Confocal microscopy

Back to stuff I should know, in theory, at least.

I had my first microscope as a child, it was a small one but I had a great time looking at little creatures that lived in dirty pond-water. I remember spending a long afternoon trying to see transparent single celled animals, and finally getting the lighting just right to see an amoeba. I also remember trying to immobilise a tiny worm with white spirit - it exploded, but I like to think it died happy.

This week I shall mostly be talking about 'confocal microscopy', this is a type of light microscopy (as opposed to electron, infra-red, x-ray, scanning tunnelling, or atomic force microscopy). The smallest thing you can see with a light microscopy is about 1 micron across, that's a thousandth of a millimetre - a human hair is about 80 micron in diameter. A normal light microscope gives you a nice focused picture of a slice of you sample at the "focal plane", but it also lets in loads of light from parts of your sample away from the focal plane which leaves you, overall, with a bit of a blurry picture. Microscopists get around this problem by slicing their samples up very thinly hence no bits to be blurry, but this is a fiddly procedure and leaves your sample very dead even if it started alive.

Confocal microscopy is a technique by which the slicing of the sample happens virtually, you can put a big fat sample in the microscope and by the use of cunning optics you only get an image from the focal plane which is lovely and sharp. You can build up a 3D picture of the sample by moving it up and down in front of the lens. Marvin Minsky was the original inventor of the confocal microscope in about 1955 but was somewhat held back by the lack of lasers, computers and stuff. Things picked up again in the 1980's as these things became readily available. Oops, I think that might have been some cod history ;-)

An interesting feature of the confocal microscope is that if there's nothing in the focal plane, you don't see anything (unlike a conventional light microscope where you can always see a big bright something, even if it's blurry) this can be disconcerting for the learner - you can't find your sample!

Every microscopy needs a contrast mechanism, a way of separating one thing from another. In confocal microscopy by far the most popular contrast mechanism is to use fluorescence via the use of a fluorescent dye to label bits of your sample.  If you illuminate a fluorescent dye with light of one colour it emits light of another colour (making it stand out particularly well). If you ask an organism nicely (okay - genetically engineer), you can get it to make Green Fluorescent Protein (GFP) which is a protein that fluoresces green (duh!).  All that remains is to find a way of  sticking the fluorescent dye to the thing in which you're interested.

In each post about science I like to add a little fact to help you wind up / avoid winding up practitioners in that field. So to wind up a microscopist: project an image onto a screen for a presentation and claim "x800" magnification (or whatever). The problem is: to what does "x800" magnification apply? Is it what the microscope told you when you looked through the eyepiece? Is it the magnification on the printed page, the computer screen or on the wall? We really doubt you know. It's scale bars all the way.

For several years I was proud keeper of a confocal microscope. I, and my students, had great fun with the microscope and it had fun with us. The pointy end of the microscope is the objective lens, the bit closest to the sample. A fancy microscope like our Zeiss LSM 510 had 5 or more objectives mounted on a turret (see the image at the top of the post), each objective gives different magnification. The Zeiss LSM 510 was fully motorised, and too clever by half. It would assume that you wanted to stay focused on the same part of the sample when you changed objectives (or it changed them for you, with it's motors). Now the problem is that for a x10 objective the focal plane is about 1cm from the front of the objective lens, and for a x40 objective lens it could be only a tenth of a millimetre. Now imagine I've just focused deeply inside my sample using an x10 objective, I switch to the x40 object on the computer..... and the microscope mashes the x40 objective lens into the sample, blithely ignoring the sound of £6000 lens smashing glass coverslip and covering it in sticky sample!

In later posts I'll show some of the results from the confocal microscope in non-mashy-lens-into-sample mode.

Here are some images, these are all slices through solid objects. I didn't really think this through in terms of explaining what's in these first three images, roughly they're what you get if you add a small amount of salt-water to Fairy liquid (although I would prefer you to use Persil washing up liquid). First up is a cross-section through an "onion-type micelle":

And these are the structures you see in a similar system but with a different concentration of water:

This is a false colour image, bit lurid - don't know what I was thinking at the time. These are known as "myelin":

Pollen-grains are always popular - I stole this one from here. Each of the images is a slice, and the inset bottom right is the result of adding all the slices together.

There may be blue skies ahead

You have to feel sorry for Lord Drayson. At a time when he is doing his best to stand up for science and the funding of science in what are very difficult economic circumstances, where every department in government must show it's worth, scientists appear to be trying to hack his legs off; by balking at the proposal that they should explain their impact on society and insisting that they should be left to do 'blue skies' research. I refer to the recent debate hosted by the Times Higher Education Supplement (THES): "Blue skies ahead? The prospects for UK science", a discussion which centred around the impact of science on society and how you might increase that impact, how impact is evaluated, the role of blue skies research and the crisis at the Science and Technology Facilities Council (STFC).

In this post I'll try to explain why scientists are so impassioned about the subject of impact assessment and make a couple of suggestions as to how we might collectively do this better.

