Olympus ScanR

The ScanR microscope allows us to quantitatively and unbiasedly monitor the dynamics of cellular responses to DNA damage in a fully automated high-content fashion. Moreover, the ability to multiplex numerous fluorophores means that we can do this at an unprecedented level of detail in single cells. The figure below demonstrates the power of the ScanR system to generate flow cytometry-like data with standard fixed samples. In this example we can segment thousands of individual cells by cell cycle phase by plotting total DNA content (DAPI) versus the single-stranded DNA binding protein RPA70. We can also use a third channel to analyse ssDNA formation through incorporation of the nucleotide analogue BrdU and map this onto the DNA versus RPA70 plot. In general, the approach can be used with targeted genetic and chemical perturbations using fixed or live cells, or can be adapted for focussed libraries, to assess their impact on cellular protein dynamics. For example, as shown above, combination treatment of ATR inhibitor (an important apical kinase in the DNA damage response) and Hydroxyurea (a dNTP synthesis inhibitor) leads to a substantial increase in single stranded DNA generation and subsequent RPA70 binding than the DNA interstrand crosslinking agent MMC, which reflects the mechanism of how each drug causes ssDNA formation.