Figure 3

The BABB-induced spectral alterations exacerbate crosstalk in multiband excitation scenarios. Single excitation and single bandpass emission spectra barely contain foreign signal in PBS and BABB (a,c,e). The foreign signal is caused by fluorophores which emit light in another filter range. Multi excitation and multi bandpass emission spectra contain a noticeable amount of foreign signal (b,d,e). Relative contributions of each fluorophore per emission filter band differ strongly between PBS and BABB (f,g). The foreign signal in multiband settings exceeds that in single bandpass setups (e). The line graphs represent the emission spectra of excited fluorophores and fluorescent dyes in PBS (a,b) and BABB (c,d) using optical single (a,c) and multi (b,d) bandpass filter sets indicated by shaded areas. DAPI (blue), Alexa Fluor 488 (green), Alexa Fluor 568 (orange), and Alexa Fluor 647 (magenta) are excited individually (single bandpass) or simultaneously (multi bandpass) with 405 nm, 488 nm, 555 nm, and 639 nm light. The bar graph (e) compares the percentage composition of the foreign signal within certain emission bands (filter bands illustrated in a–d) using single and multi-bandpass filter sets in BABB and PBS. This reflects the percentage of light that could be misattributed to an incorrect fluorophore in certain scenarios. The stacked graphs illustrate the fluorescent signal distribution within certain emission bands using optical multi bandpass filter sets in PBS (f) and BABB (g). n = 8 independent wells and measurements for each datapoint. Error bars were omitted for clarity, but are the same as in Fig. 1.