep, this happens to be some pictures that we took when we were preparing a sample from a clump of spores, a large clump of spores, so we wanted to make a sample across here for transmission electron microscopy so we used our focus ion beam to remove this material and remove this material, and then gradually thinned this sample down to electron transparency on the order of 100 nm or less, and here's our finished sample, we used the ion beam to cut it free, on all three sides, we manipulate that out of the hole, put it on a carbon support cell, and then we can look at that in the TEM.
This whole preparation technique takes about an hour, so we can prepare samples quite quickly this way - which is great if you have a semiconductor fab, because they want to know right away what's wrong with their material, or their process, and in this case it it's very nice to prepare samples quite quickly as well. So what do those samples look like? This is one of our first attempts, we just picked a couple of spores, and honestly we could STEM one spore if we wanted to, it's really straightforward. These are actually large features for what we work with.
So, here's a cross-section - we lay some platinum down on top of that, to protect the sample when we make the section, to give it a little bit of mass, and this is the annular dark field image so you can see the spores themselves, we can see the spore coat, and if you look closely there is some indication of the exosporium up here and up here.
What happens when we do the chemical analysis, microanalysis of that? Now we start to see the sample elemental distributions as we expect, we see calcium in the center of the spore, and you can see that here in the spectrum, here's the calcium peak, there''s the oxygen, so that's located in the center, out on the spore coat, again, we see this silicon and oxygen, in or on the spore coat. So, we are seeing the same information using two different preparation techniques - the traditional ultramicrotomy and the less traditional focused ion beam technique.
Let's repeat that: "We see this silicon and oxygen, in or on the spore coat." That directly contradicts FBI claims that no chemical additives were employed, doesn't it? (Bruce Ivins didn't have access to such chemical additives, or to the equipment needed to apply them to individual spores.)
One other point - now we don't have all those other elements involved, the lead, uranium, osmium, we now pick up other trace elements - so here we see tin and iron, out here in the spectrum, and that was consistent in all the spores we looked at from either the, all three mailing materials we had, this is just the blow-up of them. It's not a lot, it is a fairly low amount - of tin and silica - of tin and iron, I should say.
Note that this could be used to distinguish the Detrick Ames spores from the letter spores - but did they ever look at the RMR flask spores? The ones that the FBI claims Ivins harvested and used in the anthrax attacks? What if the RMR flasks spores had no tin or iron?
We did want to verify that with another technique, so we took it to our time-of-flight SIMS, this is a secondary ion mass spectroscopy technique, and indeed when looked at that, it doesn't have the spatial resolution we'd like to have, but it does have enough resolution that we can see the tin, and the ion, in our SIMS spectra. So, this was verification of what we had seen earlier.
QUESTION: Were those previous spectra on the previous slide, were those actual -[inaudible]- spectra, or were those multivariate factors or whatever?
These are the multivariate data that corresponds to this...
QUESTION: And so then, the second study then just reconfirms that your optical method is...
Correct. And we can actually go back now, since we do have a spectrum image, we have the original data from every pixel in this image, we can say now, let's sum all these pixels from around here and see what that spectrum looks like. And again, that reproduces this spectrum quite nicely. So there's a couple internal checks that we do here to make sure everything's working correctly.
QUESTION: Joe, can you give us a just a rough idea of how many spores were looked at with this degree of detail?
Um... on the order of thousands by the time we were done - so we looked at quite a large number of spores, to get the statistics, and I was going to get to that in the next couple of slides, because there's two signatures here that I'd like to leave you with.
One is the fact that we see silicon and oxygen on the spore coat, the other is, every spore doesn't seem to have this signature - so to produce that statistic, we had to look at a lot more data.
Sample processing artifacts?
