Scanning Microscopy


Optical fluorescence is characteristic of molecules and their environment, and dyes can be made whose fluorescence is altered by reversible binding to specific ions. By introducing these into the cytosol, fluorescence microscopy can be used to form dynamic images of ionic activities in living cells under experimental manipulation. Optical fluorescence spectra are broad-band, and if specific ion binding alters the wavelength of maximal excitation or emission, quantitative measurements can be made from the ratio of images taken at two different wavelengths, eliminating errors due to spatial variations in dye concentration and optical path-length. This method is analogous to continuum normalisation in X-ray microanalysis, and is implemented using a sensitive video camera and computer processing of digitised images. Fluorescent indicators exist for calcium, magnesium, hydrogen, sodium, zinc and chloride ions. Most imaging work has been on calcium, which is important in many cell signalling processes, and several calcium indicators are available with different spectral properties. Spatial resolution is limited to a few μm by out-of-focus blur, but repeated images can be captured with a time resolution as low as 200 msec, and by using dyes with high binding affinity, detection limits can be lower than by X-ray methods. There is a large and fast-growing literature of applications to many plant and animal cell-types.

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