Abstract
A major benefit of fluorescence microscopy is the now plentiful selection of fluorescent markers. These labels can be chosen to serve complementary functions, such as tracking labeled subcellular molecules near demarcated organelles. However, with the standard 3 or 4 emission channels, multiple label detection is restricted to segregated regions of the electromagnetic spectrum, as in RGB coloring. Hyperspectral imaging allows the user to discern many fluorescence labels by their unique spectral properties, provided there is significant differentiation of their emission spectra. The cost of this technique is often an increase in gain or exposure time to accommodate the signal reduction from separating the signal into many discrete excitation or emission channels. Recent advances in hyperspectral imaging have allowed the acquisition of more signal in a shorter time period by scanning the excitation spectra of fluorophores. Here, we explore the selection of optimal channels for both significant signal separation and sufficient signal detection using excitation-scanning hyperspectral imaging. Excitation spectra were obtained using a custom inverted microscope (TE-2000, Nikon Instruments) with a Xe arc lamp and thin film tunable filter array (VersaChrome, Semrock, Inc.) Tunable filters had bandwidths between 13 and 17 nm. Scans utilized excitation wavelengths between 340 nm and 550 nm. Hyperspectral image stacks were generated and analyzed using ENVI and custom MATLAB scripts. Among channel consideration criteria were: number of channels, spectral range of scan, spacing of center wavelengths, and acquisition time.