Biocurious

/ a biophysics blog

Fluorescence Nanoscopy Just Keeps Getting Better

Posted 1 April 2008 by Andre under

If you’ve been following the development of optical microscopy at all, you’re familiar with the recent advances that have been made in sub-diffraction limited optical microscopy. The new results keep coming fast and furious, pushing the field forward at an amazing rate. It’s interesting that the important advances are obvious even if their method of implementation is not. What I mean is that the goals of this research are clear: increase sensitivity, spatial resolution in 2D and 3D, temporal resolution, and the range of dye colours that can be simultaneously visualized. Of course, anyone can give you an important goal (fusion anyone? How about a time machine?) but having a practical approach is the challenge.

In sub-diffraction limited optical microscopy, there are currently two approaches that are driving things forward on all of these fronts.

One is the PALM/STORM* approach of Eric Betzig and Xiaowei Zhuang. PALM and STORM both take advantage of the fact that single fluorophores can be localized with nanometer accuracy when they are well separated, even if they can’t be resolved into spots when they are closer than the standard diffraction limit. This idea is turned into an imaging technique using photoactivatable dyes that can be turned on and off within the sample so that the dyes that are “on” at any given point are far enough apart to be localized before the next set of dyes are activated and in turn localized. By doing this procedure many times, the dye locations can be combined into a high resolution image:

nanometers!!1!**

The other approach is called Stimulated Emission Depletion or (STED) microscopy and has been pioneered by Stefan Hell and his group. It also takes advantage of photo-activatable fluorescence to beat the diffraction limit, but in a very different way. They use one beam to make a doughnut-shaped “depletion” patch and another to make an activating spot in the middle (we Canadians might say a Timbit excitation?). By increasing the intensity of the depletion beam, they can effectively squeeze the region where excitation is likely down to a spot smaller than the diffraction limit. This spot can then be scanned around a sample as in a confocal microscope to produce a nanoscale resolution image.

The image below compares the results of these two approaches to images taken using their most closely related diffraction-limited cousins.

STED/PALM comparisonThe image is taken from Hell’s excellent review of these techniques that appeared last year in Science (2007) 315:1153

Because PALM and STORM require the collection of a lot of speckled images to reconstruct the full distribution of dyes, it was clear that STED would have the advantage in time resolution.*** Now, in an article published online in Science [abstract, subscription required for full article], they’ve really driven that message home with a report of fluorescence imaging with 63 nm resolution at 28 frames per second.

“Video-rate imaging was accomplished by scanning the excitation and depletion beam pair in the focal plane by means of a 16 kHz resonant mirror in one direction; the second direction was scanned by moving the sample with a piezo actuator.” They also tightened their doughnut down to the point where the number of photons emitted from the hot spot was just big enough to provide the intensity needed to distinguish the signal from the background.

They used this method to image vesicles moving in neurons that are normally hard to see directly because they move in a nerve terminal that is only about 1 micron in diameter. As soon as you have a few vesicles passing each other in a space that size, a standard microscope (even a really good one) won’t be able to resolve them. Experts in the field can tell us if they learned anything interesting about vesicle traffic, but to me that’s not really the point. The paper is really about showing what can be done and getting cell biologists excited about the possibility of using STED for their favourite system.

It’s only a matter of time (I would say a few years at most) before this technology becomes available on a commercial instrument. When that happens and more biologists start to get their hands on one, we’ll really start to see its impact. In a huge understatement, Volker Westphal et al. start their paper with this:

Many questions in the life sciences could be answered if lens-based optical microscopy featured the resolution of electron microscopy, or if the electron microscope operated under physiological conditions.

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*PALM stands for photo-activated localization microscopy. STORM stands for stochastic optical reconstruction microscopy.

**The statistics of my schematic are way off. In my animated gif it is, conveniently, exactly the needed remaining pixels that are filled in. This is especially important in the last couple of frames. In a real reconstruction, because it is based on stochastic activation of dyes, really filling out the image at the end takes a very long time. This is one of the things that limits the time resolution of the technique. I think the first demonstrations of PALM and STORM used acquisition times of over an hour on fixed samples to get enough data for their images. These times have now been shortened, but video rate seems like a long shot.

