Perils of Electron Microscopy

I do a lot of work with electron microscopes. This is amazingly neat stuff, every bit as fun as I imagined it would be, and perhaps the closest thing that biologists have to being astronauts. I get to play with exotic, highly expensive equipment, and I get to see alien things that nobody has ever seen (or, in some cases, dreamed of) before. Twice in the last six months my lab has had a visiting scientist come to do work that is right up my alley, and since I am checked out on the microscope and she is not, I got to "drive" (the term that we seem to use for operating the 'scope) with her giving me instructions. It was very illuminating, seeing what is pertinent to our study and what can be skipped over, and at the same time there were several moments of both of us sitting and gaping and asking each other, "What on earth is that?!" It is fantastic stuff.

There are several kinds of electron microscope. The one that my lab uses most is the transmission electron microscope (or TEM), which operates in a very intuitive way: it works just like a regular light microscope, except that instead of shining a beam of light through the specimen, it shines a beam of electrons. The electrons are focussed using lenses, and (just as with the more expensive light microscopes) the images are captured by a camera. Of course, there are other significant differences as well: the lenses are in fact magnetic fields, and because electrons do not travel very far through air, the inside of the microscope is pumped down to a high vacuum.

Specimens for a light microscope are generally mounted on slides. This means that the specimen, usually aqueous in nature, is dropped onto a glass slide, over which is placed a coverslip. If the specimen is important enough, and stable enough not to degrade on its own, the slide may be sealed using Vaseline or fingernail polish, which can preserve the specimen indefinitely, but otherwise, that tends to be it. Of course, there are often also specific ways of preparing specimens, but I will not get into that right now.

Specimens for a TEM are analagous. The specimens are mounted not on slides but "grids", which are little (3 mm diameter) copper circles with a slot-shaped hole in the middle. (Yes, they are not actually gridlike: this is a historical term, used because other grids actually do have mesh gridwork in the holes. Many labs use those kinds as well, but my lab has so far used only the other kind, called slot grids.) The specimen is actually mounted on a very thin plastic film, on the order of 40-100 nm thick, which is suspended in the middle of the grid. Like a well-preserved slide, the specimen will last indefinitely.

There are of course other differences, many of which are obvious upon contemplation, and some of which require further explanation (which I may get to in other posts). However, one difference that is significant here is that electron microscopes (all of them, but particularly TEMs) are not just sending a beam of light through the specimen: they are shooting charged particles, which can and often do interact with the specimens. Electrons can be really harsh! In the case of my recent work, for which I was using very thin films (40 nm or so), the electrons can rip right through the specimen. This means that -- often right after discovering the perfect example of a cell amongst dozens of unusable contenders -- one can watch one's precious work tear apart, wrinkle, and wither away before one's eyes. No amount of preparation can prevent this: it just happens. Thicker films are less prone to this sort of damage, but they also impede the electron beam more, and so lose resolution. Resolution is the reason why we use electron microscopes in the first place, so we tend to use the thinnest films that we can. Obviously, I need to experiment more with this, to find a better film thickness that will not break apart in the electron beam, without losing too much resolution.

There are numerous other ways in which TEM specimens can be irreparably lost. One of the most frustrating is tweezering -- putting a hole in the film with clumsily handled forceps while moving the grid around. I have had much hapless experience with that. Another is that some of the stages (the devices that hold the specimens inside the microscope) hold the specimens in place with spring clips, which can and often do tear through the films when removed (or, on occasion, when put in). There are ways of minimising this, which I have only recently become competent at.

Unfortunately, "recently" means that I had lost several specimens while learning how to handle, mount, and dismount them. Up until last Friday, I had only one pristine specimen left, undamaged by my apprentice clumsiness. That specimen, last Friday, self-destructed in the 'scope, as the film gave way spontaneously. This is a setback, of course, but I can always make more specimens. This is of course how science works. Only now, I will try thicker films!

The Commandments of Science

I have been reading blogs more than is good for me (and I must admit that writing one is not something that I have time for, either), but every so often I come across something that reminds me that, however much time I may spend on them, I am not (entirely) wasting my time by reading blogs. This example was not a post on a blog: it was a comment. The comments section of a blog post is often more enlightening than the post itself, something not realised by people who only read what shows up on their RSS reader. This particular example was on a post on EvolutionBlog on an interview on Fox News discussing Ken Ham's new Creation Museum. The interview itself was somewhat predictable, and not very noteworthy; the post was mainly there to allow those of us unable or unwilling to watch Fox News see how each side presented itself (a service which is in itself much appreciated!). A commenter called Hoary Puccoon, however, wrote a truly remarkable piece. Since I suspect that this is a pseudonym, and since no Web site or blog was given, I fear that this will fall into undeserved obscurity. (I recognise that my own blog is not likely even more obscure than the comments section of a far better established blog, but at the very least this renders it more prominent to me, and any further dissemination that it can achieve is good.) This is the complete comment, with nothing changed except the formatting.

