acs boston update 3: thursday

August 23, 2007 at 1:18 pm | | conferences, science community, seminars

Thursday was the last day of ACS Boston. I’m tired of trying to be entertaining (you probably can’t tell the difference), so I’m just going to summarize the talks I liked:

  • Peter Lu has recently moved from PNNL to Bowling Green State University in Ohio. He talked about his fancy instrumentation that combines AFM/STM with a fluorescence/Raman microscope (with photon time-stamping and pulsed excitation, etc.).
    • First, he talked about looking at membrane-roughness changes with changes in membrane proteins: interfacial electron transfer caused changes measured by AFM and Raman.
    • Then he talked about studying LH1-LH2 FRET to probe dynamics of light-harvesting protein energy transfer. The FRET signal was rapidly fluctuating and looked like noise (not like TJ Ha’s obviously anticorrelated SM FRET time traces, for instance). Any FRET information was further obscured by Levy-flight dynamics. To glean anything from this data, he developed an analysis he called 2D cross-correlation amplitude mapping (see the new J. Phys. Chem. C paper on this analysis). He also applied this analysis to studying SM enzymatic pyrophosphorylation reaction dynamics.
  • Norbert Scherer talked about chemical-perturbation spectroscopy: watching FRET fluctuations and how they are related to periodic [Mg] jumps to probe the enzymatic reaction landscape. His experimental system is RNase P RNA labeled with Cy3/Cy5. My favorite part was when called a complicated figure on one of his slides a “ridiculogram.”
  • Jamie Boyce is a grad student finishing up in Sergei Sheiko’s lab, where I did my undergrad research. I went to her talk in the POLY division, and it was nice to see Jamie again and get an update on the lab’s work. She talked about a range of AFM studies of the shape, deformation, physics, and conformational changes of polymers with complex architectures, especially molecular “bottle brushes.”
  • Hu Cang is a postdoc in Haw Yang’s lab. They are using a scanning stage and careful setup of the confocal pinhole and detectors to track/”trap” diffusing particles in three dimensions. To get motion in the z direction, the confocal pinhole is positions slightly off the focus, so position changes are seen as intensity changes. Changes in x and y are measured using a prism mirror placed at the focus, splitting the beam to two detectors; position changes are seen as more light going to one detector. The particle (GNP, QD, fluorescent bead, etc.) is tracked by keeping it in the focus using a feedback loop with the translation stage. For a bead, they can track something with 60-nm precision in 3D. They can also learn information about rotation and shape from a dichroic beamsplitter and two detectors: the decay of the correlation reveals information about rotational dynamics. This was demonstrated by using Ag nanorods in glycerol: we were able to see correlated fluctuations. He mentioned their J. Phys. Chem. C paper on the tracking method. Here’s their J. Phys. Chem B paper about a unbiased binning method for change-point detection.
  • Peter Kapusta from PicoQuant talked about commercial implementation of Enderlein’s concept (see this ChemPhysChem paper) of using two foci to quantitatively measure the confocal spot volume for FCS.
  • Adam Cohen is a former member of the Moerner lab; he just joined the faculty at Harvard. He talked about his ABEL trap, which traps single fluorescent particles/molecules using a feedback voltage applied to the solution (pushing the dot back to the target position). He talked about his early experiments, such as DNA fluctuation dynamics. Then he talked about his “hardware trap” version, which can apply a trapping voltage with each photon measured by an APD, the trapping speed limited only by photon shot noise. He also talked about measuring dynamics of individual GroEL chaperonin molecules trapped by his machine. Finally, he talked about trying to trap single Cy3 molecules in solution. All his stuff was really impressive, but I don’t want too sound biased. (He also advertised spots in his new lab for “bright, motivated” grad students interested in building and testing novel devices.)
  • Michael Greene at NIST described some cool nanodroplets of sample in perfluorinated liquids. He could trap the nanodroplets, analyze the sample, and even bring two different droplets together. I wish I had more to say, but my brain is dead.

OK, that’s all on my reporting from ACS. Now I’ll get back to blogging about silly things…

acs boston update 2.5: posters

August 23, 2007 at 12:53 pm | | conferences, science community, seminars

I saw many cool posters at the PHYS poster session Wednesday evening. Here are my favorite:

