1 Department of Tumor Immunology, University Medical Center Nijmegen, NCMLS/187 TIL, PO Box 9101, 6500HB Nijmegen, The Netherlands
2 Applied Optics Group, Faculty of Applied Physics and MESA+ Research Institute, University of Twente, PO Box 217, 7500AE Enschede, The Netherlands
*Author for correspondence (e-mail: c.figdor{at}mailbox.kun.nl)
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SUMMARY |
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Key words: Super-resolution, Near-field scanning optical microscopy, Single-molecule detection, Distribution, Cell surface, Membrane, GFP
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Introduction |
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The ability to tailor fluorescent fusion proteins, exploiting the strong autofluorescence of the green fluorescent protein (GFP) family (Patterson et al., 2001), has fueled interest in fluorescence microscopy even further. The most successful applications of these fluorescent proteins are in gene expression, protein targeting and trafficking, and protein-protein interaction studies (Tsien, 1998; Lippincott-Schwartz et al., 2001). Employing various forms of these fluorescent proteins, in most cases using fluorescence resonance energy transfer (FRET) as a read-out (Wouters et al., 2001), highly potent cellular indicators for calcium (Miyawaki et al., 1997; Miyawaki et al., 1999), pH (Llopes et al., 1998), cyclic AMP (Zaccolo et al., 2000), cyclic GMP (Honda et al., 2001), caspases (Harpur, 2001) and small G protein activity (Mochizuki et al., 2001), among others, have been developed.
The limit to the resolution that can be reached in optical imaging techniques is directly related to the wavelength of the light. This diffraction limit originates from the fact that it is impossible to focus light to a spot smaller than half its wavelength. In practice this means that the maximal resolution in optical microscopy is 250-300 nm. Since a large body of evidence indicates that dynamic cell-signaling events start by oligomerization and interaction of individual proteins (i.e. on the molecular scale), the need for imaging techniques that have a higher resolution is growing. Traditionally, high-resolution cell biology (Table 1) is the arena of electron microscopy, which offers superb resolution but lacks the above-mentioned advantages of fluorescence microscopy. The advent of scanning probe microscopy (Table 1), and especially atomic force microscopy (AFM), in which an atomically sharp probe attached to a cantilever is scanned over the surface of interest, has made nanometer resolution also attainable on living cells (Hansma et al., 1994; Putman et al., 1994). However, although AFM produces a high-resolution topographical picture of the sample, it lacks chemical specificity. Hence, although individual molecules can be seen, their identities cannot be defined. This seriously limits the usefulness of AFM for high-resolution imaging on cells. Initially, this contrast problem was tackled by the use of immunogold-labeling approaches (Damjanovich et al., 1995; Neagu et al., 1994). A promising new way around the problem comes from work on the specific labeling of the AFM probe with biomolecules (e.g. with antibodies or ligands). This introduces a contrast mechanism based on specific interactions between the probe and certain types of molecule in the specimen (Willemsen et al., 2000). Other attempts to enhance AFM contrast involve the modification of the probe by fluorescent molecules, which introduces an optical contrast mechanism (Vickery and Dunn, 2001). Currently, however, the combination of high-resolution scanning probe and fluorescence microscopy is the realm of another scanning probe technique: near-field scanning optical microscopy (NSOM).
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NSOM |
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The optical detection sensitivity of NSOM depends largely on the extremely small excitation/detection volume set by the aperture dimensions as well as the depth of penetration of the near-field into the specimen. Together, these properties effectively reduce background fluorescence and thereby enhance detection sensitivity. Betzig and Chichester exploited this eight years ago, providing the first observation of single-molecule fluorescence under ambient conditions (Betzig and Chichester, 1993). Furthermore, employing the polarization characteristics of the near-field, they showed that it is possible to determine the full spatial orientation of fluorescent molecules by making use of polarization-sensitive fluorescence detection (see below). Subsequently, developments in instrumentation that greatly improved signal-to-background ratios allowed single-molecule fluorescence studies to come within reach of far-field methods such as total internal reflection, confocal and bright-field microscopy (see Table 2). The advantage of single-molecule studies is that they provide a way to monitor time-dependent processes and reaction pathways in non-equilibrated systems, which reveals the distribution of a given molecular property instead of a statistical average. This has already led to a whole new frontier in science, its applications ranging from basic photo-physics and material research to biology (Frontiers in Chemistry, 1999; Xie and Lu, 1999; Sako et al., 2000; Ishii and Yanagida, 2000). Nowadays almost all experiments in this field use far-field methods, mainly because they are relatively easy to use. However, the obvious disadvantage is that, because of the diffraction-limited system response, only scarcely labeled samples can be studied, but, in biology, molecules are usually present in close proximity. The combination of topographical information, optical super-resolution and single-molecule detection sensitivity therefore makes NSOM a unique tool for biological applications.