Impact means several things in this context: there is the HEFCE Impact pilot exercise, which is retrospective and in pilot phase at the moment and there are impact statements in grant applications which were introduced this year which should provide a prediction of the economic and societal impact of the work proposed. I suspect it is the impact statements in grant applications which are causing the real concern here, and I also suspect that Lord Drayson was talking primarily about the former, and the audience and panel were talking about the latter at the THES event.

Before I go on I will provide a short autobiography to provide context for my comments: I'm currently a research scientist in a large company, I've been here for 5 years but until the age of 35 I was an academic scientist, rising to the position of tenured lecturer in the physics department of what is now Manchester University. The opinions I present here are entirely my own.

Putting aside the question of how good a scientist I am; I can tell you one thing for certain: I'm an incredibly bad at writing successful grant applications! Really bad, awful, abysmal. I wrote about 8 over my relatively short period as a grant writer and they all failed (and not even by a small margin). I don't think I'm alone in this.

Exactly what grant applications mean varies from subject to subject, but in my field: experimental laboratory-scale soft matter physics it's important: in order to do research you need people and equipment. Typically grant applications are written to get 2-3 years of postdoctoral research assistant, or a PhD student and some equipment. Your department will rate itself on the grant funding it obtains, and you on your contribution to that figure. You will definitely feel that winning grants is something you have to do to succeed in your job. You can eke out an existence without grants, funding students from other sources and helping colleagues with successful grant applications, throwing yourself into teaching but it doesn't feel like the way you're supposed to do it.

I find it difficult to put into words how much I loathed the whole grant application process. A grant application requires you to describe the research you're going to do over the next few years, and how the results will be world-class. The average success rate is about 20% (disputed) in the field in which I was applying. Once written the grant applications are sent to reviewers - actually that means someone just like you - it's peer review. Reviewers know full well that if they rate an application "excellent" as opposed to "outstanding" in any one of several areas they are damning the application to failure. Reviews go forward to a panel who plough through huge numbers of these things (about 5 times as many as they are going to fund), then rank them. Funding is given to those at the top of the list, working down until the available cash is exhausted. There are serious questions as to whether we can rank schools accurately, here we try to do it for world-class research. It's a grim process for all involved.

It's in this context that the new impact statements are introduced, potentially these contribute 25% to the grant decision. As an grant application writer I need this like I need another hole in the head! Writing the science part of the grant application is a work of pure fiction (I think it might have helped if I'd appreciated that when I was doing it), writing the impact statement: "The demonstrable contribution that excellent research makes to society and the economy" is going beyond that. This is forward looking, you're being asked to quantify the future economic value of that bit of world-class research you're going to do.

The problem is that university science can take a long time to trickle out, in a case I highlighted yesterday in what is a pretty applied area, research was having a very direct, specific impact 40 years after it was done. Prior to my grant seeking days my work as a PhD student, postdoc and ADR has been funded, at least in part by three separate companies - little word of the economic impact has made it back to me from these companies.

So this brings me to some concrete points:
Point 1: For many areas of research societal, economic impacts are diffuse and long term, and actually the academic proposing the research is not in the best position to determine those impacts. As an industrial researcher I've written business cases for doing external research, this is a very revealing exercise which I know I couldn't have done as an academic because I simply wouldn't have had the required information to hand. Impact statements should not be required on a "one per application" basis, they should be for whole subject fields and written in consultation with people with actual data on the economic and societal impacts.

Moving on to a second point, Lord Drayson very generously praised scientists in Britain for the quality of the science they do, but said the problem was in how this expertise is translated into wider society:
Point 2: Impact statements are purely about scientists, applying for grants in universities. The onus seems to be on scientists to fix a problem which has two sides. What are we doing about how society and industry interact with science?

In the end impact is about communication, it's about understanding the preconceptions that other people bring to the party and addressing these preconceptions in the way you communicate. Although I described myself as a research scientist, I'm actually a science communicator in and industrial environment.

This whole blog is really about communicating what it's like to be a scientist, how it feels, the little details of the tribe. The people I follow on twitter are all very clever, they do lots of different things and from a combination of tweets and blogs you learn about their lives. This is my contribution to that discussion, it's a societal impact.

Wordless Wednesday

A short story about scientific impact

I work for a company, I won't tell you who.

40 years ago a man wrote a paper, published in a scientific journal, you won't have heard of it, it wasn't important.

A couple of years ago I read the paper, and I carried out the calculations described in the paper. I showed the results to the team I worked with. They showed that what we were doing wasn't going to work. So we stopped the project.

There were 10 people on the team, let's say they each cost my company £100,000 a year. Let's be harsh and say that my presentation only accounted for 10% of the decision to stop.

That man saved saved us £100,000.

I wish I could tell the man that wrote the paper, so he can put it in his next impact statement.

Update: Thanks to @dr_andy_russell for pointing out my rather significant typo!

Wonderful Life

You might have noticed I'm happy to go off into areas of science which are not my own with gleeful abandon, applying a physicist's mind to what I find. This week I'm off to see the biologists, Mrs SomeBeans is a zoologist - so in a sense, I sleep with the enemy. Twitter puts me in touch with so many more of the biological persuasion.

Animals are very well covered in TV documentaries but their view is somewhat partial, favouring the furry and cute, but there's so much more.

