SPEKcheck — fluorescence microscopy spectral visualisation and optimisation: a web application, javascript library, and data resource [version 1; referees: 3 approved]

Abstract

Advanced fluorescence imaging methods require careful matching of excitation sources, dichroics, emission filters, detectors, and dyes to operate at their best. This complex task is often left to guesswork, preventing optimal dye:filter combinations, particularly for multicolour applications. To overcome this challenge we developed SPEKcheck, a web application to visualise the efficiency of the light path in a fluorescence microscope. The software reports values for the excitation efficiency of a dye, the collection efficiency of the emitted fluorescence, and a "brightness" score, allowing easy comparison between different fluorescent labels. It also displays a spectral plot of various elements in the configuration, enabling users to readily spot potential problems such as low efficiency excitation, emission, or high bleedthrough. It serves as an aid to exploring the performance of different dyes and filter sets.