LIVE-PAINT: Super-Resolution Microscopy Inside Live Cells Using Reversible Peptide-Protein Interactions
Posted on: 11 June 2020
Preprint posted on 15 May 2020
Article now published in Communications Biology at http://dx.doi.org/10.1038/s42003-020-01188-6
No photobleaching, no photoconversion – a novel super-resolution technique for live-cell imaging
Selected by Xenia MeshikCategories: bioengineering, biophysics, cell biology, synthetic biology
Background
Traditional microscopy is limited by the diffraction limit of light and is therefore capable of around 250 nm resolution. As various cellular processes are studied in more and more detail, there has been much interest in developing convenient and affordable super-resolution techniques. Some techniques, like photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) are based on sequential photoactivation of fluorophores. These techniques are often limited by photobleaching of fluorophores over time. Stimulated emission depletion microscopy (STED) is based on selective deactivation of fluorophores and requires specialized instrumentation, making it impractical for many researchers. Point accumulation for imaging in nanoscale topography (PAINT) is based on transient binding of fluorescent molecules to the protein of interest, and DNA-PAINT increases the specificity of this technique by fusing complementary DNA strands to the fluorophore and the protein of interest. However, DNA-PAINT cannot be used in live cells. In this work, the authors overcome this limitation with LIVE-PAINT, a technique that allows for super-resolution imaging of dynamic proteins in live cells.
Key findings
The authors demonstrate the effectiveness of the LIVE-PAINT, which is based on the reversible binding of a non-functional protein, which is fused to a fluorescent protein (FP), and a small peptide, which is fused to the protein of interest. The protein-FP complex diffuses freely in the cytosol and is temporarily immobilized as it binds to the peptide-protein of interest complex. These localization events result in transient fluorescence spikes as the cell is imaged continuously in the plane of interest. The sum of acquired images results in a super-resolution image. The expression level of the components can be tuned based on the abundance of the protein of interest in the cell to achieve the optimal resolution.
LIVE-PAINT has a number of advantages over other super-resolution techniques. It does not rely on sequential photoswitching of fluorophores, like PALM and STORM, and does not require special instrumentation, like STED. It is not affected as much by photobleaching, since any bleached FPs diffuse away upon dissociation and are rapidly replaced by unbleached FPs. This allows for the sample to be imaged for longer periods of time. Because the protein of interest is not fused directly to a large FP, it more closely resembles its endogenous state. The authors showed that the peptide-binding protein can be fused to as many as 3 FP to achieve a better signal-to-noise ratio.
Using this technique, the authors were able to image the proteins Cdc12, actin, and cofilin in live yeast with a resolution as low as 50 nm and observe these proteins’ dynamic movement.
What I like about the paper
I like this paper because it presents a practical super-resolution technique that seems easily achievable for any lab that has the capability to perform confocal or TIRF imaging. It does not require the purchase of specialized equipment or specialized fluorescent proteins, and the necessary peptide-protein and protein-FP fusions can be achieved with standard molecular biology techniques. It seems particularly well-suited for live imaging of dynamic cellular processes.
Future directions and questions
- It would be interesting to see how well this technique works with transient transfections, where controlling the expression levels of various constructs in a cell is more difficult.
- One potential disadvantage of this technique is that it is not possible to visually confirm the expression of the protein of interest in the cell, since it is not tagged with a FP. This may make it difficult to troubleshoot the experiment if it does not immediately work. Do the authors anticipate this being a problem?
- I wonder how much initial optimization would be required when first using LIVE-PAINT to image a particular protein. A protein of interest that moves rapidly throughout the cell would necessitate a protein-peptide pair that associates and dissociates very rapidly (high Kd value). Do the authors anticipate that one protein-peptide pair would be fairly well-suited for most applications, or would the user need to try several pairs?
doi: https://doi.org/10.1242/prelights.21865
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