Abstract
We report the observation of chromatin dynamics in living budding yeast (Saccharomyces cerevisiae) cells, in three-dimensions (3D). Using dual color localization microscopy and employing a Tetrapod point spread function, we analyze the spatio-temporal dynamics of two fluorescently labeled DNA loci surrounding the GAL locus. From the measured trajectories, we obtain different dynamical characteristics in terms of inter-loci distance and temporal variance; when the GAL locus is activated, the 3D inter-loci distance and temporal variance increase compared to the inactive state. These changes are visible in spite of the large thermally- and biologically-driven heterogeneity in the relative motion of the two loci. Our observations are consistent with current euchromatin vs. heterochromatin models.
Highlights
Regulated gene expression is critical in cell development and disease progression [1]
Fluorescence microscopy is an excellent match with the challenge of live chromatin studies on the single cell level, thanks to its specificity, high precision, and typically high SNR capability
The samples imaged in this work are budding yeast (S. cerevisiae) cells mounted on agarose pads
Summary
Regulated gene expression is critical in cell development and disease progression [1]. Most studies investigating gene regulation rely on epigenomic methods [2,3], where the regulatory DNA is extracted outside of its native environment of the cell nucleus These methods provide only static snapshots of the local spatial organization of the genome and typically show data from an ensemble of cells, and they do not provide information about the complex spatio-temporal organization in single cells. We use a specially fabricated dielectric transmittance mask for phase modulation This dramatically increases photon efficiency, which is of great importance due to the challenging conditions of live-cell imaging (high background), and the large lateral extent of the Tetrapod PSF, which spreads photons over many camera pixels
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