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Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM
Published on: December 22, 2015
Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)
Andrew Nickerson1, Tao Huang1, Li-Jung Lin1
1Department of Biomedical Engineering, Oregon Health and Science University; Knight Cancer Institute, Oregon Health and Science University; OHSU Center for Spatial Systems Biomedicine, Oregon Health and Science University.
Bimolecular fluorescence complementation with photoactivated localization microscopy (BiFC-PALM) enables visualization of protein-protein interactions in cells at nanometer resolution. This technique offers high specificity and sensitivity for studying molecular events.
Area of Science:
- Cell biology
- Microscopy
- Molecular biology
Background:
- Protein-protein interactions (PPIs) are crucial for biological processes.
- Conventional light microscopy is limited by diffraction (~250 nm), hindering visualization of PPIs at high resolution.
- Existing methods struggle to provide both sensitivity and spatial resolution for studying PPIs in vivo.
Purpose of the Study:
- To develop and present a protocol for visualizing protein-protein interactions (PPIs) in cells with single-molecule sensitivity and nanometer spatial resolution.
- To combine bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM) for enhanced PPI visualization.
- To demonstrate the utility of BiFC-PALM for studying molecular interactions in live cells.
Main Methods:
- Utilized bimolecular fluorescence complementation (BiFC) by splitting a photoactivatable fluorescent protein (PA-FP), PAmCherry1, into two non-fluorescent fragments.
- Employed photoactivated localization microscopy (PALM), a superresolution technique, to achieve nanometer-scale imaging.
- Designed and optimized BiFC probes using PAmCherry1 split between amino acids 159 and 160 for efficient and specific PPI reconstitution at 37 °C.
- Applied single molecule tracking (smt-) PALM to track diffusion of interacting proteins in live cells.
Main Results:
- Successfully demonstrated BiFC-PALM by reconstituting split PAmCherry1, achieving high specificity and low non-specific binding.
- Visualized Ras-Raf interactions at the nanometer scale with single-molecule resolution.
- Showcased the ability to track protein diffusion in live cells using smt-PALM.
- Provided a detailed protocol covering fusion protein design, sample preparation, image acquisition, and data analysis.
Conclusions:
- BiFC-PALM is a powerful technique for visualizing protein-protein interactions (PPIs) with high spatial resolution, specificity, and sensitivity in intact biological samples.
- This method overcomes the limitations of conventional microscopy for studying PPIs.
- BiFC-PALM facilitates detailed investigation of molecular interactions and dynamics in cellular environments.
