Wide-field four-channel fluorescence imager for biological applications

A wide-field four-channel fluorescence imager has been developed. The instrument uses four expanded laser beams to image a large section (6 mm×9 mm). An object can be sequentially illuminated with any combination of 408-, 532-, 658-, and 784-nm lasers for arbitrary (down to 1 ms) exposure times for each laser. Just two notch filters block scattered light from all four lasers. The design approach described here offers great flexibility in treatment of objects, very good sensitivity, and a wide field of view at low cost. There appears to be no commercial instrument capable of simultaneous fluorescence imaging of a wide field of view with four-laser excitation. Some possible applications are following events such as flow and mixing in microchannel systems, the transmission of biological signals across a culture, and following simulations of biological membrane diffusion. It can also be used in DNA sequencing by synthesis to follow the progress of the photolytic removal of dye and terminator. Without utilizing its time resolution, it can be used to obtain four independent images of a single tissue section stained with four targeting agents, with each coupled to a different dye matching one of the lasers.