Near-Field Scanning Optical Microscopy in Cell Biology and Cytogenetics

Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented.