PIPE - Photo-converted Intensity Profile Expansion
To characterize protein motion in cells, researchers have developed multiple fluorescence microscopy methods. However, many of these methods do not observe protein motion directly, but measure indirect observables and fit them to models to infer the underlying motion. In addition, operationalizing these methods requires expertise that can be a barrier to their broad utilization. Here, in collaboration with our colleagues in the Kaganovich Lab we have developed PIPE – Photo-converted Intensity Profile Expansion – to directly measure the motion of tagged proteins and quantify it using an effective diffusion coefficient. PIPE works by pulsing photo-convertible fluorescent proteins, generating a peaked fluorescence signal at the pulsed region, and analyzing the spatial expansion of the signal. We believe that the direct measurement and ease of use that PIPE offers make it an appealing tool for cell biologists.
See this user manual for instructions on how to run PIPE and what types of output you may get.
While you can acquire data using many imaging systems, review this experimental protocol to see how we did it.
Download the source code.