Okay, here's the New York Post - these are the only two samples that we had powders of that we could do the thin prep of, I think they ran out of Daschle material, or didn't want to share it with us, and you can see again the same thing, calcium, in the spore body itself, silicon and oxygen co-located with the, ah, spore coat, and again we see the iron and tin signature - so that sort of links the two attacks together, maybe, they're very similar on a spore-by-spore basis, if you look at single spores. This is the thin prepared section, looking at many spores, and what you see here again is the same result, we see the calcium in the spores and we see the silicon and the oxygen on the spore coat.
What's interesting here, and what I want to point out to you, is not every spore - if you look at - I don't know, there's a couple in here like this one here - not every spore shows the same silicon-oxygen signature on the spore coat. This was the same result we got from the ultramicrotome sections as well.
QUESTION: What relationship between the amount of calcium and silicon and oxygen in the spore coat?
I didn't see any, in our studies.
QUESTION: When you just said it relates to two preparations, to the exclusion of other preparations of spores...
We had the samples the FBI sent us, which were ultramicrotomed and prepared traditionally, and then we had our focused ion-beam prepared sections. Both gave us the same answer as far as the silicon and the oxygen signature - minus the fact that in some cases with the staining and the osmium, we couldn't see the small features like the iron and tin.
QUESTION: Did you do a control that wasn't these samples? To see what elemental signatures they showed?
We looked at quite a few samples before this, and quite a few samples after this for the FBI that did not show any of these signatures. So...
QUESTION: Did you have an example of that?
Something that doesn't show anything,I didn't bring any of those with me, but we can show you blank pictures...
QUESTION: Many of the preparations don't contain silica?
Most of the ones we looked at don't contain silicon, or the iron and tin, in the same location, co-located, making that association between the two.
QUESTION: And the other samples you looked at, these were the repository samples?
Ah, you'll have to ask the - all samples came to us blind, after the fact we learned what these were, and a few others, and I'll show you...
He doesn't even know if he was ever given samples from the RMR flasks to look at!
QUESTION: The Post material was quite different from the Leahy material...
Well, physically it's different - but it's not different on the spore - if you handed me a spore from the Leahy and a spore from the New York Post, I couldn't tell you which one it came from.
QUESTION: Yeah, that's exactly what I'm getting at.
Right. And - this was another point I wanted to make, and along those lines - I'll make it later - is, if this silica had something to do with the material's ability to be aerosolized, one wonders why is the Leahy clumped up in big clumps, and the other ones are not, since they all look the same? So to me that's sort of an indication that this has nothing to do with aerosolization or intentional, uh, acts to make it more aerosolizable.
QUESTION : Right, but -[inaudible] is very interesting -[inaudible]- spores - in other respects, preparations - it conceivably could have been prepared differently?
Yeah, we were able to tell the FBI most of that - probably March 2002.
QUESTION: Early on, when you showed your low resolution data, you had the estimate of the amount of silicon. I assume that in the Leahy letter, that actually as I recall matches what they found by bulk analysis. Is that generally what you have found, and did you - when you received samples, did you have all the information as well, the low resolution - was roughly in the same ballpark as the STEM, or...
We had no information other than, "here's your sample, tell us about it" - so we knew nothing - and in fact, I only heard that number, what their bulk analysis was, was at the ASM meeting this winter. That was the first time I heard their actual numbers. Umm... one point I'd like to make that I think was important, Tom Geisbert at USAMRIID sent us on this chase to find vegetative cells during the spore forming process - he said, there's a few in there, in a grid, and go look for them. Well, it took me three days to find one, at this level, but here is a mother cell in the process of forming a spore, and if we do the chemical analysis what I'd like to point out, we are already seeing silicon and oxygen on that spore coat, in the mother cell. Okay, so...
These "vegetative cells" could have been a Bacillus subtilis contaminant, something picked up during the growth process. Or, they could have been anthrax strains deficient in the ability to sporulate - or simple vegetative anthrax cells that hadn't had time to finish sporulating before being harvested. None of that seems to fit with the Detrick RMR flasks.
QUESTION: What else could be....
Well, I believe that this, and I've been told this to be true, this is the cell..
