news and views over, Aplysia shows classical conditioning9, and much is known about the molecular and cellular mechanisms underlying this form of learning10–12. So it might be possible to compare classical and operant conditioning in Aplysia in mechanistic terms. If they have features in common, an exciting principle might emerge: evolution may have come up with a neural ‘associative cassette’ that can be used in either type of conditioning, depending on the neural circuit in which it is embedded. Of course, this is pure speculation, but the work by Brembs and colleagues will be instrumental in exploring this intriguing possibility. ■ Thomas J. Carew is in the Department of Neurobiology and Behavior, 2205 McGaugh Hall, and the Center for the Neurobiology of Learning and Memory, University of California at Irvine,
California 92697-4550, USA. e-mail:
[email protected] 1. Brembs, B., Lorenzetti, F. D., Reyes, F. D., Baxter, D. A. & Byrne, J. H. Science 296, 1706–1709 (2002). 2. Skinner, B. F. The Behavior of Organisms (Appleton, New York, 1938). 3. Thorndike, E. L. Psychol. Rev. Monogr. Suppl. No. 8 (1898). 4. Pavlov, I. P. Conditioned Reflexes (Oxford Univ. Press, Oxford, 1927). 5. Botzer, D., Markovich, S. & Susswein, A. J. Learn. Mem. 5, 204–219 (1998). 6. Nargeot, R., Baxter, D. A., Patterson, G. W. & Byrne, J. H. J. Neurophysiol. 81, 1983–1987 (1999). 7. Schultz, W. Nature Rev. Neurosci. 1, 199–207 (2000). 8. Nargeot, R., Baxter, D. A. & Byrne, J. H. J. Neurosci. 19, 2261–2272 (1999). 9. Carew, T. J., Hawkins, R. D. & Kandel, E. R. Science 219, 397–400 (1983). 10. Hawkins, R. D., Abrams, T. W., Carew, T. J. & Kandel, E. R. Science 219, 400–405 (1983). 11. Walters, E. T. & Byrne, J. H. Science 219, 405–408 (1983). 12. Murphy, G. G. & Glanzman, D. L. Science 278, 467–470 (1997).
Light microscopy
Beyond the diffraction limit? Ernst H. K. Stelzer The wave nature of light manifests itself in diffraction, which hampers attempts to determine the location of molecules. Clever use of microscopic techniques might now be circumventing the ‘diffraction limit’. he two best-known physical limitations are Abbe’s resolution limit in optical physics and Heisenberg’s uncertainty principle in quantum physics. Each defines a natural limit to the resolution or accuracy with which certain parameters can be measured. But, writing in Physical Review Letters, Marcus Dyba and Stefan Hell1 claim to have taken a step beyond one of these limits. In 1873, Ernst Abbe2 realized that the smallest distance that can be resolved between two lines by optical instruments has a physical and not just a technical limit. The distance — the diffraction limit — is proportional to the wavelength and inversely proportional to the angular distribution of the light observed. No matter how perfectly an optical instrument is manufactured, its resolving power will always have this natural limit. About 50 years later, Werner Heisenberg3 realized that the parameters describing a quantum particle, such as its location and momentum, are not independent. The accuracy with which one parameter can be determined is coupled to the accuracy with which the other can be determined: one can have an accurate idea either of the location or of the momentum, but not of both at the same time. Improvement in accuracy in one parameter will always occur at the expense of decreasing the accuracy in the other. Heisenberg’s ‘uncertainty principle’ is probably one of the most thoroughly tested relationships in physics, a firm foundation of modern quantum theory, and there is no reasonable account that suggests it is incorrect.
