ArticleeLife2022
Fixation can change the appearance of phase separation in living cells.
Article in eLife, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 88 papers.
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Who cites it
88 citing papers in PubMed, 138 citations in OpenAlex.
- A practical guide to investigating biomolecular condensates: a comment from the plant community.Science China. Life sciences · 2026Review
- Single-Molecule Dwell Times in Biomolecular Condensates.bioRxiv : the preprint server for biology · 2026Article
- Overexpressed Spindlin1 relocalizes to the nucleoli during fixation.Biochemistry and biophysics reports · 2026Article
- A Simplified Wheat Protoplast Transformation System and Guideline for Avoiding Protein Localization Artifacts.Plants (Basel, Switzerland) · 2026Article
- HAK-actin, a U-ExM-compatible probe to image the actin cytoskeleton.Cell reports methods · 2026Article
- Real-time snapping dynamics and nanoscale thickness profiling of salmon keratocyte tunneling nanotubes using partially coherent quantitative phase microscopy.Scientific reports · 2026Article
- IDR-induced CAR condensation improves the cytotoxicity of CAR-Ts against low-antigen cancers.Nature chemical biology · 2026Article
- Decoding the molecular logic of rapidly evolving ZAD zinc finger proteins inScience advances · 2026Article
- Distinct nanoscale membrane organizations of mucins andbioRxiv : the preprint server for biology · 2026Article
- Tensile expansion microscopy applies mechanical force to super-resolve fixed and image live cellular samples.bioRxiv : the preprint server for biology · 2026Article
- HCR-Proxy resolves site-specific proximal RNA microenvironments at subcompartmental resolution.Nucleic acids research · 2026Article
- Dynamic regulation of endogenous transcription factor hubs at single-molecule resolution.bioRxiv : the preprint server for biology · 2026Article
- Phase separation as an emerging regulatory framework in antibody class switching and genome stability.Frontiers in immunology · 2026Review
- Active Zone: Linking Resolution Levels of Microscopic Modalities.Advances in neurobiology · 2026Review
- Fluorescent protein and peptide tags alter condensate formation and dynamics in vivo and in vitro.EMBO reports · 2026Article
- Feeling the vibes: Vibrational spectroscopic imaging of biomolecular assemblies in their natural environment.Applied physics reviews · 2025Article
- Conformational dynamics, RNA binding, and phase separation regulate the multifunctionality of rabies virus P protein.Nature communications · 2025Article
- Review
- Protocol for characterizing biomolecular condensates through live-cell imaging and analysis.STAR protocols · 2025Article
- Biomolecular Condensates as Emerging Biomaterials: Functional Mechanisms and Advances in Computational and Experimental Approaches.Advanced materials (Deerfield Beach, Fla.) · 2025Review
28 more citing papers are in PubMed but not listed here.
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed-cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.