  • Charles Schroeder did his graduate work with Steve Chu and he’s finishing his postdoc with Sunney Xie; I did a summer REU with Chuck when he was at Stanford. For his work in the Xie lab, he used a custom promotor to incorporate fluorescein UTPs and anti-fluorescein QDs to study RNA and DNA polymerases. Looking at a DNAP/RNAP system on a chain extended from a magnetic bead using flow, he was able to track changes in position of the polymerases and chain length (using a similar technique to what Antoine van Oijen did—I summarized his stuff before). His conclusion was that DNAP moves past the position of RNAP (either over it or pushes it along the chain).
  • Stirling Churchman is just finishing up in the Spudich lab. In a collaboration with Henrik Flyvbjerg, a theorist, she has used a better fitting method than Gaussians to fit the PSF for high-precision localization. Instead of Gaussians, they use the theoretical emission pattern of a dipole emitter (taking into account the NA of the objective and the higher rate of emission into the medium with higher index of refraction) to fit the PSF. With this more accurate fitting—and a MLE algorithm—they were able to get the same localization precision using only half the photons! They’re writing up a paper now.
  • Volkan Ediz is a grad student in David Yaron’s lab. He uses QM calculations to predict some photophysical properties of a class of asymmetric cyanine dye, predicting barriers to twisting into dark states (see their JACS paper here). Their work is related to some of the work I’ve done and a previous grad student in the Moerner lab did with a different fluorophore; but their calculations are more hard-core. I met Volkan and David last year at ACS San Francisco; they’re really nice!
  • Klaus Schaper is at Heinrich-Heine-University Dusseldorf and has been synthesizing rhodamine dyes with triplet quenchers covalently attached. The concept is that the quencher will make the dye brighter by both reducing the triplet lifetime and reducing the probability of photobleaching (from excitation from triplet states or from reactive oxygen produced by interactions with the dye triplet state). He used azobenzene on a sulforhodamine B, and found that he could get at 2.5-fold increase in the maximum emission rate (before saturation and bleaching) in an FCS experiment!
  • Franziska Luschtinetz, from the University of Potsam, looked at the changes in the photophysics of biotinilated dyes (called DY-635B and DY-647B) upon binding to streptavadin. She found different effects on the absorption and fluorescence emission spectra, ranging from dye rigidization and H-type excimer behavior, upon the binding. Finally, she also did some time-dependent fluorescence anisotropy and FCS measurements with these dyes. I didn’t actually get to talk to Franziska, but she helpfully provided printouts of her poster!

One more day of ACS coming up!

acs boston update 2: wednesday

August 22, 2007 at 8:13 pm | | conferences, science community, seminars

Today started with the T Red Line turning around back toward Cambridge* two stops from my station. Then there were delays. All because of some switching problems. Or something. Well, I took a cab (slightly more expensive than the ones in San Francisco, but not bad). In the end, I actually made it to the seminar room before the first talk.

acs_bost_graf1.jpg

Also, I saw some wicked awesome graffiti on my walk to the convention center. I guess someone really like 2-methylpentane! And there was more than that. I saw a methylacetylene somewhere, too! And whatever this is:

acs_bost_graf2.jpg

Here are some highlights from the talks on Wednesday:

  • Sunney Xie talked about “old stuff” (SM Michaelis-Menton kinetics and reaction theory using data from beta-gal—see the Nat. Chem. Biol. paper here) and new in vivo work looking at fluorescently labeled lac repressor binding to DNA.
    • For the first part, Sunney mentioned a new SM enzyme kinetics theory he developed (with Haw Yang and others) based on Marcus theory of electron transfer; they’ve submitted a paper to J. Phys. Chem. B (first author is Min).
    • The second “half” of his talk was so fast it was hard to follow. But he discussed using very fast imaging (5-ms frames and excitation pulses) to differentiate proteins (lac repressor) specifically bound to DNA and those that are nonspecific or diffusing (because only bound proteins are imagable as a bright spot, while diffusing spots blur out). They can also do kinetics studies of protein binding or unbinding to DNA: by finding how long it takes for spots to appear or disappear, respectively. This is reported in his recent Science paper.
  • Peter Sims is a grad student in Sunney’s lab at Harvard. He talked about some molecular motor experiments in live cells. Using dark-field microscopy, he imaged the scattering of gold nanoparticles (endocytosized into vescicles, then transported by kinesin or dynein) onto a quandrant photodiode. Using this fast detector and many photons scattered by the GNPs, he was able to get 1.5-nm spatial precsion with very fast temporal resolution. In order to track the cargos throughout the cell, they used a piezo translation stage to keep the signal on the QPD. The results: “kinesin” (those dots moving consistently toward membrane) took 8-nm steps; “dynein” (dots moving toward nucleus) took steps of 8, 12, 20, 24, 32 nm and other factors of 4; and dynein also showed smaller steps when load was added. I suspect they’ll write a paper soon.
  • Paul Alivisados spoke during Daniel Chiu‘s slot; I don’t know why. The must have swapped slots, but I don’t know because I missed the previous slot. Paul talked about plasmon coupling of his nanoparticle pairs and different colors of scattering from transverse vs. lateral coupling modes of the pairs (and triples and different groupings). He also talked about his nanorods in which atoms sort into dots or bands, depending on the doping. He gave a similar talk at Stanford a few weeks ago.
  • Antoine van Oijen did his postdoc with Sunney I think; now he’s at Harvard Medical School. Anyway, he talked about replisomes and other replication machinery, trying to understand how lagging-strand synthesis works while the polymerases move in the other direction. He used flow cells to pull strands of DNA (attached on one end to the surface and on the other end to a bead), and watched the workings of the proteins. He used the fact that ssDNA is much shorter than dsDNA at the same flow rate (i.e. force) to convert the extension of the chain to percentage that is single-stranded. He could see replication loops being formed and move down the chain; he also saw pauses in the leading strand while primers were being synthesized, presumably allowing the lagging strand to catch up the leading strand. He also did some work on a Xenopus cell-free system to determine the distance between origins and pre-replication complexes on single DNA strands.
  • Both Stephen Kowalczykowski and Chirlmin Joo (a TJ Ha student) spoke about SM imaging of RecA filaments on DNA.
    • Steve uses laminar-flow channels and optical tweezers to move DNA from a region with proteins and ATP (or other sets of components). He watched fluorescently labeled RecA nucleate and grow on stretched DNA chains. He talked not only about RecA, but also other DNA-associated proteins: RecBCD, Rad54, and Tid1 (see new paper here). He watched stretched DNA strands to learn about speed and direction of DNA motor proteins (using a fluorescent bead attached to the protein).
    • The Ha lab uses SM FRET fluctuations to learn information about nucleation and dynamics of RecA fibers. They also noted faster FRET dynamics of systems locked in lipid vesicles, concluding that fully formed filament nuclei bind and unbind to the DNA (because the filament can find the DNA again in the vesicle, and one-at-a-time monomer addition shouldn’t change the FRET signal in the vesicle). Chirlmin also mentioned that they use a hidden Markov model in finding transitions in FRET levels.
  • Richard Ebright discussed careful FRET measurements of RNA polymerase movements and mechanisms. Some rigorous evidence for certain mechanisms or RNAP (and strong evidence against mechanisms). He also mentioned that he used non-natural amino acids (like Peter Schultz) to add azides to proteins and specifically label with fluorophores using the Staudinger reaction (see Bertozzi’s reviews here and here).

