Multichannel Hyperspectral Imaging and Characterization of Far-Red Fluorophores using a Thin-Film Tunable Filter

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Multicolour fluorescence imaging has traditionally been limited to the use of fluorophores with non-overlapping emission spectra. The advent of hyperspectral imaging has made it possible to distinguish signals with partial overlap by acquiring fluorescence over narrow emission bands. We report here the implementation of one such approach, using the Semrock Versachrome™ thin film tunable filter integrated via a custom-built insert into an Olympus IX-83 dual-deck inverted TIRF microscope. We demonstrate the ability to distinguish and resolve the emission spectra of two far-red fluorophores, Alexa 647 and ATTO 680, by varying the angle of incidence of the emitted light on the Versachrome. We have implemented a galvanometer based solution that provides fast and precise control over the transmission band. The high transmission coefficient of the thin film filter allowed us to reduce the exposure time and acquire a full-spectrum sequence in less than 100ms. Deconvolution of the fluorescence signals allowed determination of the relative fluorophore population maximizing the recovery of information. We demonstrate the application of the technique to imaging multiple sub-cellular targets with well-characterized supra-molecular morphology, including cytoskeletal, mitochondrial, plasma membrane and nuclear proteins. This technique for high-speed spectral imaging will bring new applications into focus and improve performance in already established approaches.