Fig. 2

Quantification of Itgα5 Dynamics in the Presomitic Mesoderm

(A) A schematic of FCS data collection for fluorescently tagged proteins in the PSM. For FCS, the confocal volume (drawn approximately to scale in blue in A and B) was statically positioned in a cell membrane oriented orthogonally to the optical axis.

(B) Confocal image of a zebrafish embryo expressing membrane RFP (mem-RFP). The scale bar represents 10 µm.

(C) Autocorrelation curve of cytosolic GFP (cyt-GFP), fit to 3D diffusion (Equation 1 in the Supplemental Information). The inset shows a histogram of the measured number of Itgα5-GFP molecules.

(D) Representative set of ten autocorrelation curves (gray) of Itgα5-GFP with the averaged curve used in fitting (green). The inset shows the intensity fluctuations and binned intensities from this measurement.

(E) Autocorrelation measurements of mem-RFP, Itgα5-RFP, and Cdh2-RFP. The fits to 2D diffusion and fluorophore fluctuation (solid line, Equation 3 in the Supplemental Information) and to 2D diffusion only (dotted line, Equation 2 in the Supplemental Information). To facilitate the comparison, the diffusion components are normalized to one.

(F) Autocorrelation curves of mem-GFP-RFP illustrating the fast decay component is fluorophore specific, but the slow decay, i.e., diffusion, is identical in both autocorrelation curves, as well as the cross-correlation curve.

(G) Table of measured diffusion times and calculated diffusion coefficients ± SD.