The whole is-there-a-god thing bores me. But why doesn't anyone emphasize how ethical science is-- and how unethical the creationists look to scientists?


The Six (and counting) Commandments of Science


1. Thou shalt not lie. Fudging data is a mortal sin, enough to terminate one's career.


2. Honor thy fathers. You must give credit to the previously-published work of other scientists.


3. Thou shalt not bear false witness against thy neighbor. Misrepresenting another scientist's work merits public exposure and condemnation. (The creationists never understand just how immoral their quote-mining seems to scientists.)


4. Love thy neighbor. Ad hominem arguments are not acceptable in scientific discourse.


5. By their works ye shall know them. If Linus Pauling is a legend and Watson and Crick are complete unknowns, whose model of DNA is accepted? W&C's-- because theirs is right and Pauling's was wrong.


6. Let your yeas be yeas, and your nays, nays. Scientists must define their variables explicitly, and not fudge and say, 'oh, I really meant something else' if their hypothesis is disproven. (This is actually why theism versus atheism doesn't much matter in practicing science. God, whether he/she/it exists or not, is too fuzzy a variable to produce clear results.)


This could probably be worked up to ten commandments, but the point is, scientists, whether theists or atheists, do have strict rules of ethics-- which the creationists constantly violate. This point needs to be hammered into the public discourse. The creationists aren't just getting a few dry facts wrong-- they are undermining the entire ethical basis of science. Letting the fundies get away with claiming they represent morality is, in my opinion, morally wrong.

While obviously aimed towards the creation/evolution controversy rampant in the USA, and something that the creationists would do very well to read and understand, it is also absolutely right about how science works. The only change that I would make is one of ordering; some of the more important issues in discussing creationism are almost trivial (or taken for granted) in discussions of science. Each of these points deserves further elaboration, here in an order that makes more sense outside of the creation/evolution discussion.

1. "Thou shalt not lie." This is absolutely binding in science. There is in science, as anywhere, room for cynicism, but never when one reports data. This is one of the reasons -- the main reason -- why most papers have separate "results" and "discussion" sections. The "discussion" section is interpretation, and there the scientist may be dead wrong (although s/he must support every significant assertion with data, either hir own or someone else's through citation), but the "results" section is pure fact. It is for this reason that scientific data are never published without an accounting of how the data were acquired. Should the data in fact be wrong, repeating the experiments or observations that produced those data will show that. Scientists demand transparency and accountability. Anything worthy of inclusion in the scientific canon must pass through editorial and peer review before publication, the latter process being undertaken by specialists, often the reporting scientists' competitors. Because of this, it is very hard to get away with a deliberate tampering of data (although it does -- very occasionally -- happen). Incorrect data resulting from a misreading of experimental results, or from the application of an incorrect analysis to such results, are generally dealt with graciously by both the discoverer and the scientists reporting those incorrect data. Should something -- anything -- in the "results" section prove to be a conscious fabrication, however, sooner or later, someone will find out, and their perpetrators' careers are over: nobody will take them seriously again. Scientists are unforgiving of frauds.

   
   It is worth noting here that faked-data scandals (including the creationists' favourite, "Piltdown Man") are invariably brought to light by scientists. It is for this reason that science is called "self-correcting".



2. "By their works ye shall know them." There is a status system in science. This is based, among other things, on credentials, networking, position, seniority, and awards. Status in science is never inherited or bought or bestowed. Everyone in science must work for their status, producing and interpreting data and hypotheses that withstand the most exacting scrutiny. Status ultimately comes from one's ability to do that work. Since anybody -- even a high-school dropout -- can challenge any piece of work (assuming that they have novel interpretations or data, that these make sense, and that they are articulated intelligibly), science is in a sense the ultimate classless society. In other words, one must prove oneself, but anyone and everyone is given the opportunity to do so. If proving oneself means showing that a highly-regarded scientist is wrong, so be it. Science values truth* more than status.


3. "Thou shalt not bear false witness against thy neighbor." This actually follows from the first two points. One must be honest, and one must acknowledge one's sources honestly. Scientists take a dim view to having words put in their mouths. Intentionally misrepresenting others' work is not as bad as making up one's own, but it is still a grievous breach of ethics. For the most part, it does not happen in science, but when it does, it is quickly established as such, and is thereafter ignored.

