The tutorial initializes with the spectral profile from 450 to 600 nanometers of a calcium biosensor (YC3.6) being presented in the graph on the left in the window. A cartoon illustration of the fluorescent protein biosensor appears to the right of the graph with green calcium ions beneath. The cyan fluorescent protein (ECFP) is represented by a cyan barrel and the yellow fluorescent protein (EYFP) is represented by a yellow barrel when FRET occurs (it is otherwise gray). Images from a lambda stack composed of sequential 10-nanometer wavebands are presented beneath the main window. In order to operate the tutorial, use the FRET Progress slider to increase the calcium ion concentration and evoke a structural change to the biosensor that produces FRET. Note changes to the intensities of the nucleus in the lambda stack wavebands as the degree of FRET increases.

The complex signals encountered in FRET microscopy are confounded by the excessive amounts of spectral overlap that are required by the fluorophores in order to undergo resonance energy transfer. Therefore, in addition to the ability of spectral imaging to separate fluorophore spectra in multicolor fixed and living cells, the technique is uniquely suitable for unraveling the emission contributions from donor and acceptor fluorophores in FRET imaging. Modern high-performance confocal microscopes equipped with multianode detectors are particularly suited for FRET analysis due to their high rate of image capture, which is often necessary when investigating fluorescent protein biosensors that operate on the millisecond timescale. With 32-channel multianode detectors, spectral imaging confocal microscopes such as the Nikon A1 HD25/A1R HD25 and C2+ systems can acquire the entire spectral response from both FRET fluorophores in a single scan. However, even though spectral imaging is capable of simultaneously detecting both fluorophore emission signals in FRET, the technique is incapable of distinguishing between acceptor emission generated through energy transfer and signal that originates from direct excitation. Therefore, proper controls using donor and acceptor proteins expressed separately are necessary for quantitative analysis. In cases where spectral imaging is used to evaluate FRET in fluorescent protein biosensors expressed as a single polypeptide, controls are less important.