Prof. Karl Duderstadt
Max-Planck Institute of Biochemistry, Germany
The Duderstadt Group are interested in understanding the organization and dynamics of macromolecular complexes, such as the replisome which is responsible for DNA replication.
Successful DNA replication is critical for cell survival, and errors within this process have been implicated in many disease pathologies. The Duderstadt Group studies this process by introducing fluorescently labeled enzymes, such as DNA polymerase and RNA polymerase, into bacterial model systems. This allows them to visualize and track the polymerase enzymes within the replisome as they move along a DNA substrate during replication. They can then use kymograph analyses to quantify the movement along the DNA strands to better understand these processes.
The group primarily use a home built single molecule micromirror TIRF imaging system in their investigations. The micromirrors remove the need for a dichroic mirror and permit switching colors quickly, also removing the need for a filter wheel, making it an ideal system for live imaging.
Professor Duderstadt told us, “Our biggest challenge is how many photons to collect and the corresponding lifetime of the fluorophore we are looking at. This limits our ability to track sometimes.” For this reason, the group needs the highest camera sensitivity to ensure the dyes do not bleach and to permit the collection of as much light as possible to allow tracking.
Faster frame rates and lower costs were also strong factors in the decision to purchase a new camera. Professor Duderstadt told us, “Ideally, we want to put the least amount of excitation light in to allow just enough signal to track the polymerase over time. That’s why, in the past, we used an EMCCD camera, but it appeared to us that the Prime BSI could match the EMCCD for sensitivity.”
The chip size is large, giving us a large field of view, which is really fantastic, and the pixel size is small at 6.5 microns, which permits Nyquist matching on our 60x lens, maximizing resolution in our experiments.