   
   Unfortunately, this is not how mass media operates. Non-scientifically-trained writers and editors tend to take things out of context, or to reword things in manners that they may think are paraphases but actually have significantly different meanings scientifically. Corrections in the popular literature often go unregarded, so that a single misinterpretation may plague a scientist for the rest of their career. (The classic example of this is the tendency for creationists to use quotes by the prominent evolutionary biologist Stephen Jay Gould out of context to suggest that he did not believe that evolution was a real thing.) Given the complex and often-conditional nature of their work, many scientists simply refuse to discuss their work with the media. The irony of this is that scientists in general value communication -- which is why they tend to be eager to publish and to teach.



4. "Let your yeas be yeas, and your nays, nays." Although it may not seem that way to the uninitiated, science demands clarity and simplicity. Much of scientific jargon is essential because it is unambiguous; new terms are always carefully defined before they are used. Of course, words do get redefined, or used in different ways by different scientists; but in such cases, the scientists are always careful to indicate the meanings that they employ.

   
   Scientists are expected to stand by, and to defend, what they present to the scientific community. They may, and often do, change their minds, but they lose respect if they say that they really meant something that they did not say. To use a metaphor often mentioned in the creation/evolution debate, the goalposts may not be moved. Once terms are agreed upon, they may not be changed, and if an explanation fails to account for any given phenomenon under those terms, it must be discarded. The only exception to this is when additional data can be produced to account for the phenomenon under question.



5. "Honor thy fathers." Citing others' work goes beyond courtesy. If one did not acquire a given datum, one should acknowledge its source. This allows for the data supporting original ideas to be investigated, to determine whether or not they actually apply to the new idea. If one is arguing for or against an idea already articulated in the scientific literature, one should indicate whose idea it is, which allows for arguments both for and against older ideas to be interpreted in the context of the ideas' original construction. It also allows for those whose ideas have been misunderstood or misinterpreted to present their arguments explaining that.

   
   All of this pertains to an aspect of science not commonly appreciated amongst the lay: it is a social enterprise. Many scientists are intensely competitive, but all acknowledge that, ultimately, science is a collaborative process. Everybody has access to, and can use and interpret, everybody else's published data. Progress cannot be made otherwise.



6. "Love thy neighbor." This is actually not that important in science itself; it is more a question of style than anything else. The expectation actually goes further than this, though. In the scientific literature, one never refers to others except through their publications. All that matters in science is science; the personal lives of individual scientists are irrelevant. Scientists have friends, but friendships are acknowledged at most in the (invariably brief) "acknowledgements" section of scientific papers. Everything else in a scientific publication is regarded as timeless, outside the scope of personal activities and allegiances, and anything that does not directly affect the work being published is omitted. Such matters impede papers' being relevant and understandable indefinitely, which is as close to immortal as any scientist can hope their work to be.

I am fairly certain that I will want to re-order these, probably soon after I hit the "post" button. (Arguments about this order will be gratefully accepted!) I make exceptions for the first and last points, which will always be first and last. But overall, it is a good list (and much tidier than mine in its original presentation). I will be happy to see it get more widespread dissemination.


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* "Truth" is a somewhat dangerous word to use in descriptions of science for the layperson. In science, "truth" is always provisional, "proof" always conditional. This is not to say that scientists are necessarily postmodernists; indeed, I would argue that science is inherently opposed to postmodernism. Rather, scientists accept that the world around them is a real place, and that it operates in a consistent fashion. What scientists regard as "true" is their best understanding of how independently verifiable observations and/or experimental results fit with one another. New observations or experiments may require the re-evaluation, and sometimes indeed the replacement, of established understandings, but such understandings did not become established without having already proven themselves consistent many times over.

The Meaning of the Word "Opisthokont"

The name that I have chosen for this blog is bound to be an unfamiliar term for most people. It probably represents an unfamiliar concept as well, so I will start gently. It is a term that denotes a specific, large, and (to humans) very important group of organisms. The importance of the group is probably understandable once one understands that humans are opisthokonts. So, however, are all other animals. So are all fungi! Rounding out the group are a number of single-celled organisms known to be related to either animals or fungi or both. Notably absent from this group are plants, algae, and quite a few other organisms, including all bacteria.

