Figure 2

TagRFP and mKalama1 are an optimal pair for dual-color imaging. (a) 2PA spectra of tagRFP (red circles) and mKalama1 (blue squares) and the fluorescence emission spectra of tagRFP (red line) and mKalama1(blue line). The y-axis represents the cross section (GM). The top x-axis represents the transition wavelength and the bottom x-axis represents the laser wavelength used for excitation. The different transitions of each FP chromophore are marked with arrows. Fluorescence emission spectra are shown in arbitrary units. (b) Jablonski diagram showing the electronic transitions that tagRFP and mKalama1 experience upon excitation with 760 nm light. (c) To test this approach we mixed HEK293 cells expressing either tagRFP or mKalama1 and imaged them with 780 nm excitation. (d) Scatter plot graphing red vs. blue pixel intensity of the image found in (a). With the settings used, none of the red signal is picked up by the blue detector. However, because of the broad red-shoulder in the mKalama1 emission spectrum (Figure 2) a small amount of the signal in the red detector is due to blue emission, as indicated by the slight linear slope found in the blue cells.