ArticleNature protocols2017
Quantifying transcription factor-DNA binding in single cells in vivo with photoactivatable fluorescence correlation spectroscopy.
Article in Nature protocols, 2017. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 13 papers.
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Who cites it
13 citing papers in PubMed.
- Mapping Protein Diffusion from the Plasma Membrane to the Nucleus: Insights from Fluorescence Correlation Spectroscopy.Biomolecules · 2026Review
- Celebrating 50 years of fluorescence correlation spectroscopy (FCS): Advancing live-cell massively parallel FCS studies with photostable GFPs, mStayGold and StayGold/E138D.Biochimica et biophysica acta. General subjects · 2025Article
- Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2.ACS central science · 2023Article
- Phase Separation-Mediated Chromatin Organization and Dynamics: From Imaging-Based Quantitative Characterizations to Functional Implications.International journal of molecular sciences · 2022Review
- Application of Genetically Encoded Photoconvertible Protein SAASoti for the Study of Enzyme Activity in a Single Live Cell by Fluorescence Correlation Microscopy.Materials (Basel, Switzerland) · 2022Article
- Of numbers and movement - understanding transcription factor pathogenesis by advanced microscopy.Disease models & mechanisms · 2020Review
- From whole-mount to single-cell spatial assessment of gene expression in 3D.Communications biology · 2020Review
- Superresolution imaging reveals spatiotemporal propagation of human replication foci mediated by CTCF-organized chromatin structures.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
- Specification of the First Mammalian Cell Lineages In Vivo and In Vitro.Cold Spring Harbor perspectives in biology · 2020Review
- Understanding biochemical processes in the presence of sub-diffusive behavior of biomolecules in solution and living cells.Biophysical reviews · 2019Review
- Using enhanced number and brightness to measure protein oligomerization dynamics in live cells.Nature protocols · 2019Article
- Monomerization of the photoconvertible fluorescent protein SAASoti by rational mutagenesis of single amino acids.Scientific reports · 2018Article
- Optimal fluorescent protein tags for quantifying protein oligomerization in living cells.Scientific reports · 2018Article
Corrections and comments
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Authors and funding
6 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Probing transcription factor (TF)-DNA interactions remains challenging in complex in vivo systems such as mammalian embryos, especially when TF copy numbers and fluorescence background are high. To address this difficulty, fluorescence correlation spectroscopy (FCS) can be combined with the use of photoactivatable fluorescent proteins to achieve selective photoactivation of a subset of tagged TF molecules. This approach, termed paFCS, enables FCS measurements within single cell nuclei inside live embryos, and obtains autocorrelation data of a quality previously only attainable in simpler in vitro cell culture systems. Here, we present a protocol demonstrating the applicability of paFCS in developing mouse embryos by outlining its implementation on a commercial laser-scanning microscope. We also provide procedures for optimizing the photoactivation and acquisition parameters and determining key parameters describing TF-DNA binding. The entire procedure can be performed within ∼2 d (excluding embryo culture time), although the acquisition of each paFCS data set takes only ∼10 min. This protocol can be used to noninvasively reveal cell-to-cell variation in TF dynamics, as well as critical, fate-predicting changes over the course of early embryonic development.
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