Figure 2

ME-MPM improved signal-to-noise ratios in whole cells. Images of 293T cells transfected with either hrGFP or DsRedII were collected using sequential single photon excitation at 488 nm and 543 nm for CLSM (a), simultaneous ME-MPM at 920 nm and 1040 nm (b), or SE-MPM at 970 nm (c). Signal intensity was held constant at the saturation point in the red channel over all experiments (a-c). Note that there is more bleed-though from hrGFP signal into the red emission channel for SE-MPM than for either ME-MPM or CLSM (yellow arrows in b-c). Scale bar: 50 μm. All MPM images were acquired under identical VBF settings. The red and green emission signal generated from each excitation source in ME-MPM are shown in Additional file 3 and indicate reduced cross-excitation of the fluorophores in the sample. (d) Quantification of signal-to-noise ratios that were achieved for green signal and red bleed-through in the green emission channel as well as red signal and green bleed-through in the red emission channel.