I think my appreciation for the wonders of the tree of life was first stimulated by "Wonderful Life" by Stephen Jay Gould. This book describes the fossils uncovered in the Burgess Shale a collection of fossils, from almost the very earliest life in the record - 500 million years ago. Gould is making two key points in his book, the first is that the so-called Cambrian explosion of species threw up a very diverse range of body plans; his second point is that the ones that survived did so almost by chance, there wasn't anything obviously superior about them. Richard Dawkin's book, "The Ancestor's Tale" is also well worth a read.

I should explain body plan: this is the overall layout of the animal. So for the tetrapods (including mammals, reptiles, amphibians, birds) you get four appendages. Insects: the plan is six legs, three body parts and an exoskeleton. I'm reliably informed that snakes are tetrapods, although I'm struggling with this particularly since one of my advisers previously tried to persuade me that mohair came from mo's. Basic rule seems to be that you can lose bones, fuse bones but not gain bones.

Once you appreciate this body plan stuff you start to get offended by representations of mythical beasts like angels and centaurs: they are clearly mammalian so angels can either have wings or they can have arms, they can't have both! Similarly centaurs can either have two pairs of horsey legs and no arms or a pair of horsey legs and a pair of arms, what they can't have is two pairs of horsey legs and two arms. The only reason I'm letting off the fairies is that I suspect they might be insects.

As a physicist, I really like this approach. Physicists basically have poor memories which shapes their approach to science, they like nice simple rules that encapsulate as much information as possible. You also spot them looking for "the simplest possible" model. So being freed from the requirement to learn lots of animal names by the simple expedient of calling them all 'tetrapods' is great. It's true that the tree of life is more complicated than that but the principle is there. If you want to go into this in more depth, the technical name for this study is phylogenetics... *time passes as I get completely distracted*

At this point I originally made my usual error by referring to other living animals as being further down the tree of life: they're not, we're all leaves on the surface of the tree. A good way to wind up a biologist is to refer to another currently living species as 'primitive'. They don't like this because, as they point out, they've been evolving for just as long as us! So, rather than referring to them as 'primitive' here are a couple of distant leaves: Hagfish are the only vertebrates to have a skull, but not a spinal column. They evade capture by covering themselves in slime. And as for tunicates, they start with a notochord (a precursor to a spinal cord) as larva but give it up as adults which indicates a certain bloody-mindedness that I admire. (it's a tunicate that decorates this post, at the top).

Rather more interesting than yet another furry animal...

Wordless Wednesday

Not Waving but Drowning

I thought I'd write a little post about Google Wave, and more generally the uptake of new software.

Wave is a recent innovation from Google, currently in restricted beta. It's a combination of e-mail, instant messaging and wiki. So if you even feel the need to utilise e-mail, instant messaging and wiki functionality simultaneously then this is the app for you. Actually, that's a little unfair.

This is what it looks like:

So, broadly it looks like an e-mail client. I won't describe the details here but you can get a better idea from the very fine Complete Wave Guide.

At the moment the problem with Google Wave is that the a relatively small number of people have been given the equivalent of e-mail addresses and told to randomly e-mail each other. Unsurprisingly they're spending a lot of time "e-mailing" each other about Wave. However, there is serious potential in Wave, aside from the traditional Waving about Wave I got lucky and managed to have a non-Wave useful discussion on Wave. It started with a thing that looked like an e-mail (I had a bunch of questions), my interlocutor started answering the questions in real time. Wave allows you to launch chat at any point in an e-mail, not only that it always you to see  your colleague typing character-by-character. In earlier forays this had been irritating but for a 'real' question it was actually rather useful - I could see my colleague getting the wrong end of the stick and put him right promptly. We managed to usefully flit between several conversations, adding things as they occurred to us and the end result is a nice record of a branching conversation.

As a wiki / document preparation system I'm less convinced. The core formatting available in Google Wave is fairly basic, although it can be extended significantly using gadgets and robots, but I can't see it being a comfortable way of working together on anything other than quite a short document.

I can see this being a great replacement for interminable e-mail threads between multiple participants, and even a way of writing minutes for a bunch of people sitting in a room with each other. If Google Wave were ubiquitous and people were willing to use it, then it would be a significant improvement over e-mail. Ubiquity may be attained in the future for the domestic user - I could imagine it becoming available as an option in Google Mail. To be honest I see far more applications for the business user and there, for larger organisations, uptake will be much slower.

This leads to another issue, even if they have access will people use such a tool? I'm dubious about this, I work in the research arm of a large commercial organisation. So you'd expect this group to be more tech savvy than average, but uptake of new software is pretty slow, people have taken to instant messaging but not wikis, reference management software, revision control, all things you might expect them to find useful. Once you get past the standard business suite of Word, Excel, PowerPoint and Outlook enthusiasm peters out and arguably mastery of even these applications is limited. This isn't a criticism of my colleagues, it highlights how difficult it is to gain traction with new software. When it comes down to it remembering how to use software is  hard and unnatural so not surprisingly we don't do it very well.

Ultimately Google Wave is another tool, in a toolbox that is overflowing.

Wordless Wednesday