Under NSF support we have developed several generations of visualization and analysis software. We have developed a program for interactively scanning through a 3D or 4D image one 2D image at a time while simultaneously displaying corresponding electrophysiology data and interactively performing a variety of measurements on both. We have also pioneered in the development of volume visualization in fluorescence microscopy. Our research in visualization and analysis has resulted in the Data Visualization and Analysis Environment (DAVE) that can interactively visualize, in stereo, 3D single, double, or triple labeled images, simultaneously display volume data (fluorescently labeled plasma membrane pumps or channels) and surface data (e.g., the membrane itself ), automatically locate, size and sort, by total fluorescence intensity, discretely distributed sites, and automatically locate, count and highlight the colocalization sites of any two labels. DAVE has the ability to step through a time-series of 3D images and visually compare each with chosen "reference" time points in order to better understand temporal changes. DAVE supports viewing and analyzing images collaboratively over the Internet. Since we spend more time viewing and analyzing images than acquiring them, the capabilities of DAVE are an indispensable part of the DIM.

We have developed methods for measuring the degree of colocalization of two fluorophores in 3D. We have developed Monte Carlo methods for estimating the statistical significance of that colocalization and we have studied the effects of sub-pixel mis-registration of the two images. We have developed a deformable model shrink wrap method for locating the plasma membrane in smooth muscle cells and have used it to greatly reduce the labor required to analyze the relationship of Protein Kinase C to the cell membrane proteins. We have also developed methods of analysis for detecting and counting single copy mRNA probes.

We have developed a sophisticated data analysis and visualization environment (DAVE) which provides interactive visualization of, single, double, or triple labeled images in 3D and over time. DAVE is over 100,000 lines of ANSI-C code, based upon standards (OpenGL, X11) and public-domain tools (XForms), and contains hundreds of capabilities, many uniquely optimized for our instruments and biological problems and not found in other visualization packages. For instance, it permits simultaneous viewing of "surface" data (e.g., a plasma membrane found via our deformable model) and volume data (fluorescently labeled pumps, channels, etc.). It can also aid in the visualization of temporal changes. This is accomplished by permitting any two 3D images from a time series to be displayed simultaneously (as different colors in the same image). For example, total mitochondrial motion can be appreciated by fixing the red channel in the displayed image to be the initial mitochondrial image (for a reference) and permitting the green channel to cycle through all of the other images in the time series. DAVE can also automatically identify and display colocalized structures in dual-labeled images. The degree of colocalization can be quantified. Data can also be quantified at the voxel level by displaying individual voxels as cubes with its numeric value. Capabilities such as these are critical for understanding complex structural and functional relationships within and between images of molecular distributions measured with our novel instruments. A few of its major capabilities are the ability to render single, double, or triple-wavelength images and display them together; the ability to display colocalization of multiple dyes; 4D time series playback, with looping; full motion stereo, zoom, and pan. In addition, DAVE provides a number of interactive data analysis tools that we have found useful for 4-D images.