Cell biology beyond the diffraction limit: near-field scanning optical microscopy

Throughout the years, fluorescence microscopy has proven to be an extremely versatile tool for cell biologists to study live cells. 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. A drawback of light microscopy is the fundamental limit of the attainable spatial resolution - similar to 250 urn - dictated by the laws of diffraction. The challenge to break this diffraction limit has led to the development of several novel imaging techniques. 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. 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