NANOVID TRACKING: A NEW AUTOMATIC METHOD FOR THE STUDY OF MOBILITY IN LIVING CELLS BASED ON COLLOIDAL GOLD AND VIDEO MICROSCOPY HuGo GEERTS, MARK DE BRABANDER, RONNY NUYDENS, STAF GEUENS, MARK MOEREMANS, JAN DE MEY, AND PETER HOLLENBECK* Division of Cellular Biology and Chemotherapy, Department of Life Sciences, Janssen Pharmaceutica Research Laboratories, B-2340 Beerse, Belgium; *Medical Research Council Cell Biophysics Unit, London WC2B 5RL, United Kingdom ABSTRACT We describe a new automatic technique for the study of intracellular mobility. It is based on the visualization of colloidal gold particles by video-enhanced contrast light microscopy (nanometer video microscopy) combined with modern tracking algorithms and image processing hardware. The approach can be used for determining the complete statistics of saltatory motility of a large number of individual moving markers. Complete distributions of jump time, jump velocity, stop time, and orientation can be generated. We also show that this method allows one to study the characteristics of random motion in the cytoplasm of living cells or on cell membranes. The concept is illustrated by two studies. First we present the motility of colloidal gold in an in vitro system of microtubules and a protein extract containing a kinesin-like factor. The algorithm is thoroughly tested by manual tracking of the videotapes. The second study involves the motion of gold particles microinjected in the cytoplasm of PTK-2 cells. Here the results are compared to a study using the spreading of colloidal gold particles after microinjection. INTRODUCTION During the last few years, some elegant techniques for studying the lateral movement of molecules have been developed (Axelrod, 1976; Elson and Magde, 1974; Magde and Elson, 1974; Kapitza, 1985; Peters, 1984; Barak and Webb, 1982; Smith, 1979). They all use fluorescently labeled molecules, the time evolution of which can be followed by a variety of means. One of the main features of spectroscopic techniques is the use of concentrations of molecules, i.e., quantities that are related to the global number of molecules in a certain area, defined within the resolution of the chosen optics. This means that the global mobility behavior of a set of molecules is studied. Ideally one would like to follow individual molecules to obtain exact information on this mobility. For this one needs a particular marker. Such a marker is colloidal gold, which has been very popular for morphological studies. One can indeed couple antibodies to specific molecules with colloidal gold probes and study the localization at the ultrastructural level (electron microscopy). Recently, a new approach, called NANOVID (nanoparticle video microscopy), has coupled the colloidal gold technology with light microscopy (De Brabander et al., 1985). It turns out that, despite the very small size of the gold probes (20-40 nm), single probes can be made visible in the light microscope by video-enhanced contrast techniques (Allen et al., 1981; Inoue, 1981). Essentially, the technique uses BIOPHYS. J. a Biophysical Society * 0006-3495/87/11/775/08 Volume 52 November 1987 775-782 the potential of video microscopy to enhance contrast greatly by electronic subtraction of background intensity. Individual gold probes (20-40 nm) are observed with bright field illumination at maximal aperture. They are invisible to the eye but appear as clearly defined black dots on the video screen after contrast enhancement. They are unambiguously discerned from cellular organelles which produce no contrast at correct Kohler illumination because they are phase objects. The gold particles are visible because they scatter light out of the aperture of the optical system. We are now able to follow the motion of individual colloidal gold markers in living cells. Furthermore, by coupling this concept to the power of image processing technology and minicomputers, we can study statistics of different mobilities in great detail. This work presents the automatic tracking approach and its application to two different experimental systems. The first application is the study of the mobility of 40-nm gold probes in a microtubule system in vitro. The aim was to investigate the mechanism of saltatory motion, induced by a kinesin-like protein extract. This model system was chosen because we could easily check the results by manual tracking and because all motions (salta- tory and random) turned out to be mainly one-dimen- sional, a very exciting result. The second application is a demonstration of the mea- surement of cytoplasmic diffusion. Here we followed the spreading of colloidal gold particles shortly after microin- $2.00 775