News
LABORATORY PROFILE Beastly robots and glowing microscopes Where to start in talking about the illustrious Laboratory of Molecular Biology (LMB) in Cambridge (U.K.)? Perhaps, a focus on one of the fifty-year-old lab's ten Nobel laureates or the achievements of countless molecular biologists? Or, the goings-on in numerous spin-off companies? From the analytical perspective, methods in genomics that avoid messy cloning techniques are being developed, and new microscopy methods 8FC opening up the possibility of observing ions in living specimens
A living glow "Ordinary light cannot reach an object without passing through the intervening space," explains Brad Amos. A seemingly obvious statement, but he has a point to make. "This," he continues, "causes problems in microscopes, because all parts of the specimen are lit up, even those parts that are out of focus, which adds nothing but glare to the image." To this ubiquitous problem, he and his colleagues have found a solution. Amos and John White developed confocal microscopy as a scientific and commercially successful technique in the 1980s. The confocal microscope reduces glare by using a pinhole to eliminate light not in the focal plane, producing what Amos describes as, "a beautifully sharp image corresponding to the structure at one particular level in the specimen." He points out that even this technique has problems, the most serious being that the intense laser light can damage living specimens. Watt Webb's group at Cornell University came up with an advance on confocal
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microscopy that uses the LMB scanning apparatus but adds a pulsed laser as the light source. The pulses are lOXM)x brighter, with each pulse lasting a mere 50 fs, so the beam does not burn the specimen. Photons arrive at the sample simultaneously, and their energies are summed so that two red photons effectively have the energy of a blue or ultraviolet photon. "This means that far-red or IR light, which is kind to living cells, can be used instead of the damaging ultraviolet light," explains Amos. The two-photon effect is proportional to the intensity squared, so it declines rapidly off focus, leading to a cleaner scan with far less bleaching. "The reduction in lightinduced damage also makes it possible to image living tissues for long periods, even days," says Amos. Amos and his team are now using the advance to image living cells stained with fluorescent antibodies, which display important parameters such as calcium ion concentration. "Hie longer-wavelength beam gives an improvement in penetration, exactly like yellow foglights, so it works better—even with nonliving prepared specimens," says Amos. "But we are beginning to use it to find out how living nerve cells and junctions work," he adds. His team is working on numerous tissues, including the almost crystalline arrangement of nerve cells in the fish brain. "Paradoxically, the most powerful laser has opened up the possibility of the gentlest and truly noninvasive studies in many areas," muses Amos. Genomic beasts Paul Dear has a robotic "beast" in his laboratory at the LMB, which can literally move hundreds of micropipettes with speed and accuracy well beyond even the most skilled technician. Hie beast runs on custom-built software and, says Dear, is taking the burden out of human genome analysis. "The routines we have written to control our robot simplify the task of telling the robot what to do—we can say, in effect, 'Go to sample number 3f>C, takeout 10 pi. of liquid, and transfer it to tube 121)'. The software works out the details without us needing to tell it each movement... which would be very tedious." Because it is customizable, the researchers can build up complex routines. "At the highest level, we can just say 'set up KXX) samples with this standard set of ingredients', and the software handles every-
Analytical Chemistry News & Features, May 1, 1998
thing else." adds Dear. In DNA research, most scientists have used cloning techniques to pro duce sufficiently large samples of DNA. Dear, however, believes there are too many drawbacks with this biological approach. For instance, the bacterial hosts can respond negatively to having alien genes spliced into their nucleic acids: They reject some, scramble some, and may be killed by others. The LMB team uses a "ludicrously simple process" to isolate fragments of a particular human DNA—dilution. In effect, they take a bulk sample of DNA, break it at random, then dilute it until they have a few molecules per microliter. Instead of then splicing and dicing with microbes, they analyze the single fragments directly using the powerful polymerase chain reaction. "Compare this with cloning human DNA into bacteria, which can take months or years to fine-tune," says Dear. "Our fragments are anywhere from 100,000 to several million base pairs long tiny compared to tile liuiirii) trt'iionic but pretty big compared to most cloned fragments" This brings a lot of pressure to bear on the analytical methods needed; they are, after all. working with molecules one by one. The technique has been rather successful, however, and Dear's team has mapped' human chromosome 14, which is about 1(X) million base-pairs long, wiihout the distorting whims of microbial assistants. "It's a fairly typical-sized chromosome, and it was a good choice to 'cut our teeth' on," ponders Dear. David Bradley
Paul Dear with "the Beast."