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Biological applications of NSOM |
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So far, most applications of NSOM in biology involve systems that are more or less isolated (Table 3) for example, studies on fluorescently labeled chromosomes (Moers et al., 1996), DNA (Ha et al., 1996; Garcia-Parajo et al., 1998) and purified fluorescent proteins (Garcia-Parajo et al., 1999; Garcia-Parajo et al., 2000). Cell biological studies include fluorescence imaging of cytoskeletal components in 3T3 fibroblasts (Betzig et al., 1993) and colocalization of malarial and host skeletal proteins on malaria-infected erythrocytes (Enderle et al., 1997). Furthermore, sub-wavelength-sized membrane patches in human skin fibroblasts (Hwang et al., 1998) and activation-dependent receptor clustering on a human breast carcinoma cell line have also been studied (Nagy et al., 1999). Although these studies show a resolution well beyond that of a confocal microscope, to the best of our knowledge no study showing single-molecule detection sensitivity in a cell membrane by NSOM has been reported.
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Single-molecule detection on cells by NSOM |
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Single-molecule localization accuracy by NSOM as compared to other microscopical techniques |
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These intrinsic advantages of NSOM might revolutionize the life sciences. But why is NSOM hardly used in cell biology, despite its development almost two decades ago? First, as an imaging tool, NSOM is a complex technique that requires well-trained operators. Second, as we point out above, the resolution and sensitivity of a near-field microscope depend strongly on the quality of the probe aperture and the accuracy of the feedback system, necessitating full control of the technology. Moreover, the top-grade near-field microscopes are still under development in experimental physics laboratories and are therefore not widely accessible. Perhaps most important is the fact that, despite great efforts of various groups including our own, the standards set by the initial work of Betzig and Chichester cannot yet be reached in a liquid and more physiological environment. This is the technical challenge that has to be faced in the coming years.
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Conclusion/perspectives |
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In the past decade, especially the past two years, several other approaches to break the diffraction limit have been developed. These include interferometric microscopy methods such as 4Pi confocal microscopy (Hell and Stelzer, 1992), I5M (Gustafsson et al., 1999), standing-wave-total-internal-reflection fluorescence microscopy (Cragg and So, 2000) and harmonic excitation light microscopy (Frohn et al., 2000). These methods, however, do not have single-molecule detection sensitivity, and all require extended electronic post-processing of the images. Recently, a novel technique involving point spread function (PSF) engineering, which exploits stimulated emission depletion (STED), has been described (Klar et al., 2000). This method involves the induced quenching of fluorescence by stimulated emission at the rim of the diffraction-limited focal spot, thereby squeezing it to an almost spherical shape of 100 nm in diameter. Thus far, this is the only technique that seriously rivals the small excitation/detection volume and therefore sensitivity of NSOM. The maximal resolution obtained with aperture-type NSOM relates to the limited energy throughput of the near-field probe. This limits the minimum size of the aperture to be used and hence the resolution of the microscope to
20 nm. A possible way around this involves exploiting single-molecule emitters, attached to a scanning probe, which act as light source to excite molecules in the sample (Michaelis et al., 2000).
The most important technical challenge that remains is the construction of an NSOM instrument that operates under physiologically relevant conditions and allows the study of soft, rough and motile surfaces, such as the plasma membrane of living cells. When combined with single-molecule detection sensitivity and an optical resolution that is comparable to transmission electron microscopy, this will prove to be an invaluable tool in cell biology. These are truly exciting times.
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ACKNOWLEDGMENTS |
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Footnotes |
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* Here, we focus on fluorescence-mode NSOM. Applications of transmission-type NSOM to biological systems have also been described; however, for these the reader is referred to Van Hulst and Moers (Van Hulst and Moers, 1996).
* Sample preparation will be detailed elsewhere.
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