Imaging DNA: from the molecule to the cell

Abstract

Cells function through the complex temporal and spatial interplay ofions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level. The same arguments apply to multicellular biological questions in fields such as developmental biology and neurobiology. There has been a renaissance and revolution in the use of light microscopy in the biological sciences over the last five years. The renaissance has been caused by the biologists' need to acquire chemical and molecular information from living cells and tissues in time and space. The revolution has been fueled by the integration of the heretofore distinct fields of fluorescent probe chemistry, biochemistry/molecular biology, physical optics, engineering and computer science. There are now large numbers of specific "probes" of cell chemistry and molecular activity, as well as machine vision light microscopes that can quantify the chemistry and molecular dynamics of cellular processes. Together, new classes of reagents and machine vision microscope systems can yield the temporal and spatial information that is needed to define the chemistry of cell functions. We-have developed a machine vision light microscope, the Multimode Cytometer, that is capable of performing many distinct modes of light microscopy on living cells and tissues on the same instrument. Video-enhanced contrast, ratio imaging, serial plane deconvolution-based 3-D microscopy, multicolor imaging, reflection interference, uncaging of caged compounds can all be accomplished by computer control on the present generation system that is based on the Macintosh platform. In addition, we have developed fluorescent reagents that yield information on calcium binding to calmodulin, the pore size of cytoplasm, dynamic distribution of contractile proteins and the phosphorylation of myosin. The combination of reagents and instrumentation has enabled us to define the chemical and molecular dynamics of cell locomotion. This same technology will be valuable in defining the chemical and molecular dynamics responsible for any cellular and intercellular function.