Real-time multi-wavelength fluorescence imaging of living cells.

We describe a new real-time fluorescence video microscope design for capturing intensified images of cells containing dual wavelength "ratio" dyes or multiple dyes. The microscope will perform real-time capture of two separate fluorescence emission images simultaneously, improving the time resolution of spatial distribution of fluorescence to video frame rates (30 frames/s or faster). Each emission wavelength is imaged simultaneously by one of two cameras, then digitized, background corrected and appropriately combined at standard video frame rates to be stored at high resolution on tape or digital video disk for further off-line analysis. Use of low noise, high sensitivity image intensifiers, coupled to CCD cameras produce stable, high contrast images using ultra low light levels with little persistence or bloom. The design has no moving parts in its optical train, which overcomes a number of technical difficulties encountered in the present filter wheel designs for multiple imaging. Coupled to compatible image processing software utilizing PC-AT computers, the new design can be built for a significantly lower cost than many presently available commercial machines. The system is ideal for ratio imaging applications; the software can calculate the ratio of fluorescence intensities pixel by pixel and provide the information to generate false-color images of the intensity data as well as other calculations based on the two images. Thus, it provides a powerful, inexpensive tool for studying the real-time kinetics of changes in living cells. Examples are presented for the kinetics of rapidly changing intracellular calcium detected by the calcium indicator probe indo-1 and the redistribution kinetics of multiple vital dyes placed in cells undergoing cell fusion.