Tonight is the PHYS poster session, so I’ll probably write a post later about the coolest papers I saw. Stay tuned!

________________

* I’m staying with a friend in Cambridge (thanks Stephen!). It’s really convenient and free. In fact, he’s treating me like a king. Which is the appropriate treatment.

acs boston update 1: tuesday

August 21, 2007 at 5:12 pm | | conferences, science community, seminars

 Although I missed Sunday and Monday, I did see some cool talks today:

  • Watt Webb‘s talk was a typical Webb talk: cool pictures, great voice, and a hodgepodge of info. He spoke mostly about lipid rafts in cell membranes.
  • Stefan Hell had some spectacular images from his super-resolution work (STED and RESOLFT and other acronyms). His newer work on fast “PALMIRA“—stochastic single-fluorophore photoswitching imaging, like PALM/STORM—and multicolor imaging is really impressive. He added a square-root factor to the denominator of the Abbe diffraction limit: (1 + I/Is)1/2, where Is is the threshold intensity above which you get sub-diffraction imaging. Hell also talked about 3D PALMIRA using slicing 2-photon excitation of new photoactivatable rhodamines they developed!
  • palm_3color.jpgBo Huang is a postdoc in Xiaowei Zhuang’s lab; I know him from when he was a graduate student in Dick Zare’s lab. He showed some truly beautiful three-color PALM images of microtubules and clathrin in cells (fixed, I think). They have a brand-new paper in Science with all these great images, check it out! Viewing just the TIR image, one would conclude that some clathrin pits were associated with microtubules; the PALM image revealed that the clathrin was only near the microtubles, but not actually colocalized (see image above). As far as the mechanism of cyanine dye photoswitching, Bo said that it was triplet related (dependent on heavy-ion concentration), thiol related (BME, L-glutathione, and cysteine all worked), and not isomerization (not dependent on viscosity). Xiaowei said that Roger Tsien thinks that it is nucleophilic attack by the reductants in the oxygen scavenger.
  • Michel Orrit used some sensitive interferometric and heterodyne experiments to measure the absorption of single gold nanoparticles (~10 nm) at room temperature. These particles absorb a lot of light, but are not very luminescent: so they efficiently heat the surroundings, allowing for photothermal detection. Some cool work, but hard to relate to cellular imaging.

I’ll update more tomorrow. Prepare for Sunney Xie, Daniel Chiu, Antoine van Oijen, and much more!

And you can always check out the C&E News bloggings about the Boston conference. Or Mitch. Or Paul. Or Kyle.

acs boston update 0: sunday and monday

August 21, 2007 at 4:37 pm | | conferences, science community, seminars

Because a bunch of my family was in town, I missed the first two days of ACS Boston. I thought it would be better to see some friends and family whom I rarely see than hear about science that I listen to everyday. After a few days, that decision starts to feel like a dumb move. (Just kidding, Dad.)

So I missed some talks I was excited about:

  • Steve Quake
  • Arne Gennerich
  • Alice Ting (but W.E. gave me a good synopsis—some cool stuff!)
  • Paul Barbara
  • Jim Spudich
  • Erdal Toprak

I’ve already seen recent talks by the most of the other speakers on Sunday and Monday, so I’m not as sad about missing those other great talks (e.g. Yildz, Zare, Alivisados, Dickson, etc.).

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