Wedge prism approach for simultaneous multichannel microscopy

Abstract

Multichannel (multicolor) imaging has become a powerful technique in biology research for performing in vivo neuronal calcium imaging, colocalization of fluorescent labels, non-invasive pH measurement, and other procedures. We describe a novel add-on approach for simultaneous multichannel optical microscopy based on simple wedge prisms. Our device requires no alignment and is simple, robust, user-friendly, and less expensive than current commercial instruments based on switchable filters or dual-view strategies. Point spread function measurements and simulations in Zemax indicate a reduction in resolution in the direction orthogonal to the wedge interface and in the axial direction, without introducing aberration. These effects depend on the objective utilized and are most significant near the periphery of the field of view. We tested a two-channel device on C. elegans neurons in vivo and demonstrated comparable signals to a conventional dual-view instrument. We also tested a four-channel device on fixed chick embryo Brainbow samples and identified individual neurons by their spectra without extensive image postprocessing. Therefore, we believe that this technology has the potential for broad use in microscopy.