QUESTION: It's not an exosporium?
Well, I don't believe so because there's another chain down here...
QUESTION: That thing down there - well, I don't know what that is.
Well, isn't that the section through a string of these things? You remember, this is a thin section.
QUESTION: -[inaudible]- A chain of cells halfway through the process?
And even then we see the silicon and oxygen signature on that spore, okay? We only found one of these, since I spent three days finding this one, and we sort of stopped at that point.
QUESTION: So the fact that you see the silicon inside a vegetative cell is what really allows you to conclude that this is probably a natural process?
One, one of the things, sure, right, I believe that's true - and by natural, we can argue what natural means all day long.
This may be the most ridiculous statement in the entire talk - possibly.
QUESTION: Such as mineralization?
Right, right, umm... Just a comparison of the Leahy material, the Daschle, and the New York Post - from - In terms of the silicon and oxygen signature, again, the tin and iron, and here we see the iron but our tin is obscured by, I think it's an osmium peak, and again there's that point - the silicon and oxygen layer does not seem to prevent the New York Post powder from clumping, okay.
Well, that's because it was autoclaved, most likely.
QUESTION: Joseph, you mentioned in passing anti-foam agents that were used. Could silicon-based...
I believe they are silicon-based...
QUESTION: What kind of materials are those and what kind of molecular weights... do you know what they are?
You'll have to ask someone that grows bacteria.
QUESTION: Yeah, we got a few of those... We used them long ago but we don't use them now but I don't remember what the form of the silicon is.... Could be a silane of some sort.... But there are small molecules, silicon based materials that... If you're shaking large cultures of bacteria, you tend to get foam building up... Especially in fermenters, where like, you're bubbling air through... It could be a surfactant like material... Yes, that's an interesting point. It suggest that growing them -[inaudible]-
Damn it, didn't any of them read the "reference paper" this was based on? They suggest that it was the silicon vacuum oil in the centrifuge that may have contaminated their samples.
I think we looked at a lot of samples, with various preparations - and sometimes they mentioned they used anti-foams and sometimes not - and it wasn't - we couldn't correlate this sort of appearance with the use or not use of the anti-foam.
QUESTION: So, along the same lines, have you looked into any other Bacillus species, spores?
Yeah, I've looked in the literature...
QUESTION: -[inaudible]- have seen all this, kind of silicon, -[inaudible]- similar spores, have you seen those things?
We certainly don't grow our own spores and our own samples, so we were dependent upon whatever the FBI gave us to look at.
And that's a major problem, isn't it?
QUESTION: -[inaudible]- You have not seen anything else?
Right. I have not. But I do have, and I'll show you some literature, from the eighties, when people have done this sort of experiment on other species and shown the same sort of things.
QUESTION: I'm reading ahead - it says there tin is somewhat -[inaudible]- by the stain. Are there other elements that might have been obscured by the process?
When we actually made our - that was just referring to the Daschle material, because Daschle only came to us as pre-prepped ultramicrotomed fixed and stained sections. So they always came with the uranium, the osmium, the lead, the tungsten - all these nice heavy elements that the biologists love to stick in their samples that we microanalysts hate to see, because they have this huge range of characteristic peaks that obscure lots in the spectrum. I think, you know, as an aside, you microbiologists just might want to consider STEM instead of bright-field TEM because in STEM, you can see lots of the features without staining, a little aside.
-[inaudible]-
The question of spore count came up, how many spores we looked at, this is just our estimate of what the error bar would be if we looked at a total of N spores, and X showed a particular chemical feature, what would be our confidence, our 95% confidence level. And you can see if we look at ten spores, we really have a difficult time putting a confidence level on there that is small. up a hundred spores and a thousand spores, we bring that confidence level way down, so with this data in mind we went off and started doing more statistics, and this is the result of that - this is the number of spores we imaged, the number with the Si-O on the spore coat, or in the spore coat, and this is the percentage. So, the top one's Leahy, the next one's Daschle, and the next one's New York Post.