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Although not a great surprise, it has only recently been shown that Heisenberg’s and Abbe’s formulae are related4. Over the past few years, several groups have claimed to have achieved image contrasts that have taken the resolution of light microscopes beyond that of classical instruments, and beyond Abbe’s limit. For example, confocal fluorescence microscopy has had an enormous impact in biology. Using lasers to induce emission from fluorescent chromophores (the molecules responsible for the colour of the material), it combines point-bypoint illumination with synchronous pointby-point detection. This technique has proved especially useful for imaging biological objects because of its optical sectioning capability: deep inside optically dense objects (such as embryos), it is possible to record fluorescent images that show the chromophore distribution in just a single focal plane. The advantage with this type of instrument comes from its nonlinear behaviour — it is essentially sensitive to the square of the light intensity, not to the intensity itself, and this discriminates against light that is out of focus. On the basis of the work of Lukosz5, Gustafsson and colleagues6 have achieved excellent resolution with their images of actin protein networks, collected by illuminating the object with light patterns whose intensity varied in a sinusoidal fashion. These and several other contributions to the field have pushed the image resolution down to about 100 nm — which does not contradict Abbe’s resolution limit. © 2002 Nature Publishing Group
Imaging the common soil bacterium Bacillus megaterium with focused light of wavelength 760 nm, Dyba and Hell1 claim to have observed excited molecules with a resolution of 33 nm — that is, down to a distance considerably smaller than Abbe’s limit. The authors’ technique relies on two essentially independent technologies. The first is ‘4Pi confocal fluorescence microscopy’, which uses two opposing lenses of high numerical aperture to illuminate an object coherently from two sides7. This creates an interference pattern that is modulated along the optical axis and reduces the observed volume by a half. The main maximum in the pattern of diffracted light — one ‘volume element’ in the image sought — is surrounded by other peaks of light intensity, which can generally be removed by further computations and by using knowledge gained about the object with other means. The second technique is more complicated. First, the fluorophores are excited with a regular, well-focused beam of light. Picoseconds later, a second beam operating at the emission frequency of the fluorophores but with a light-intensity distribution that has a gap in its centre, stimulates the emission of the fluorophores outside the gap region. A few more picoseconds later, the regular fluorescence emission, stemming from the volume defined by the gap, can be observed. Dyba and Hell used the 4Pi technique to create the gap pattern for the stimulated emission. The question is what the observed spot width of 33 nm tells us. Have Dyba and Hell achieved a spatial resolution of 33 nm? Although the experiments shown in their paper seem convincing at first, I question whether this is actually a demonstration of resolution improvement. Their images of membranes of Bacillus megaterium show a two-dimensional view of well-spaced membranes that are already well resolved in the straightforward confocal image. The images recorded with the new technique seem to localize the two membranes more exactly. This is at best an improvement in the precision of their locations, but not actually in the resolution of the image, which is defined as the minimum distance between two features that can be resolved with a certain contrast8. As already seen in the work of Toraldo di Francia and others9,10, the central diffractive peak can be made much narrower, but this always generates huge side ‘lobes’ of light, which appear outside the area of interest. This effect is equally visible in the images shown by these authors1. Extensive computations are required to discriminate the information in the central lobe from that in the side lobes. So the improvement is not entirely due to the instrument but in fact stems from combining the imaging techniques and from using a priori knowledge. Another problem is that the signal-to-noise ratio becomes worse. NATURE | VOL 417 | 20 JUNE 2002| www.nature.com/nature
news and views Nevertheless, although the usefulness of the method introduced by Dyba and Hell is not yet clear, the development is quite fascinating. For the moment one thing is still true: Heisenberg was right and Abbe’s limit was certainly not broken. ■ Ernst H. K. Stelzer is in the Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany. e-mail:
[email protected]
1. 2. 3. 4. 5. 6.
Dyba, M. & Hell, S. W. Phys. Rev. Lett. 88, 163901 (2002). Abbe, E. Arch. Mikrosk. Anat. 9, 413–468 (1873). Heisenberg, W. Z. Phys. 43, 172–198 (1927). Stelzer, E. H. K. & Grill, S. Opt. Commun. 173, 51–56 (2000). Lukosz, W. J. Opt. Soc. Am. 57, 932–941 (1967). Gustafsson, M. G. L., Agard, D. A. & Sedat, J. W. J. Microsc. 195, 10–16 (1999). 7. Hell, S. & Stelzer, E. H. K. J. Opt. Soc. Am. A 9, 2159–2166 (1992). 8. Stelzer, E. H. K. J. Microsc. 189, 15–24 (1997). 9. Toraldo di Francia, G. Atti Fond. Giorgio Ronchi 7, 366–372 (1952). 10. Martinez-Corral, M., Caballero, M. T., Stelzer, E. H. K. & Swoger, J. Opt. Express 10, 98–103 (2002).
