Analysis of fast dynamic processes in living cells: High-resolution and high-speed dual-color imaging combined with automated image analysis

The generation of spectral mutants of the green fluorescent protein (GFP) s et the stage for multiple-color imaging in living cells. However the nse of this technique has been limited by a spectral overlap of the available GFP mutants and/or by insufficient resolution in both time and space. Using a new setup for dual-color imaging, we demonstrate here the visualization of small, fast moving vesicular structures with a high time resolution. Two GF P-fusion proteins were generated: human chromogranin B, a secretory granule matrix protein, and phogrin, a secretory granule membrane protein. They we re tagged with enhanced yellow fluorescent protein. (EYFP) and enhanced cya n fluorescent protein (ECFP), respectively. Both fusion proteins were co-tr ansfected in Vero cells, a cell line from green monkey kidney. EYFP and ECF P were excited sequentially at high time rates using a monochromator. Charg ed coupled device (CCD)-based image acquisition resulted in 5-8 dual-color images per second, with a resolution sufficient to detect transport vesicle s in mammalian cells. Under these conditions, a fully automated time-resolv ed analysis of the movement of color-coded objects was achieved. The develo pment of specialized software permitted the analysis of the extent of coloc alization between the two differentially labeled sets of cellular structure s over time. This technical advance will provide an important tool to study the dynamic interactions of subcellular structures in living cells.