Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.
Bottom Line: In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells.We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins.We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.Show MeSH
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Mentions: For experiments in Figures 1B and 2A, mEos3.1 fluorophores were activated with sequential pulses of the 405-nm laser at power densities ranging from 0.1 to 10 W/cm2, adjusted manually to ensure single-molecule activation. Each activation cycle was followed by imaging of the sample using the 561-nm laser under incident intensities of the order of 1 kW/cm2. Iterative cycling continued until no further in-focus nuclear fluorescence could be detected. Samples for Figures 3 and 4 were imaged using continuous wave illumination of 405 nm (0.1–1 W/cm2) and 561-nm (1 kW/cm2) laser light, in order to reduce experiment duration. Control experiments showed no significant difference in the total number of localization using the pulsed activation method or the continuous wave approach (data not shown). All experiments were carried out at 17.0 ± 0.5°C. The typical number of frames (350 ns): 6000–11 000.
Affiliation: Genome Damage and Stability Centre, School of Life Sciences, University of Sussex, Falmer, Sussex, UK.