Figure 3

ME-MPM improved signal-to-noise ratios and spectral separation in subcellular structures. Images of fixed Cos7 cells stained with FITC-tubulin and Alexa Fluor 594-phalloidin were collected using simultaneous CLSM at 488 nm and 543 nm (a), simultaneous ME-MPM at 920 nm and 1040 nm (b), and SE-MPM at 950 nm (c). Green and red arrows correspond to distinct signal from FITC and Alexa Fluor 594, respectively, and dim green arrows correspond to bleed-through green signal into the red emission channel. Note that ME-MPM achieved comparable spectral separation to CLSM, whereas SE-MPM was hindered by false positive bleed-through of green emission signal in the red channel (middle panel, haze). Scale bar: 50 μm. The red and green emission signal generated from each excitation source in ME-MPM are shown in Additional file 3 and further verify that ME-MPM achieved better spectral separation by decreasing cross-excitation and false-positive bleed-through compared to SE-MPM.