***But people are clever and there could always be surprises.

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Hot off the Press: Single-ribosome translation

Posted 26 March 2008 by PhilipJ under &

While we’ve been watching trippy 70s-era translation videos on YouTube, the Bustamante lab has been busy measuring this process at the single-molecule level, at least indirectly. This was one of those surefire Nature papers, whenever someone figured out how to measure translation at the single molecule level using optical tweezers, it would show up in Nature, and sure enough….

In Following translation by single ribosomes one codon at a time (closed access, PDF), we get to see for the first time the march of the ribosome along mRNA. I don’t think we’re anywhere near the end of single-molecule ribosome work, and part of the reason why is the setup for the experiments outlined in this paper. Reproducing part of Figure 1 from the paper:

single ribosome experiments setup

What’s being measured is not the action of the ribosome itself, but the extension of the RNA/DNA construct as translation occurs. RNA/DNA handles are coupled to mRNA hairpins, and an initially stalled ribosome is then re-activated (by supplying the appropriate tRNAs), and the extension vs time of the entire construct is monitored at very high resolution. Examples of their extension vs time traces look like this:

ribosome steps

The left hand plot shows a typical trace as the ribosome carries out its work, and the right hand side shows what’s called a pairwise difference distribution, or basically the difference between data points. If there is some periodicity in the dynamics, this would show up as a peak in the pairwise differences, and indeed they see peaks at 2.7nm (and multiples thereof). This corresponds to three basepairs being broken in the hairpin, and the ribosome translocating by 3 bases (where three bases form a codon, the unit which specifies for an amino acid). While not a surprising result (despite the authors claiming this was “striking”), this is still significant.

The tricky bit, however, is that this doesn’t really get at the heart of the ribosome’s interesting mechanochemistry. We still know nothing about the force-velocity relationship in single ribosome molecules, and while there are some interesting experiments in this paper involving dwell times at Shine-Dalgarno sequences, and some discussion on changing the ribosome’s reading frame, the paper is a little short on really new measurements.

Technically this is impressive, but the new and interesting data on ribosomes that single-molecule techniques can measure are still to come.

(Hat tip: in singulo)

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Hot off the Press: Omnidirectional sensing of prey in weak electric fields by fish

Posted 20 December 2007 by PhilipJ under &

Blogging on Peer-Reviewed ResearchThe November issue of PLoS Biology has a very neat article (open access, of course) on the mechanism by which the South American black ghost knifefish Apteronotus albifrons senses prey. A self-generated electric field surrounding the fish is coupled with some 14,000 electrically-sensitive organs, which can measure perturbations in voltage induced by local changes in electrical conductivity in the water, such as when prey enter what is called the sensory volume, or where this electric field is active.

For A. albifrons, it was found that the sensory volume is actually roughly a cylinder which encompasses the entire body of the fish, shown in Figure 3 from the paper and reproduced here:

This is in contrast to most other active sensing mechanisms, such as echolocation by bats and the use of sonar by dolphins, as the sensory volume is more often roughly cone-shaped and necessarily in the forward direction. Unlike dolphins or bats, for which changing direction quickly is not necessarily easy, A. albifrons has, along with pectoral fins, a long ribbon fin that runs the length of it’s body (See above, in figure A), which allows the fish to swim forward, backward, or upward, and change pitch or roll its body. It has also been observed that the fish comes to a near stop when consuming or “handling” its prey, so the combination of a versatile swimming mechanism and observed behaviour suggested a motor volume, or roughly, the volume through which the fish can react to a stimulus in its sensory volume, that should match the sensory volume. This makes some intuitive sense: there is no point sensing for prey in areas that are too far away for you to catch them given the energy costs of creating this electric field.

The determine motor volumes (not shown here, see the text for details) are a good match to the sensory volumes, or, related to known behaviour, the sensory volume is roughly equal to the volume required for the fish to come to a stop, and that these volumes changes as the ambient conductivity in the water changes.

