Prof. Erwin Peterman
Dr. Andreas S. Biebricher
LaserLaB – Physics of Living Systems, Vrije Universiteit
Amsterdam, The Netherlands
Prof. Peterman and team research protein structural and functional dynamics using combinations of optical tweezers and single molecule fluorescent microscopy. One branch of this research involves the study of proteins that bind to DNA and are involved in DNA repair and replication, in this case a human protein complex of topoisomerase IIIα, RMI1 and RMI2 that is related to DNA replication and a rare genetic disorder known as Bloom’s syndrome.
Using a custom-built inverted microscope that combines widefield fluorescence with dual-trap optical tweezers, a length of DNA (approx. 16 μm) can be stretched out between two points and imaged, observing the protein complexes bound to the length of DNA. These proteins are counted and potentially tracked using the mCherry red fluorescent protein, with an image taken every second at high magnification.
While the advanced experimental set-up does allow for images of proteins on captured DNA to be taken, it introduces several imaging challenges. The mCherry fluorophore gives a low signal and bleaches quickly, meaning that high sensitivity and low intensity/exposure times are required.
The imaging is also limited by a 1.2 numerical aperture water immersion objective with lower light collecting efficiency, resulting in multiple lenses required to reach a 120 nm pixel size for imaging DNA and proteins.
The Prime BSI had a larger field of view than our EMCCD and was straightforward to set up and get running… we didn’t have to play around with the gain and it was good for our quantitative signal-critical applications