Taekjip Ha (UIUC, HHMI) gave the applied-physics seminar yesterday, and talked about his single-molecule FRET on helicases and sm-FRET combined with force experiments on Holliday DNA junctions. It was a normal Ha talk: with long SM time traces (with the help of oxygen-scavengers) and sensitive FRET measurements. It was interesting to see what Ha calls “repetitive shuttling” of the rep-helicase—sliding on ssDNA, then jumping back to the end of the strand.
He also mentioned something funny at the beginning of his talk. He searched PubMed for the term “single molecule” in the title and saw an exponential growth since the late 1980s, doubling every 2.2 years. He also noted that the total number of titles in PubMed only doubles every 20 years. Thus, he reasoned, by 2035 AD, every paper published in the field of biomedicine will be a single-molecule paper. Ha! Well that should make some people pretty upset.
Cornell has some cool videos of Hans Bethe talking about quantum mechanics. Here’s his intro: “Quantum theory is the most important discovery of the 20th century. And it has been presented to the public in a completely distorted way, what you hear normally has very little to do with quantum theory.”
There are three 50-minute talks, so you can watch them while waiting for the laser to warm up, the cells to divide, the coffee to percolate, or whatever you do…
What is going on here? I woke up the last two mornings to look out the window only to see pigeons in queues in the field across the street:
This is strange. Upon closer inspection, it was revealed that they were eating along an old faded white line on the soccer field. I tried finding some mention of this phenomenon in The Google, but to no avail. The closest we found were blue jays eating house paint (here and here) and sparrows eating grit. Maybe this is the same thing: pigeons eating field chalk for grit or minerals. But they were only on one older faded line. Maybe the new lines are a different paint (to avoid this problem). Or maybe the field paint made some yummy type of grass grow there. Still unclear to me. Anyone else know what’s going on here?
You can read Part I for techniques such as STED, PALM, STORM, etc.
In Part 2, we’ll explore the wide-field structured-illumination approach to breaking the diffraction limit of light (BTDLOL). Structured-illumination (SI)—or patterned illumination—relies on both specific microscopy protocols and extensive software analysis post-exposure. But, because SI is a wide-field technique, it is usually able to capture images at a higher rate than confocal-based schemes like STED. (This is only a generalization, because SI isn’t actually super fast. I’m sure someone could make STED fast and SI slow!)The main concept of SI is to illuminate a sample with patterned light and increase the resolution by measuring the fringes in the Moire pattern (from the interference of the illumination pattern and the sample). “Otherwise-unobservable sample information can be deduced from the fringes and computationally restored.”1
So SI enhances spatial resolution by collecting information from frequency space outside the observable region. The figure below shows this process in reciprocal space: (a) the Fourier transform (FT) of a normal image and (b) of an SI image, with arrows pointing to the additional information from different areas of reciprocal space superimposed; with several images like in b, it is possible to computationally (c) separate and (d) reconstruct the FT image, which has much more resolution information. The reverse FT returns d to a super-resolution image.
But this only enhances the resolution by a factor of 2 (because the SI pattern cannot be focused to anything smaller than half the wavelength of the excitation light). To further increase the resolution, you can introduce nonlinearities, which show up as higher-order harmonics in the FT. In reference 1, Gustafsson uses saturation of the fluorescent sample as the nonlinear effect. A sinusoidal saturating excitation beam produces the distorted fluorescence intensity pattern in the top curve of (a) in the figure below. The nonpolynomial nonlinearity yields a series of higher-order harmonics in the FT, as seen in the top curve of (b) below.
Each higher-order harmonic in the FT allows another set of images that can be used to reconstruct a larger area in reciprocal space, and thus a higher resolution. In this case, Gustafsson achieves less than 50-nm resolving power (solid line), more than five times that of the microscope in its normal configuration (dashed line).
The figure below demonstrates the high resolution, going from (a) the normal microscope image, to (b and c) increasing order of harmonics used in the reconstruction, to (d and e) a high-res image from nine frames of 50-nm fluorescent beads.
The main problems with SI are that, in this incarnation, saturating excitation powers cause more photodamage and lower fluorophore photostability, and sample drift must be kept to below the resolving distance. The former limitation might be solved by using a different nonlinearity (such as stimulated emission depletion or reversable photoactivation, both of which are used in other sub-diffraction imaging schemes); the latter limits live-cell imaging and may require faster frame rates or the use of some fiducial markers for drift subtraction. Nevertheless, SI is certainly a strong contender for further application in the field of super-resolution microscopy.
- Gustafsson, M. G. L. Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution. PNAS 2005, 102(37), 13081–13086.
- Gustafsson, M. G. L. Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy. J. of Microsc. 2000, 198(2), 82–87.
- Bailey, B.; Farkas, D. L.; Taylor, D. L.; Lanni, F. Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation. Nature 1993, 366, 44–48.
I wish I had taken this one: www.1-800-dotcom.com
I recently found a cool website: CiteULike. It allows you to easily post scientific articles that you find interesting. To post an article you find, you simply stay on the page with the article (or abstract) and click a CiteULike bookmark, which automatically imports the various fields (e.g. authors, title, journal, etc.) into your library; then you simply add your own “tags” (or categories) and submit the post. I love it!
This site has a two-fold benefit: (1) you can share this library with others who are in your field (see my Watchlist), and (2) you can rank them according to how much you want to read them and remember to read papers.
CiteULike was written by Richard Cameron in 2004 and is run by him. And it’s free! I was so happy to find CiteULike, because I came up with the idea a few weeks ago, but I don’t have enough programming experience to write such a website (I took a C++ in high school, but didn’t really do the homework).
There are probably several other sites like this one, but the only other one I’ve found is Connotea, which was commissioned and now part of Nature Publishing Group. I checked out Connotea, and even imported my CiteULike library (tags include). But I prefer CiteULike’s style and features, especially the ability to rate and sort how interested I am in reading each article (maybe that feature exists in Connotea, but I can’t find it). I hope that CiteULike eventually becomes an open-source, collaborative effort; but until then, I’m still really happy with it.
So start you own library and send me a link!
Ever wonder how many licks it takes to get to the centre of a tootsie roll pop? How about what is the meaning of life? I’m guessing no, because you were too busy trying to figure out how many condoms will fit on a simulated phallus, and the ramifications thereof. Well, in a seminal work, researchers at myscienceproject.org have set a lower bound on the critical condom parameter of 625. They’ve also characterized potential side effects of, uh, 625-bagging, of which priapism is evidently absent.