by PhilipJ on 24 April 2006
I obviously didn’t manage to do any live updates from the workshop in Japan, but now that I’m back in Vancouver I can do a proper recap. This post will focus on tweezers and fluorescence talks, with a cryo-EM post coming next.
Toshio Yanagida started the workshop off with the talk “Single molecule nanobioscience: learning nanotechnology from biology.” He talked at length about how molecular motors (for example, ribosomes) work in chaotic environments with large thermal fluctuations, but are required to carry out extremely important functions that will in some cases literally kill their host cells if they go wrong. In contrast, man-made machines work in a very different world where thermal fluctuations aren’t important and the environment is well behaved, so biological machines and man-made machines work in fundamentally different operating environments. His ultimate goal is to learn the engineering principles behind biological machines so that we might use them in designing our own nanomachines. The way he’s been studying these machines is via fluorescence imaging (his lab helped develop TIRF microscopy), and by manipulation through optical tweezers and scanning probe microscopes. The take home message (from a detailed analysis of myosin) was that molecular motors use thermal fluctuations instead of fighting them, and that this is something that can’t be overlooked when trying to design new nanotechnology.
Atsushi Miyawaki gave a talk on his extensive work on Green Fluorescent Proteins and extending them to emit in different areas of the spectrum via chemical modifications. The particulars of the chemistry were often beyond me, but the power of these tools was obvious when he showed an image of a cell stained by six different GFP mutants at once, which spanned virtually the entire visible range, all combined into a single image from only a couple of excitation sources.
Yale Goldman spoke about his lab’s work on the various myosin motors, and gave a very nice explanation of the polarisation dependent fluorescence measurements they do in their lab. By chemically modifying the light chain region of myosin with a fluorophore and changing the input polarisation of a TIRF excitation, the fluorescence output from the fluorophore allowed measurements of the angular orientation of the light-chain region relative to the actin filament that myosin walks on. Their experiments showed two individual orientations consistent with the two orientations available while bound to the actin filament, either as the leading or trailing head.
Down the coast from me from the University of Washington, Xiaohu Gao gave a lecture on his work on quantum dots as fluorescent markers. Unlike the organic molecule probes, quantum dots have a variety of advantages that make them ideal for imaging applications. Their emission wavelengths are tunable with the size of the dots, going from about 1.5-5.5 nanometres. Extremely broad absorption and very narrow emission lines make them easy to excite all by the same source, and are easy to distinguish from one another. They are significantly brighter (up to 50x over the same concentration of organic dyes), and over 1000x more stable (that is, constant emission intensity for up to longer than a full day, while dyes stop emitting after roughly an hour). Examples of their use in imagine applications were as single-molecule detectors that I’ve talked about before, and imaging in cancer cells. When testing for cancer, it is often particularly hard to get a large sample of cells to test due to the discomfort caused to patients (think prostate cancer), but there are a huge number of particular tests you would like to have done. With the variability of quantum dot emission wavelengths, it is possible to tag a huge number of different receptors in the same cell and have them photostable for significantly longer than traditional dyes have previously allowed.
With all that being said, they aren’t going to replace GFP- and dye-based fluorescence just yet, as there are some other problems unique to qdots. The entry mechanism of qdots into living cells is currently poorly understood, and they have to be attached to short peptides that somehow find their way across the cell membrane. As well, they blink. This isn’t a problem for imaging applications, but when you want to do single-molecule spectroscopy, a blinking signal is not an ideal source. Finally, the GFP variants have the distinct advantage of being made in vivo—you can place your gene of interest next to the GFP gene and express this fused construct in living cells using their own translation machinery. Qdots obviously can’t do this in any way, so both tools are needed depending on the science you’re trying to do.
The last fluorescence talk I’ll mention is a historical account of the development of fluorescence correlation spectroscopy (FCS) by one of its creators, Rudolf Rigler. While highly technical, he discussed taking the instrument from bulk measurements down to the single molecule level by changing to confocal microscopes, and also showed an image of the original FCS instrument built in his lab some 30 years ago. He’s still actively involved with the development of new instruments that you can buy from Zeiss, so it was very neat to see the changes in the state of the art FCS instruments.
The talk most similar to my kind of research was an introduction to magnetic tweezers by Vincent Croquette from the ENS. Similar in application as optical tweezers, by using a microbead embedded with magnetic particles and functionalised as mentioned before, it is possible to apply force and torque to these beads using permanent magnets atteched to a rotation stage. They can then be used to study the rotational motion induced in DNA by enzymes like the topoisomerases.
These talks were all excellent, and even though this is the area of single molecule analysis that I’m more familiar with, I still learned quite a bit. Up next will be a post on a technique that I knew virtually nothing about, cryo-electron microscopy!