Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

In its active form, plasma membrane-associated Ras monomer ― a guanine nucleotide-binding protein ― recruits Raf, an inactive protein kinase that binds to Ras via its Ras-binding domain.

This binding results in a functional Ras-Raf complex that triggers the downstream signaling process.

To visualize the intracellular Ras-Raf interaction, begin with a suspension of genetically-engineered cells in a well-chambered slide. These cells express two non-fluorescent fusion proteins ― one comprises Ras linked to one-half of a photoactivatable fluorophore via its N-terminal, and the other is Raf fused to the other half of the same fluorophore at its C-terminal.

Inside cells, Ras-containing fusion proteins dock at the plasma membrane and interact with Raf-containing fusion proteins, reconstituting the fluorescent protein's fragments to a complete photoactivatable fluorophore. The absence of Ras and Raf binding keeps the two fluorophore halves separated.

Add a fixative reagent to the slide. The fixative molecules form cross-linkages with the proteins and stabilize the protein complexes.

Remove the fixative reagent and add an imaging buffer. Place the slide under a fluorescence microscope set for photoactivated localization microscopy, PALM. Illuminate the cells with a high-intensity laser, activating a small subset of fluorescent molecules.

Image the randomly-originating fluorescence produced by each nano-sized, single fluorophore-tagged Ras-Raf complex, visible as high-resolution fluorescence dots.

To prepare a sample for imaging, plate about 5.5 x 10⁴ stable expression cells per well of an 8-well glass-bottomed chamber slide, in 350 microliters of phenol red-free DMEM. Use fresh paraformaldehyde with glutaraldehyde to fix the cells, and after replacing the fixative with PBS or imaging buffer, vortex 100-nanometer gold particles, and add 35 microliters per well for tracking stage drift during imaging.

Approach the microscope and power on the 405 and 561-nanometer lasers. Keeping the shutters closed at this point, ensure the 561-nanometer dichroic mirror and notch filter are in place. Open the image acquisition software.

Turn on the EMCCD camera and allow it to cool down, and set the exposure time to 100 milliseconds and the EMCCD gain to an appropriate value. Add immersion oil to the objective, and secure the sample on the microscope stage. Next, with either brightfield or the 561-nanometer laser, bring the sample into focus.

For imaging Ras and other membrane proteins, use a 60X apochromat TIRF objective with a 1.49 numerical aperture, and bring the microscope into TIRF configuration.

Adjust the excitation laser so that it is off-centered when hitting the back aperture of the TIRF objective, which will cause the laser to deflect upon reaching the coverslip buffer interface. Keep adjusting the laser incident position until the critical angle is reached and the laser is being reflected back.

Search for a cell to image with several gold particles in view. Then, set a region of interest that encloses the cell or a region of it and the gold particles.

In case sufficient activation is already occurring because of high expression levels, begin acquisition with the 405-nanometer laser off and the 561-laser on. Otherwise, turn on the 405-nanometer laser at the lowest factory power setting, and increase it gradually until there are tens of molecules per frame or so that single molecules are well-separated.

As data acquisition continues, gradually increase the 405-nanometer laser power to keep the spot density roughly constant. Continue image acquisition until high 405 power does not initiate any more activation events.