So all those three materials had quite high numbers of spores that showed the silicon and oxygen signature. When we looked at other materials - this is a sample produced I believe at Dugway, we saw things like this, 26%, 11% and 29%, this is RMR-1030, I'm sure you are familiar with that flask, now that was another one grown at USAMRIID, it showed about 6% of the spores with the same signature, and when we get down here to RMR-1029, we saw no silicon signature on the spore coat.
Again, they did not prepare these samples themselves. Who did? And doesn't this contradict the earlier claim about samples coming to them blind?
QUESTION: So,um -inaudible- level 0402055 level 258 - are those - do we know much about those specimens?
I have some of the data on how they are grown, I do not know what levels 2, 5 and 8 mean. I believe they were from - I think what they were trying to do was take a capsule and section different parts through the material that's been spun down into it, but I'm not sure of that. You'd have to ask the people who made the sample.
QUESTION: I thought you were saying that you hadn't seen any other spore preparations that had silicon...
I think the question was other species?
QUESTION: Oh, I thought earlier, I thought Richard had asked you were there any other spore that showed a silicon coat, and you said you looked at thousands...
Well, maybe I misunderstood the question.
QUESTION: Maybe I misunderstood.
We have seen, as you can see here, and I'll show you some of this data. We did see other samples with similar - on a spore basis, similar appearance and similar signatures, but again when you start to look at the statistics, they're not the same sort of account, okay.
QUESTION: Just so I understand - the RMR-1029, the presumption is that was the source - or some of those three samples at the top - so that would imply that those were a culture, to get them to the point where they actually had some kind of silicon -[inaudible]-
That would be my understanding of it was well.
QUESTION: Because the primary culture or the stock culture did not have any - anything - but that was a liquid culture, that presumably had to be washed and dried and prepared, right?
I would expect that to be true.
Just an image from RMR-1030, here's the spores, a little bit dark, but you can again see the silicon and oxygen signature on the spore coat, very much like we've seen before. This is that sample from Dugway, this is the one that had different levels. Again, here's our sample, and here's the spores that had different - that had the silicon and oxygen on the spore coat, and again, here is the same signature that we saw. And again, note the variability here. We see a couple spores in this field and we see a lot of spores in this field that have the silicon and oxygen on the spore coat. And as far as I can find out, this is Ames grown via fermentation at Dugway using a L-D media. That's all the information I had about that sample.
Another sample that came to us in May 2008 was quite fascinating to me, these are the numbers, I was told the sample was to be described as 'evidence.' That's all they gave me, and that's all they would give me about this sample, but I think this is a fascinating series because you can see up here, I should also add we've developed better techniques so we get better statistics over the past eight years - when we first started doing this, it took us two or three minutes per spore, to actually do this analysis, now we're down to about 0.2 seconds per spore, so now we can really start to build up statistics. And that was with a nice grant from DHS that we managed to pull that off.
If you look at this series of samples, though, we've analyzed quite a few spores, the interesting thing is here, this would be a great sample to mine for conditions to see why it went from 18% of the spores with the silicon and oxygen signature, down to 1.2. And there were others in this series that had nothing, in that batch of samples. So, just to bring this to your attention, this is a great sample that you might want to pull the string on a little bit if you can.
QUESTION: Pull the string - what are we pulling for?
I don't know anything about these or where they came from. Okay - and if they know the growth conditions, you may be able to associate this change in silicon content, or silica - the number of spores with silica - with the growth conditions.
QUESTION: It could also be a calibration curve, where two things are being mixed, to see if it scales linearly?
I don't know what it was.
QUESTION: Many of these also have the tin and iron signatures?
I don't think we ever saw tin and iron again in these. We've seen it in other sample's that we've gotten from a DHS study that we published in that - the reference I gave previously when we looked at the weaponized material. In some cases we've seen tin, and iron, in those materials as well. And again, that's documented in one of the publications we've put out, and I think it's in your publication list.
