A novel dual-color bifocal imaging system for single-molecule studies

In this paper, we report the design and implementation of a dual-color bifocal imaging (DBI) system that is capable of acquiring two spectrally distinct, spatially registered images of objects located in either same or two distinct focal planes. We achieve this by separating an image into two channels with distinct chromatic properties and independently focusing both images onto a single CCD camera. The two channels in our device are registered with subpixel accuracy, and long-term stability of the registered images with nanometer-precision was accomplished by reducing the drift of the images to ∼5 nm. We demonstrate the capabilities of our DBI system by imaging biomolecules labeled with spectrally distinct dyes and micro- and nano-sized spheres located in different focal planes.