Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Abstract

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples. Video LinkThe video component of this article can be found at

Superresolution Imaging of Clinical Formalin Fixed Paraffin Embedded Breast Cancer with Single Molecule Localization Microscopy

“…Raw SMLM movies were processed using both ThunderSTORM (for 3D data sets)40 and a custom-written MatLab package (wfiread and palm for 2D data sets)41. For 3D data processing, a calibration curve was generated using images of 40 nm fluorescence beads scattered on a coverslip taken at a series of focal positions, and the resulting calibration file was imported into ThunderSTORM to convert the measured widths of individual single molecule images into axial coordinates.…”

“…Color-coded 3D SMLM images were subsequently generated using ImageJ. Processing of 2D data sets using the wfiread and palm software packages have been previously described41.…”

Sample preparation and imaging procedures for fast and multiplexed superresolution microscopy with DNA-PAINT-ERS

“…Here we describe . More details can be found in our recent publication by Nickerson et al 6 3. Turn on the microscope, lasers and EMCCD camera.…”

“…Engage the auto-focus system and start the acquisition using micromanager 7 or another acquisition software of choice 7. Extract the localizations, sorting, and rendering by using in-house Matlab scripts 6 Troubleshooting Time Taken Anticipated Results…”

Simultaneous Multicolor Single-Molecule Tracking with Single-Laser Excitation via Spectral Imaging

“…Acquisition of raw images was performed using the open source micromanager software suite (https://micro-manager.org/). Image analyses for extracting single-molecule localization, spectra, and trajectories were all performed with custom MATLAB (The MathWorks, Natick, MA) scripts as described previously (26,27).…”