Quarterly Reviews of Biophysics 9, 1 (1976), pp. 35-47 Printed in Great Britain
Chemical Kinetics and Fluorescence Correlation Spectroscopyf D. MAGDE Department of Chemistry, University of California, San Diego, Lajolla, California 92093, U.S.A.
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. They and later workers, including Svedberg (1928), were able to investigate particles as small as 6 nm. Usually the light emitted by the particle was simply elastic scattered light, but it was recognized also that fluorescent labelled particles could be studied. With this ultramicroscope, individual macromolecules could be counted and their diffusion through a defined sample volume could be studied. Note, however, that those workers were not really interested in observing single particles. In fact, they had to make many tedious observations to develop statistics for the average behaviour which were the real concern. Concentration correlation analysis may be thought of very accurately as a sort of ultra-ultra microscope, wherein we make a second sacrifice. We not only give up trying to resolve the shapes of the individual particles but we no longer even try to distinguish one particle from another. We t This paper was presented at the symposium on Dynamics of Macromolecules in Solution at the 5th International Biophysics Congress in Copenhagen, Denmark, 4-9 August 1975. [35]
36
D. MAGDE
Fig. 1. Schematic of the optical apparatus for an FCS experiment. D, detection system (photomultiplier); M, laser intensit ymonitor; DA, differential amplifier; COR, correlator.
retain only the ability to monitor as a function of time the total number of particles within a carefully denned volume. From this record we develop the same statistics for the average behaviour without ever 'seeing' an individual particle. It is just a question of when we perform the ensemble average. The newer method represents very little sacrifice compared to the ultramicroscope while extending significant benefits. Most importantly it permits us to work with even smaller molecules; they need not be macromolecules at all. Using fluorescence rather than scattering, Professors Webb, Elson and I were able to work with molecules of completely arbitrary size (Magde, Elson & Webb, 1972; Elson & Magde, 1974; Magde, Elson & Webb, 1974). Moreover, it is possible to study in this way not only such hydrodynamic processes as diffusion, but also chemical dynamic behaviour, the latter being surprisingly more difficult. Another way to probe chemical dynamics by analysing concentration correlations using a conductometric method was demonstrated by Feher & Weissman (1973). Two technical advances made possible the development of concentration correlation methods. First, lasers offer an incredibly bright light source which is also extremely convenient. Early ultramicroscope studies relied on sunlight which is bright by conventional standards but pales next to a laser and is most inconvenient. Secondly and just as dramatic has been the advance in electronic data processing which permits the extraction of the required statistical parameters from the noise-like experimental record. Fluorescence correlation experiments can be performed with an apparatus such as that shown in Fig. 1. A laser beam of appropriate colour, green for the experiments discussed today, is focused into a sample
Chemical kinetics andfluorescencecorrelation spectroscopy 37
Fig. a. Definition of the mean square fluctuations, (Si2}, around the mean current and of the correlation function G(T). = [«(O]mean>
Chemical Kinetics and Fluorescence Correlation Spectroscopyf D. MAGDE Department of Chemistry, University of California, San Diego, Lajolla, California 92093, U.S.A.
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. They and later workers, including Svedberg (1928), were able to investigate particles as small as 6 nm. Usually the light emitted by the particle was simply elastic scattered light, but it was recognized also that fluorescent labelled particles could be studied. With this ultramicroscope, individual macromolecules could be counted and their diffusion through a defined sample volume could be studied. Note, however, that those workers were not really interested in observing single particles. In fact, they had to make many tedious observations to develop statistics for the average behaviour which were the real concern. Concentration correlation analysis may be thought of very accurately as a sort of ultra-ultra microscope, wherein we make a second sacrifice. We not only give up trying to resolve the shapes of the individual particles but we no longer even try to distinguish one particle from another. We t This paper was presented at the symposium on Dynamics of Macromolecules in Solution at the 5th International Biophysics Congress in Copenhagen, Denmark, 4-9 August 1975. [35]
36
D. MAGDE
Fig. 1. Schematic of the optical apparatus for an FCS experiment. D, detection system (photomultiplier); M, laser intensit ymonitor; DA, differential amplifier; COR, correlator.
retain only the ability to monitor as a function of time the total number of particles within a carefully denned volume. From this record we develop the same statistics for the average behaviour without ever 'seeing' an individual particle. It is just a question of when we perform the ensemble average. The newer method represents very little sacrifice compared to the ultramicroscope while extending significant benefits. Most importantly it permits us to work with even smaller molecules; they need not be macromolecules at all. Using fluorescence rather than scattering, Professors Webb, Elson and I were able to work with molecules of completely arbitrary size (Magde, Elson & Webb, 1972; Elson & Magde, 1974; Magde, Elson & Webb, 1974). Moreover, it is possible to study in this way not only such hydrodynamic processes as diffusion, but also chemical dynamic behaviour, the latter being surprisingly more difficult. Another way to probe chemical dynamics by analysing concentration correlations using a conductometric method was demonstrated by Feher & Weissman (1973). Two technical advances made possible the development of concentration correlation methods. First, lasers offer an incredibly bright light source which is also extremely convenient. Early ultramicroscope studies relied on sunlight which is bright by conventional standards but pales next to a laser and is most inconvenient. Secondly and just as dramatic has been the advance in electronic data processing which permits the extraction of the required statistical parameters from the noise-like experimental record. Fluorescence correlation experiments can be performed with an apparatus such as that shown in Fig. 1. A laser beam of appropriate colour, green for the experiments discussed today, is focused into a sample
Chemical kinetics andfluorescencecorrelation spectroscopy 37
Fig. a. Definition of the mean square fluctuations, (Si2}, around the mean current and of the correlation function G(T). = [«(O]mean>
