Part III: Microscopy Techniques

Chapter List

Optical microscopy, including wide-field microscopy, scanning microscopy and micromanipulation, has become an indispensable tool in cell biology and photo-biology. The present article is concentrated on wide-field microscopy and covers transillumination and fluorescence microscopy. Basic principles are described as well as advanced techniques, e.g. fluorescence lifetime imaging (FLIM), energy transfer spectroscopy (FRET) and total internal reflection fluorescence microscopy (TIRFM). A few applications are depicted, which may give some insight into a fascinating microcosmos.

13.1 Introduction

The theory of optical microscopy was established in the second half of the nineteenth century by Ernst Abb (1840 1905), although the first microscopes had been built much earlier. Usually, a microscope is characterized by a highly magnifying objective lens used for imaging a sample with a half-angle a of the incident cone and a numerical aperture A = n sin ?, where n is the refractive index of the medium between the sample and the objective lens. The numerical aperture is an important parameter for the quantity of light taken up by a microscope (which is proportional to A ²), for the lateral resolution ? x = ?/ A and for the depth of focus ? z = n ?/ A ², with ? being the wavelength of optical radiation.

Epiillumination...