What can animals and fungi have in common that plants do not? Well, think of a sperm cell. This is a tadpole-like thing, with a roughly spherical cell body and a single, tail-like flagellum trailing behind. The cell swims by wiggling its flagellum, again much like a tadpole swims by wiggling its tail. As it happens, this is a very unusual cell type. Most other flagellated organisms swim with their flagella in front, pulling themselves through the surroinding medium. Only the flagellated cells found in animals, fungi, and related microbes swim with their flagella behind. This gives rise to the name: "opistho-" means "behind", and "-kont" refers to the flagellum.

One could easily be forgiven for finding this a minor difference, given everyday experience. To most of us, animals are things that move around and eat things, and plants and fungi are rooted in the ground. However, there are animals that are rooted to the ground as well (including sponges, corals, and sea squirts), and animals that do not eat (such as some of the worms living near deep sea hydrothermal vents). There are fungi that do not grow in the ground (yeasts are fungi, for instance, and do not root themselves in anything). Everyday experience, it turns out, is insufficient to categorise life; science has moved well beyond that.

Science has taken its time to get to where it is today, though. Decades ago, fungi were classed amongst the "lower plants" because their cells are surrounded by rigid cell walls, a characteristic then thought to define a "plant". However, it has since been shown that the materials that make up those cell walls are completely unrelated (they are derived from sugars in plants and from proteins in fungi, for instance). More importantly, the organisms indisputably most closely related to each, which look like single-celled versions of their better-known counterparts, lack any vestige of the cell wall. In other words, the common ancestor of plants and fungi did not have a cell wall; this character is a homoplasy, something that evolved more than once in the history of life.

Genetic analysis confirms this. Most hypotheses of evolutionary history (of extant organisms, anyway) are now made by having computers analyse the DNA of comparable genes from different organisms, and these tend to connect animals to fungi, to the exclusion of plants. (There are exceptions -- there are always exceptions -- but those are from genes with a lot of evolutionary "noise". In other words, such genes are either not large enough or evolve too quickly to retain enough information to resolve the animal/plant/fungus relationship with any reliability. There are statistical tests that indicate the trustworthiness of these computer analyses, and those which are judged acceptable almost always support the close relationship between animals and fungi.)

Analysis of genes goes beyond using them to reconstruct evolutionary history directly. For instance, there is an insertion into one of the genes used in the replication of DNA, an extra stretch of about fifty nucleotides (the "letters" of DNA's "alphabet"), which is found in animals, fungi, and their close relatives, and nothing else. This might not seem particularly important, but the gene in question is important enough that it is not prone to change easily (in scientific parlance, it is "evolutionarily conserved"), and perhaps more importantly, the insertion is itself conserved. In other words, the same nucleotides (or some obvious derivation of them) are present in the same place in all opisthokonts.

Non-genetic data helps link the two groups as well. The architecture of individual cells in the single-celled relatives of animals and fungi is strikingly similar, both inside and out. This was not apparent until the advent of electron microscopy; many of the features that link the two groups are either too small to be seen with a light microscope (the "regular" kind) or are easily overlooked in favour of other, more striking features, many of which (like the cell walls already mentioned) can be taken to imply connections that do not hold up when investigated through other techniques.

These features include the arrangements of the components of the cytoskeleton, a set of protein-based rods and tubes that gives a cell its shape. The arrangement and replication of the flagella is also thought to be a conserved trait. A substantial part of my graduate work is investigating these things; while the coherence of the opisthokonts as a group is nowadays almost beyond question, the uniqueness of some of its defining characteristics is simply not known. Electron microscopy has not been around long enough for much data to have been generated, and most of what has been observed focuses on a few well-known organisms. Those organisms that can tell us the most about the relationships of living things are often obscure and poorly studied, a situation that holds perhaps nowhere more strongly than in this case.

But there is one feature that is readily observed and consistent, and that is the number and position of flagella. Like I mentioned, most organisms have flagella at the front ends of their cells, and pull themselves through their surroundings with them; opisthokonts are unusual in pushing their cells through their surroundings. Furthermore, most non-opisthokont cells have flagella that appear in twos, or are obviously derived from ancestors that had flagella in twos, while all opisthokonts' flagella appear without any others associated with them. These may not seem like significant things, but one must bear in mind that, when discussing the divergence of animals and plants and fungi, we are discussing the evolution of single-celled organisms. In that context, seemingly unimportant things like the position and number of flagella can be highly significant.

So, classifying something as an opisthokont is not a natural thing for most people. It may seem like an obscure and unimportant distinction. Classifying animals and fungi as each others' closest multicellular relatives has (so far) no known consequences to medicine or agriculture or anything else that most people would notice. But the opisthokont hypothesis is, as far as we can tell, an accurate description of the relationships of living things: it is our best understanding of the relevant facts, and the closest that science can come to the truth.