LIVE-PAINT: Super-Resolution Microscopy Inside Live Cells Using Reversible Peptide-Protein Interactions
Preprint posted on 15 May 2020 https://www.biorxiv.org/content/10.1101/2020.02.03.932228v2.full
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?
Posted on: 11 June 2020
doi: https://doi.org/10.1242/prelights.21865
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the bioengineering category:
Also in the biophysics category:
Phosphorylation controls spatial and temporal activities of motor-PRC1 complexes to complete mitosis
Embryo mechanics cartography: inference of 3D force atlases from fluorescence microscopy
Flat clathrin lattices are linked to metastatic potential in colorectal cancer
Also in the cell biology category:
Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice
Coxiella burnetii actively blocks IL-17-induced oxidative stress in macrophages
Complete male-to-female sex reversal in XY mice lacking the miR-17∼92 cluster
Also in the synthetic biology category:
Genetically encoded multimeric tags for intracellular protein localisation in cryo-EM
Dissecting aneuploidy phenotypes by constructing Sc2.0 chromosome VII and SCRaMbLEing synthetic disomic yeast
Synthetic memory circuits for programmable cell reconfiguration in plants
AND
Synthetic genetic circuits enable reprogramming of plant roots
preListsbioengineering category:
in theEMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Advances in microscopy
This preList highlights exciting unpublished preprint articles describing advances in microscopy with a focus on light-sheet microscopy.
List by | Stephan Daetwyler |
Also in the biophysics category:
66th Biophysical Society Annual Meeting, 2022
Preprints presented at the 66th BPS Annual Meeting, Feb 19 - 23, 2022 (The below list is not exhaustive and the preprints are listed in no particular order.)
List by | Soni Mohapatra |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
Biophysical Society Meeting 2020
Some preprints presented at the Biophysical Society Meeting 2020 in San Diego, USA.
List by | Tessa Sinnige |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Biomolecular NMR
Preprints related to the application and development of biomolecular NMR spectroscopy
List by | Reid Alderson |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
List by | Joseph Jose Thottacherry |
Also in the cell biology category:
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
List by | Dey Lab |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
List by | Sandra Franco Iborra |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
List by | Dey Lab, Amanda Haage |
Also in the synthetic biology category:
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
List by | Alex Eve |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
List by | Jesus Victorino |
Antimicrobials: Discovery, clinical use, and development of resistance
Preprints that describe the discovery of new antimicrobials and any improvements made regarding their clinical use. Includes preprints that detail the factors affecting antimicrobial selection and the development of antimicrobial resistance.
List by | Zhang-He Goh |
Advances in Drug Delivery
Advances in formulation technology or targeted delivery methods that describe or develop the distribution of small molecules or large macromolecules to specific parts of the body.
List by | Zhang-He Goh |