Fig. 5
From: Spectral phasor imaging on a commercial confocal microscope without a spectral detector
4C-spectral phasor unmixing of green and yellow fluorescent proteins expressed in HEK293T cells. (a) 4-channel imaging of HEK293T cells expressing only mEmerald-actin and corresponding phasor plot. (b) 4-channel imaging of HEK293T cells expressing only EYFP-tagged CAAX (EYFP-CAAX) and corresponding phasor plot. (c) 4-channel imaging of HEK293T cells co-expressing mEmerald-actin and EYFP-tagged CAAX (EYFP-CAAX) and corresponding phasor plot. The elongated phasor plot reflects the presence of two distinct fluorescent species in the image. (d) Emission detected in 4 channels covering the range between 495 and 615 nm, with a spectral detection window of 30 nm for each channel. (e) The phase wavelength image reveals cells with different spectral fingerprints, such as a cell predominantly expressing EYFP-CAAX (indicated by a star) or a cell predominantly expressing mEmerald-actin (indicated by an arrowhead). (f) Phasor decomposition provides spectrally unmixed images corresponding to the two fluorescent proteins.