QUESTION: I've probably confused myself - so the - the Dugway, and one of the -[inaudible]- the actual Leahy -[inaudible]- even though the preps were superficially different, it was a common chemical signature, that was the tin?
The tin and the iron and the silicon, and the oxygen, on the spore coat.
QUESTION: You've shown silicon in other, unrelated spore preparations, so it's not distinctive to the, uh, materials that were used in the attack.
That's correct.
QUESTION: But the tin?
The tin was very distinctive in this case. And that was...
QUESTION: So the tin...
and the iron, they're very small peaks, they're not...
QUESTION: The tin, and the iron, were found uniquely, or distinctively, in those preps and not unrelated preparations.
JM : Although we did, like I said, we have seen tin in some other preps that we got from other laboratories, in an unrelated study for this, or a sort of related study, but again those samples though didn't have the silicon and the oxygen in the same way,so.
QUESTION: So if you take all of these...
JM: These were unique.
QUESTION: ...elements, you've got a certain profile...
JM: Correct.
QUESTION: ...that is characteristic of the attack preparations, and not other preparations that you've seen?
JM: That's correct.
QUESTION: Does this technology allow you to get a sense of the ratios of isotopes, so that - what I'm thinking of is, with the minor elements, from different parts of the country with different isotope compositions, that it may be possible for example to track down something? - [inaudible]-
Great question!
JM: One of the problems is that the amounts of the material are quite small, so you can go back and look at that SIMS data, and you can see the isotopic ratio in the tin peak, for example. It looks very much like a naturally occurring - what you'd expect from nature for tin. Trying to geolocate from that data would be really tough. It wasn't something we thought about at the time because the signal was so low. It really doesn't lend itself to that sort of work - and it's not the right tool to use for that anyway. There are better tools to do that sort of analysis in.
Translation: No, we didn't do any isotopic analysis. Remarkable, considering that that would be the best "fingerprint" option - and you need tiny amounts of material to do this. You don't look at the tin, you look at the carbon and oxygen. Seems like a high level of ignorance here.
QUESTION: -{inaudible]- the silde of RMR-1030 and RMR 1029 - the level, 2, 5, 8, 40255? You said that was from Dugway?
JM: Yes.
QUESTION: - And was that examined under SEM or STEM to see whether that was silicon on the coat or in the exosporium?
JM: That was - this data right here. So, again, if we look closely it's on the spore coat.
QUESTION: And on those samples, did you find tin and iron?
JM: No we did not.
QUESTION: The tin and iron were close to the detection limits, I recall?
JM: That's correct.
QUESTION: How close to the limit? -{inaudible]-
JM: Actually, um, down here you can clearly see their peaks. I'm guessing that these peaks are three or four times our detection limit.
QUESTION: Would you see those routinely on different spores? -[inaudible]- like silicon?
JM: If the silicon and oxygen were co-located with the spore coat, we saw the tin and the iron. The reverse was not true.
QUESTION: And the Ames strain, which you just showed us a moment ago, -[inaudible] - we know that never had silica? Or do we know anything about what that ever had?
JM: I don't know. That's the number, and you can query the FBI on that question. My presumption is it didn't, because they gave me this information about it, and they didn't say it had been treated in any specific way.
QUESTION: Did you have the opportunity to do the same assessment on multiple specimens that had been weaponized?
JM: Yes, we did. And that's - of course, it wasn't Ames, because nobody's weaponizing Ames, it was always a surrogate. Just a comment about how we could get all these large numbers of spores, in this example. We transferred the STEM technique from the expensive STEM instrument to the slightly less expensive SEM instrument, developed a new type of X-ray detector during the eight years we were doing this, and this was under DHS funding, and that enabled us to do extremely large fields of view, you can see a couple hundred spores in one image, then we can go ahead and find the ones that contain silicon on the spore coat, and count those up quite easily, and here's a blow-up of what one of them would look like. We went back and validated this technique against our previous STEM work with the same samples and got the same answers, within statistics. So this was a nice added thing that we added with DHS funding, and this allowed us to go from two or three minutes per spore down to a couple tens of a second per spore in analysis time.
