For FRAP experiments, the mCherry images were collected using the MetaFluor 6.2 software (Molecular Devices, Sunnyvale, California) on an epifluorescence microscope (Olympus IX81) with excitation at 572DF35 and emission at 632DF60 using 1% of the light source power. During imaging, the cells were kept in RPMI 1640 medium without serum at 37°C, and the objective was focused at the bottom of the cell. The cells were monitored before photobleaching to confirm that the fluorescence signals were stable during imaging. Photobleaching was conducted by exciting mCherry at 560DF20 in a region of interest with full power of the light source for 15 s. Subsequently, the recovery process was imaged at 5-s intervals for the mCherry fluorescence signals (47).

For FRAP analysis, we used a finite element–based diffusion analysis method, which was previously developed and rigorously characterized by us to allow estimation of diffusion coefficients based on images collected by wide-field microscopes. The methods and results have been published in PLOS Computational Biology (47). With this method, the photobleach experiment protocol is simple and does not require the usage of a confocal microscope. The analysis method can be applied to cells of complex geometry without restrictions on the photobleaching pattern and protocol. Only one fluorescence intensity image before photobleaching and two images after are required for estimating the diffusion coefficient without the need of a complete time course.

For FRET experiments, the images were collected with a 420DF20 excitation filter, a 450DRLP dichroic mirror, and two emission filters controlled by a filter changer (480DF30 for CFP and 535DF25 for FRET). The pixel-wise images of the CFP/YFP emission ratio were computed to quantify the FRET signals, which represent the concentration of the phosphorylated Lck biosensor and, hence, Lck activity in space and time.

A Nikon Eclipse Ti inverted microscope installed with a 300-W Xenon lamp (Atlas Specialty Lighting), an electron multiplying (EM) charge-coupled device camera (QuantEM:512SC, Photometrics), and a 100× Nikon microscope objective (numerical aperture, 1.45) were used to capture all imaging data with the MetaMorph 7.8 software (Molecular Devices). Analysis of all acquired images was conducted on FluoCell, an image analysis software tool developed in the Wang Lab (unpublished results, S.L. at the University of California).

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