Fluorescence spectroscopy offers particularly sensitive tools for the investigation of physical properties of macromolecules in solution. Differences in molecular structure under varying experimental conditions are often reflected in quantum yield and lifetime of natural or synthetically inserted fluorescent probes. inserted fluorescent probes. Energy transfer between fluorescent groups at known places of a primary sequence can be used to measure distances between the label sites (Eisinger, 1976) as well as to investigate their relative motion, e.g. measurements of the kinetics of variations in the end to end distance of a polymer (E. Haas & I. Steinberg, personal communication).

It is only some 50 years ago that biophysicists obtained reliable experimental methods for estimating the molecular weights of biological macromolecules, chiefly as a result of Svedberg's work in developing the ultracentrifuge as an analytical instrument (Svedberg & Pedersen, 1940). Having gained some understanding of the size of proteins, interest soon thereafter turned to the shape and rotational relaxation times of these molecules, and Perrin's work on fluorescence depolarization helped to lay the foundations there (Perrin, 1929). Biophysics had to wait for the development of X-ray spectroscopy of proteins and nucleic acids to provide a picture of the interior structure of biological macromolecules.

The dynamics of macromolecules, the subject of this symposium, are most directly studied by simply looking through a microscope and observing the molecular motion. With a microscope, we can resolve the size and shape of large particles, as well as monitor dynamic motion. For smaller particles, particularly single macromolecules, we cannot resolve the size or shape; but it is still possible to observe the motion, if we can make the particles appear as bright points of light sprinkled dilutely over a dark background. Siedentopf & Zsigmondy (1903) demonstrated this fact with a device which came to be called the ultramicroscope.

The preceding paper by Douglas Magde has recounted the basic principles of Fluorescence Correlation Spectroscopy (FCS) as originally described (see Magde, Elson & Webb, 1972; Elson & Magde, 1974; Magde, Elson & Webb, 1974 Elson & Webb, 1975; referred to collectively as MEW), and has described the first application to chemical kinetics. In this paper I shall first illustrate the same principles of FCS with a simple graphical demonstration model based on the scheme for application to lateral diffusion in membranes as it was developed in our laboratory by Dr T. J. Herbert; I shall then proceed to discuss some current research in our group organized jointly with Professor E. L. Elson at Cornell.

A quantitative relationship between polarization properties of fluorescence light and molecular rotational diffusion was first derived by Perrin (1926). His results, which concerned spherical particles, have later been refined to the more complex rotational motion of asymmetric bodies (Memming, 1961; Chuang & Eisenthal, 1972; Ehrenberg & Rigler, 1972; Belford, Belford & Weber, 1972).

In the initial period of application of photon correlation spectroscopy to molecular phenomena, attention has mainly been focused on the improvement of experimental approaches and instrumental techniques, on feasibility studies of certain cases where theoretical considerations seemed to indicate the prospective usefulness and on various applications that are relatively straightforward, such as the measurement of translational or rotational diffusion coefficients, or the determination of flow velocity by Doppler anemometry.

About 11 years ago Ford & Benedek (1964) and Cummins, Knable & Yeh (1964) developed some fascinating new light-scattering techniques for the study of macromolecular motions in solution. Enthusiastic researchers have over the years endowed these new methods with a variety of colourful names, including optical mixing or light-beating spectroscopy, heterodyne or homodyne beating, intensity fluctuation spectroscopy, Rayleigh spectroscopy, and quasielastic or dynamic light scattering.