Videomicroscopic measurements in living cells: dynamic determination of multiple end points for in vitro toxicology.

Abstract

The possibility of digitization and processing of microscope images in "real time" (i.e., at video rates) has opened a variety of ways to dramatically improve the quality of microscopic images and to create new applications of light microscopy. One of the new techniques, video-enhanced contrast (VEC) microscopy, enables one to increase contrast and magnification to the extent that positions and movements of biological objects as small as 15-20 nm (e.g., small membrane-bounded vesicles and microtubules) can be analyzed in living cells. Organelle motion can be quantitatively described by a number of parameters such as velocity, straightness of path, and reversals of direction. The second group of videomicroscopic techniques is based on the measurement of fluorescence intensities of intracellular compounds by monochromatic light microscopy or using other low-light signals (video-intensified microscopy, VIM). The resulting images are two-dimensional arrays of fluorescence or absorption spectroscopy measurements and contain information on the amounts of intracellular metabolites or exogenous agents. Typical parameters for VIM measurements include Ca2+ concentration, pH value, metabolites and membrane potentials. Specific nontoxic dyes are also available to verify the identity of the organelles seen by the VEC techniques and to quantitate their abundance. The whole battery of new tests based on videomicroscopy can be applied at selected time intervals to a given set of cultured cells to obtain simultaneous measurements of multiple end points. However, since quantitative data for these parameters can be calculated from the live images in real time and encoded in the form of gray-shaded or pseudocolor images, they can also be continuously recorded and yield video films of the complete sequence of intracellular events during and after exposure to toxic or pharmacological agents. Videomicroscopy allows multiparametric studies to be performed with cultured cells, yielding a wealth of information that could be reached in the past only in animal experimentation. In addition, videomicroscopy enables us to observe directly and quantitate metabolic, physiological, and morphological parameters in the living cell that were not accessible by animal experimentation. It is therefore to be expected that videomicroscopy in the near future will catalyze a major shift from animal to in vitro experimentation in the fields of toxicology, pharmacology, and experimental pathology.