Real Time Imaging of 3D Chromosome Reconfiguration by Multicolor Point-Spread-Function Engineering

Many cellular functions within the nucleus are influenced by chromatin structure, dynamics and reconfiguration. An example for this is the repair mechanisems of DNA double-strand-breaks (DSB), such as homologous recombination (HR) to prevent chromatin aberrations. During HR repair chromosomes undergo spatial reconfiguration to allow close proximity between relevant loci. Characterization of this process in single live cells requires 3D imaging, which is typically slow, thus dynamics over short time scales have not yet been elucidated.

We employ Multicolor Point Spread Function (PSF) engineering, a microscopy technique in which the image of a point source is modified by utilizing a spatial light modulator based phase mask in the optical path, to encode its depth and color in a widefield image. This enables simultaneous tracking of different emitters in 3D. We demonstrate our technique by simultaneous imaging of fluorescently labeled DNA loci in live cells, aiming to visualize mating type switching process in live Saccharomyces Cerevisiae yeast cells. Process is initiated by induction of DSB, followed by simultaneous 3D tracking of the fluorescently tagged loci and is validated by a reporter gene which notify DSB repair completion.

Utilizing Multicolor PSF engineering reduces image acquisition time dramatically relative to other techniques such as confocal microscopy, and holds the potential to reveal dynamics that were not detected before, including transient and unstable conformation of the chromosome, which are statistically less frequently observed.

Finally, we demonstrate very high throughput 3D colocalization of labeled loci, by incorporating PSF engineering into an imaging flow cytometer.