Dugway was weaponizing Ames for use in the biological threat assessment program - that's clear enough.
Just a couple comments on previous studies that are out there... so this is some work done by Johnston back in 1980, previous generation piece of equipment, not quite as high-resolution as what we have today, here's their specimen prep technique, and here's their background signal, this is an X-ray spectrum, here's the spore coat, and we go right there and there's a nice silicon signal on the spore coat, and again, resolution allows that to go over to the cortex, and we don't see that signal in the core. So again, this is an example from Bacillus megatherium. So, it's a different species where they actually saw silicon on the spore coat. There was another study, this is the Stewart study, back in July of 1980. These are their images...here is their silicon signature... here is the spectrum... we actually had this sample in our laboratory, and were able to go back and analyze this same sample... and again we saw this same result, just a little higher resolution. So there are multiple examples from the literature where they found silicon. On both of these examples, none of them mentioned any silicon antifoam agents, that I could find.
QUESTION: Is there any -[inaudible]- why there's -[inaudible]- in the same preparation?
JM: Yeah, they made the statement that the variation in silicon, both within and between spores, they saw this same variation that we saw, and they marked it down as an observation, and they also did two different kind of preps... and saw the same result again. So they were saying it wasn't the prep technique... maybe pickup from glassware, or something like that. Something in the environment. And both people made the same sort of observation - both papers I cited. So I'd like to stop here. I think the conclusions are:
*The New York Post, Leahy and Daschle materials are basically indistinguishable, elementally, at the spore level, and they both have a similar fraction of the spores with the silicon and oxygen in or on the spore coat.
*We found the silicon and oxygen signature on an endospore in the New York Post material, which leads me to believe that this is something that is happening during the spore formation process, not something that's added later on as an additive, once the spores have been formed.
*I believe that the letter powders are not unique. We've seen examples in the literature and examples from other samples grown for the FBI that show the silicon and oxygen signature - they do differ, though, in the percentage of spores that have the silicon and oxygen on the spore coat. And finally, I think that STEM and SEM are really the two tools that really helped us out in this study. I'll be happy to answer any questions.
QUESTION: That was really good.
JM: Thank you.
QUESTION: It looks like you've done a lot of work on addressing the sampling statistical problem with respect to the portion of the sample that you're looking at - lots of numbers of spores of the particular portion that's being analyzed - but, because some of these samples are grossly heterogeneous, I'm wondering if there were also efforts to sample different, distant parts of "the sample"? And then, the same kind of statistical, intra-portion analysis of lots of spores from that section as well?
JM: We didn't make any specific effort to do that. We were given I think 1.4 milligrams of Leahy material and about 14 milligrams of New York Post and were told to be careful how we used it... So, that is a subsample, so we really can't say much about what was in the vial... But the samples that the FBI had prepared at USAMRIID or NBFAC presumably came from another part of the vial, because we had that part in our lab. So, maybe that an indication that these are fairly consistent throughout the bulk of the material.
QUESTION: Although I guess we can't say. It may be that certain parts of a heterogeneous sample are more amenable to either picking and processing, etc. so it may be biased in a way.
JM: It may, but that's going to be a tough one for us to answer.
QUESTION: Related to that, -[inaudible]- sample preparation? do you generally do that on most samples, or just a few samples...
JM: We did that on a few samples, we didn't do that on most of them. Generally, one of the reasons we didn't do it on most of them was because they came as ultramicrotomed thin sections - so we didn't have a lot of powders that came to the lab. Most everything came already prepared for us.
QUESTION: So when you did that, it was mostly ion beam -[inaudible]- attack samples, and then a few samples early on?
JM: That's right. We did a few samples early on as well. I think we probably did have some powders early on of Bt and didn't see signatures of.
-end transcript-
