![]() ![]() Initially, this contrast problem was tackled by the use of immunogold-labeling approaches ( Damjanovich et al., 1995 Neagu et al., 1994). This seriously limits the usefulness of AFM for high-resolution imaging on cells. Hence, although individual molecules can be seen, their identities cannot be defined. However, although AFM produces a high-resolution topographical picture of the sample, it lacks chemical specificity. 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). 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. on the molecular scale), the need for imaging techniques that have a higher resolution is growing. ![]() Since a large body of evidence indicates that dynamic cell-signaling events start by oligomerization and interaction of individual proteins (i.e. In practice this means that the maximal resolution in optical microscopy is ∼250-300 nm. This diffraction limit originates from the fact that it is impossible to focus light to a spot smaller than half its wavelength. The limit to the resolution that can be reached in optical imaging techniques is directly related to the wavelength of the light. NSOM allows detection of individual fluorescent proteins as part of multimolecular complexes on the surface of fixed cells, and similar results should be achievable under physiological conditions in the near future. One of them, near-field scanning optical microscopy (NSOM), allows fluorescence imaging at a resolution of only a few tens of nanometers and, because of the extremely small near-field excitation volume, reduces background fluorescence from the cytoplasm to the extent that single-molecule detection sensitivity becomes within reach. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution – ∼250 nm – dictated by the laws of diffraction. Its high sensitivity and non-invasiveness, together with the ever-growing spectrum of sophisticated fluorescent indicators, ensure that it will continue to have a prominent role in the future. Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. ![]()
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