telomere complex or part of the telomereassociated nuclear membrane and matrix. Parkinson et al. also observed the same parallel-stranded alignment in crystals of the two-repeat quadruplex in the presence of K& ions. This quadruplex is formed when one two-repeat sequence pairs up with another. It seems that this requires the formation of A•T•A•T tetrad planes, thus expanding the tetrad alphabet beyond G•G•G•G tetrads. Such mixed tetrads might serve a valuable role in cells: they could direct the pairing of homologous chromosomes to enable the chromosomes to ‘recombine’, swapping segments of DNA. So the two-repeat structure sheds light on how homologous chromosomes might bind to each other during recombination. The four-repeat structure shows how the basic topology could protect the ends of individual chromosomes. Interestingly, the same fourrepeat human telomere sequence adopts a completely different quadruplex architecture in a solution of Na& ions5 (Fig. 2b). Here, the opposing GGG columns are antiparallel, and there are one diagonal and two edgewise TTA loops. Different monovalent cations can therefore alter the four-repeat quadruplex topology. This might be because K& cations (which have an ionic radius of 1.51 Å) are invariably sandwiched between adjacent guanine tetrads6, whereas Na& cations (ionic radius 1.18 Å) can sometimes be coordinated within a tetrad7. Single-nucleotide changes within the telomere repeats might also have a say in cation-mediated folding topology. In mammalian cells, however, telomere repeats are more likely to form K&-coordinated quadruplexes as the intracellular K& concentration greatly exceeds that of Na& (140 mM versus 5–15 mM).
Structural biology
A molecular propeller Dinshaw J. Patel Telomeres are protein–DNA structures protecting the ends of chromosomes. The crystal structure of a four-stranded stretch of human telomere DNA, bound to K& ions, has implications for the design of anticancer drugs. he ends of the linear chromosomes of eukaryotes are capped by telomeres — protective structures composed of repetitive DNA bound to proteins. Every time a cell divides it replicates its genetic material, but the replicating enzymes cannot copy the extreme ends of chromosomes. If genes were present right up to the chromosome ends, they would gradually be eroded with each cell division. Telomeres prevent that from happening and, although they are themselves eroded, they can, under some circumstances, be renewed by the enzyme telomerase1. Among their other functions, telomeres also stop chromosomes from fusing end to end. The DNA of human telomeres consists of repeats of the nucleotide sequence TTAGGG, ending in a single-stranded segment that overhangs at the end of the double-stranded DNA helix. On page 876 of this issue, Parkinson and colleagues2 describe striking crystal structures of singlestrand sequences — consisting of two or four TTAGGG repeats — in the presence of K& ions, revealing topologies that could readily be incorporated into a higher-order DNA architecture. In these structures the single-stranded repeats fold up into four-stranded (quadruplex) topologies2. The basic building block of any quadruplex is a G•G•G•G tetrad, composed of four hydrogen-bonded guanine nucleotides in a horizontal planar arrangement3 (Fig. 1a). Individual tetrads stack up on each other, with monovalent cations (Na& or K&) sandwiched between them4. With TTAGGG repeats, the GGG sequence forms a vertical strand (column) of the quadruplex; the TTA sequence loops over, joining into the GGG sequence of the next repeat, and so on until there are four vertical GGG columns, three horizontal tetrads deep. The GGG columns can adopt either parallel or anti-
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parallel alignments (they are oriented in the same or opposite directions), depending on how the connecting TTA loops are oriented (Fig. 1b–d). Edgewise loops connect adjacent antiparallel strands; diagonal loops connect diagonally opposite antiparallel strands; and double-chain-reversal loops connect adjacent parallel strands. So how do Parkinson et al.’s crystal structures look2? In the four-repeat structure, the GGG columns are all parallel to each other, and the three connecting loops are of the double-chain-reversal type2 (Fig. 2a, overleaf). Moreover, all three loops are splayed out like a propeller from the main body of the quadruplex (see Fig. 3b on page 878). They can thus be recognized by proteins that are either components of the a
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Figure 1 Basics of the quadruplex topology of human telomeric repeat sequences. a, The planar G•G•G•G tetrad alignment, viewed from above, in chemical (left) and schematic (right) form. Left, hydrogen bonds between adjacent guanines are shown by dashed lines. Right, individual guanines are represented as rectangles, and attached sugars as circles. b–d, Stacked tetrads, showing the vertical columns of guanine sequences and horizontal tetrad planes. The DNA backbone of the guanine columns and connecting loops is shown by black and red lines, respectively. Directionalities are shown by arrows. Edgewise loops connect adjacent antiparallel strands; diagonal loops connect opposing antiparallel strands; double-chain-reversal loops connect adjacent parallel strands. © 2002 Nature Publishing Group
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