Chapter

Science of Microscopy

pp 790-834

Nanoscale Resolution in Far-Field Fluorescence Microscopy

  • Stefan W. HellAffiliated withDepartment for NanoBiophotonics, Max Planck Insitute of Biophysical Chemistry
  • , Andreas SchönleAffiliated withDepartment for NanoBiophotonics, Max Planck Insitute of Biophysical Chemistry
  • , A. Van den BosAffiliated withDelft University of Technology

The discovery of the diffraction barrier in 1873 by Ernst Abbe (1873) has shown that, relying on propagating light waves and regular lenses, the traditional light microscope cannot resolve spatial structures that are smaller than about half the wavelength of the focused light. This physical insight has been accepted as an unalterable limitation of focusing light microscopy and has consequently triggered the invention of nonoptical imaging techniques, such as electron and scanning probe microscopy. In spite of the tremendous improvement in resolution brought about by these methods, light microscopy has maintained its importance in many fields of science. The reasons are mainly a number of exclusive advantages, the most prominent of which is the ability to noninvasively image (living) specimens. Light microscopy also entails the possibility of using fluorescence as a highly specific signature of the specimen features of interest. Fluorescence is particularly attractive when provided by endogenous fluorescence markers, i.e., proteins in physiologically intact cells. Mapped with a confocal or multiphoton excitation microscope (Sheppard and Kompfner, 1978; Wilson and Sheppard, 1984; Denk et al., 1990), fluorescence emission readily yields protein three-dimensional (3D) distributions, or that of other fluorescently labeled molecules from the strongly convoluted inside of biological specimens.