ArticleNature protocols2019
Using enhanced number and brightness to measure protein oligomerization dynamics in live cells.
Article in Nature protocols, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 19 papers.
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Who cites it
19 citing papers in PubMed.
- Highly dynamic assemblies of glycolytic enzymes by quinary determinants.Nature communications · 2026Article
- Structural Characterization of Protein-Nucleic Acid Complexes: An Overview of the Recent Innovation in the Analytic Methods.International journal of molecular sciences · 2025Review
- Quantifying biomolecular organisation in membranes with brightness-transit statistics.Nature communications · 2024Article
- Investigations of membrane protein interactions in cells using fluorescence microscopy.Current opinion in structural biology · 2024Review
- Utility of FRET in studies of membrane protein oligomerization: The concept of the effective dissociation constant.Biophysical journal · 2023Article
- Advanced imaging techniques for studying protein phase separation in living cells and at single-molecule level.Current opinion in chemical biology · 2023Review
- Quantitative fluorescence emission anisotropy microscopy for implementing homo-fluorescence resonance energy transfer measurements in living cells.Molecular biology of the cell · 2023Article
- Review of data processing of functional optical microscopy for neuroscience.Neurophotonics · 2022Review
- Phase Separation-Mediated Chromatin Organization and Dynamics: From Imaging-Based Quantitative Characterizations to Functional Implications.International journal of molecular sciences · 2022Review
- Radial pair correlation of molecular brightness fluctuations maps protein diffusion as a function of oligomeric state within live-cell nuclear architecture.Biophysical journal · 2022Article
- Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations.Molecular biology of the cell · 2022Article
- The timing of Start is determined primarily by increased synthesis of the Cln3 activator rather than dilution of the Whi5 inhibitor.Molecular biology of the cell · 2022Article
- Evaluation of sted super-resolution image quality by image correlation spectroscopy (QuICS).Scientific reports · 2021Article
- Biological Phase Separation and Biomolecular Condensates in Plants.Annual review of plant biology · 2021Review
- Sequence determinants of in cell condensate morphology, dynamics, and oligomerization as measured by number and brightness analysis.Cell communication and signaling : CCS · 2021Article
- Probing Membrane Protein Association Using Concentration-Dependent Number and Brightness.Angewandte Chemie (International ed. in English) · 2021Article
- Determination of G-protein-coupled receptor oligomerization by molecular brightness analyses in single cells.Nature protocols · 2021Article
- Number and Brightness Analysis: Visualization of Protein Oligomeric State in Living Cells.Advances in experimental medicine and biology · 2021Article
- Chemokine receptor CXCR4 oligomerization is disrupted selectively by the antagonist ligand IT1t.The Journal of biological chemistryArticle
Corrections and comments
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Authors and funding
13 authors.
Funding
Abstract
Protein dimerization and oligomerization are essential to most cellular functions, yet measurement of the size of these oligomers in live cells, especially when their size changes over time and space, remains a challenge. A commonly used approach for studying protein aggregates in cells is number and brightness (N&B), a fluorescence microscopy method that is capable of measuring the apparent average number of molecules and their oligomerization (brightness) in each pixel from a series of fluorescence microscopy images. We have recently expanded this approach in order to allow resampling of the raw data to resolve the statistical weighting of coexisting species within each pixel. This feature makes enhanced N&B (eN&B) optimal for capturing the temporal aspects of protein oligomerization when a distribution of oligomers shifts toward a larger central size over time. In this protocol, we demonstrate the application of eN&B by quantifying receptor clustering dynamics using electron-multiplying charge-coupled device (EMCCD)-based total internal reflection microscopy (TIRF) imaging. TIRF provides a superior signal-to-noise ratio, but we also provide guidelines for implementing eN&B in confocal microscopes. For each time point, eN&B requires the acquisition of 200 frames, and it takes a few seconds up to 2 min to complete a single time point. We provide an eN&B (and standard N&B) MATLAB software package amenable to any standard confocal or TIRF microscope. The software requires a high-RAM computer (64 Gb) to run and includes a photobleaching detrending algorithm, which allows extension of the live imaging for more than an hour.
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Registered trials
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