Simultaneous Nomarski and fluorescence imaging during video microscopy of cells

A video microscope designed to allow low light level fluorescence imaging of cells during simultaneous high-resolution differential interference contrast (DIC) imaging, without the fluorescence light losses of 60-90% normally associated with this contrast-enhancement technique, is described. Transmitted light for DIC imaging, filtered at greater than 620 nm, passes through standard DIC optical components, (1/4 lambda-plate, polarizer, and Wollaston prism) before illuminating the cells. Transmitted light and fluorescence emission pass through a second Wollaston prism but not through the analyzer, which is repositioned more distally in the optical path. Prisms designed to reflect light out a side port of the microscope to a video camera have been replaced with a dichroic mirror. This mirror reflects fluorescence emission out the side port to a low light-sensitive video camera. The spectrally distinct transmitted light continues through the dichroic mirror to an overhead camera through a polarizer (analyzer), which completes the DIC optical path. The fluorescence and DIC images can be viewed simultaneously on side-by-side video monitors, examined sequentially by an image-processing computer, or examined simultaneously using a video splitter/inserter. The ability to image cells with high resolution simultaneously with low light level fluorescence imaging should find wide applicability whenever it is necessary or desirable to correlate fluorescence intensity or distribution with specific cell structure or function.