That the sensory volume encompasses the entirety of the fish also implies that it isn’t solely for the search of prey. Analysis of the “mouth” motor volume was (not surprisingly) biased towards the front of the sensory volume, indicating where in the search volume prey can be sensed and caught. That the sensory volume is larger than this suggests that sensing could also be used to detect other obstacles in the fish’s environment.

To read the rest of the study (and see some neat interactive, 3d renderings of the sensory and motor volumes), click here.

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Hot off the Press: Single-Molecule Chemistry and Biology Special Feature in PNAS

Posted 31 July 2007 by PhilipJ under &

Lots of interesting articles this week in PNAS, which is featuring single-molecule chemistry and biology.

W. E. Moerner talks about new directions in single-molecule imaging and analysis (we should get Sam to chime in too), Sunney Xie’s group has a paper on the fluorescence of a functionally important conformation of T7 DNA polymerase, Xiaowei Zhuang and co-workers dissecting the multistep reaction pathway of an RNA enzyme, while T. Ha’s lab fueled protein–DNA interactions inside porous nanocontainers (vesicles), among others (unfortunately all requiring a subscription).

Non-single-molecule feature papers of interest include A closer look at energy transduction in muscle (open access!), Fast-scan atomic force microscopy reveals that the type III restriction enzyme EcoP15I is capable of DNA translocation and looping, and energy transfer in peptide helices, the latter two requiring a subscription as well.

Happy reading!

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Life-Science Fiction

Posted 4 July 2007 by Andre under &

There’s been some recent discussion sparked by Peggy’s and PZ’s posts on how biology gets treated poorly in science fiction, particularly compared to physics. Rob Knop and Chad Orzel respond that physics is treated poorly enough, thank-you very much, and Razib has some reasonable things to say.

Now, this week’s issue of Nature includes a timely interview with four science fiction writers with backgrounds in biology to get their opinion on the role science fiction can play in communicating good science, how even bad science fiction can play a positive role, and how they use aliens and artificial intelligence in their work.

So, the only question you should be asking is did the Nature editors use their time machine to get this story out on the heels of the discussion in the blogosphere or did they just use their mind control rays to make sure it was discussed once they had the feature ready?

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Will you ever be able to buy fitness in a bottle?

Posted 4 June 2007 by Andre under &

The diet pill industry is worth billions per year in the US alone so there’s huge incentive for companies to market miracle pills and the 1994 Dietary Supplement Act makes it easy. Just ask Bob Park. This means that neither safety nor efficacy need to be proven for “natural” remedies and this has predictable results.

mice and man plus drugs

Despite this, the idea that drugs can help with weight loss and even improve fitness is plausible. Indeed, the possibility that some future drug might live up to the claims made by these pill pushers seems ever more likely. Take the recent announcement from Ronald Evans’s lab at the Salk Institute. They are “now able to chemically switch on PPAR-d, the master regulator that controls the ability of cells to burn fat. Even when the mice are not active, turning on the chemical switch activates the same fat-burning process that occurs during exercise.” As we get better and better at controlling metabolic activity with drugs, we will undoubtedly see more applications like this. If life is just chemistry (albeit exquisitely complex and subtle chemistry) there’s nothing stopping us from controlling biology with clever pills. Jake puts it this way at Pure Pedentry:

True, the signaling cascades activated by exercise are just that — signaling cascades. Exercise works because it changes the molecular biology of cells, and there is no reason that we can’t enforce that same type of activation sans all the running around.

It also seems probable that something similar will eventually work for muscle building, but do these effects require some exercise or could someone lie in bed for a few months, pop pills, eat chocolate, and emerge a body builder? That’s an interesting question to me not only because I like lying in bed, but also because its answer might reveal an essential role for force in fitness. Put another way, do some signaling cascades critical for fitness involve more than just chemical but physical signals? Maybe that’s why they call it physical activity.

We already know of examples of cryptic sites in proteins involved in signaling that are exposed in response to force (check out this interesting work from Harold Erickson’s lab available freely from Pubmed) and if some of these processes are also activated during exercise it might not be possible even in principle to use pills to develop good overall fitness. But don’t despair, maybe a combination of fat burning, muscle building drugs and a therapeutic stretching rack would do the trick…

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