Increasing the Resolution of Far-Field Fluorescence Light Microscopy by Point-Spread-Function Engineering

Abstract

Microscopy plays a key role in many areas of modern science. This is probably because for humans visual perception is the most important way of obtaining information. Therefore, the visualization of minute structures has contributed a great deal to the better understanding of many phenomena in nature. The most important property of the microscope is the resolution, which is the ability to distinguish closely positioned objects. The resolution determines the smallest observable structure of a specimen. Therefore, most of the develop-ments in microscopy aimed at higher resolution, and increases in resolution have always led to new discoveries in science. A good example is the improvement of resolution in light microscopy at the end of the 19th century. In those days, the resolution of the light microscope was limited by chromatic and spherical aberra-tions. Mastery of aberrations improved the resolution by a factor of 2, thus allowing the first observation of chromosomal behavior during cell mitosis. (1) This progress was primarily due to the efforts of Ernst Abbe working in close collaboration with Carl Zeiss. Studying the image formation in light micro-scopy, Abbe realized the importance of the wave nature of light and its central role in the resolution issue. (2) He found a fundamental limit that still bears his name. When imaging a pointlike object with a lens, the point is imaged into a blurred spot whose radius depends on the wavelength of the light and the angular aperture of the lens. Abbe argued that objects 'closer than about half the wavelengths should not be distinguishable in a light microscope'. In fact, a