  Here we describe the coupled standardisation of two complementary
fluorescence imaging techniques and apply it to liquid-liquid phase separated
condensates formed from an EGFP fluorescent reporter of Flowering Control Locus
A (FCA), a protein that associates with chromosomal DNA in plants during
epigenetic regulation of the flowering process. First, we use home-built
single-molecule Slimfield microscopy to establish a fluorescent protein
standard. This sample comprises live yeast cells expressing Mig1 protein, a
metabolic regulator which localises to the nucleus under conditions of high
glucose, fused to the same type of EGFP label as for the FCA fusion construct.
Then we employ commercial confocal AiryScan microscopy to study the same
standard. Finally, we demonstrate how to quantify FCA-EGFP nuclear condensates
in intact root tips at rapid timescales and apply this calibration. This method
is a valuable approach to obtaining single-molecule precise stoichiometry and
copy number estimates of protein condensates that are integrated into the
chromosome architecture of plants, using confocal instrumentation that lacks de
facto single-molecule detection sensitivity.
