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. 2020 May 22;9:e55286. doi: 10.7554/eLife.55286

Figure 4. Extraction of correlation functions from axial lsFCS intensity time traces on live NCI-H1703 cells stably transfected with LRP6-mCherry and immersed in CM containing 5 nM DKK1-eGFP.

(a) Kymograph of the membrane scan recorded in the green (ligand, left) and red (receptor, right) color channels, showing the fluorescence from the membrane during 60 s of axial scanning (500 ms binning time). Thick white lines trace the membrane positions. (b) Time traces of the raw ligand (IG,raw) and receptor (IR,raw) intensities from the membrane and the corresponding background, IG,bg and IR,bg, determined ±10 pixels away from the membrane (thin white lines in panel a). Lines through, IG,bg and IR,bg are sums of six sine functions fitted to the data for noise filtering. (c) Autocorrelation (cyan, DKK1-eGFP; magenta, LRP6-mCherry) and cross-correlation (blue) functions after background and photobleaching corrections.

Figure 4.

Figure 4—figure supplement 1. Examples of autocorrelation (cyan, DKK1-eGFP; magenta, LRP6-mCherry) and cross-correlation (blue) functions after background and photobleaching corrections, calculated from individual 60 s axial lsFCS intensity time traces.

Figure 4—figure supplement 1.

(a) Correlation curves at various different receptor densities due to different cell types and receptor expression techniques, all at 0.1 nM DKK1-eGFP. (b) Correlation curves measured on transiently transfected NCI-H1703 cells at various DKK1-eGFP concentrations indicated in the individual panels.
Figure 4—figure supplement 2. Control experiments demonstrating proper functioning of the axial lsFCS method and analysis, using HEK293T cells transiently transfected to express fluorescently labeled moieties and dual-color fluorescence excitation (PIE) at 470 and 561 nm.

Figure 4—figure supplement 2.

Symbols (cyan and magenta, autocorrelations; blue, cross-correlation) represent averaged data (mean ± SEM) over at least nine axial lsFCS measurements of 60 s each; lines are fits with Equation 1 (a) Correlation functions from cells expressing LRP6-mCherry (magenta) and Mem-eGFP (cyan). Mem is a membrane marker peptide consisting of the 20 N-terminal residues of neuromodulin, which contains a palmitoylation sequence. As expected, the cross-correlation amplitude is zero because both labeled moieties diffuse independently in the plasma membrane. (b) Correlation curves of LRP6 fused to both mCherry and eGFP. The autocorrelation amplitudes are different and reflect the greatly different maturation yields of the two fluoresscent proteins. The cross-correlation amplitude is significant, revealing a sizeable fration of constructs carrying both fluorophores. Notably, the function Γ (Equation 5) equals 0.14 ± 0.02 and is thus identical to the value obtained for HEK293T cells transiently transfected with LRP6-mCherry and exposed to saturating concentrations of DKK1-eGFP (see Figure 6—figure supplement 1, Table 1), as we should expect. (c) Correlation curves of LRP6-tdTomato, with tdTomato excited with 470 nm (4–5 µW) and 561 nm (<1 µW) laser light; emission was detected in the red channel with a bandpass filter 600/37 nm (center/width). Using the same fluorophore for dual-color excitation (PIE), photobleaching is identical for both color channels and perfect cross-correlation is ensured. The cross-correlation amplitude is expected to be (GG(0) + GR(0))/2 for complete overlap of the green and red foci, as we observe in the data.
Figure 4—figure supplement 3. Axial lsFCS - data analysis.

Figure 4—figure supplement 3.

(a) The flow chart lists the individual steps of the data analysis procedure. Tasks in light gray boxes are performed using Matlab. Tasks in dark gray boxes are processed in Origin Pro. (b) Source data files illustrate the individual steps. (c) To obtain reliable and reproducible results, data selection occurs according to the stringent criteria given here.