Yandong Yin, PhD, Postdoctoral Fellow
Eli Rothenberg, PhD, Associate Professor
New York University, School of Medicine
The laboratory of Dr. Rothenberg at the New York University School of Medicine focuses on new optical methods to study biological molecules and processes at real time and nanometer scale. The Rothenberg research team studies the mechanisms of enzymes and proteins that participate in repair of DNA damage leading to cancer, and develops new imaging methods that will enable them to visualize the behavior of individual biological molecules. STORM Microscopy is used to localize and track DNA as it replicates in the cell. “We try to look at the nucleus of cancer cells as they are replicating the DNA. The DNA and proteins involved in DNA replication are labelled so we can understand what is going on when replication happens,” Yandong Yin, PhD. Postdoctoral fellow states.
One of the challenges of imaging replicating DNA is that inside the nucleus of the cell there are many labeled components crowded together, as well as very small components that need to be clearly resolved. To determine how each component is organized spatially, the lab often performs STORM imaging using three or four colors sequentially, which makes resolution, sensitivity, and localization accuracy a great concern. “The DNA replication fork is very small. We can’t image it without super resolution,” says Yin. The laboratory calibrates their STORM post-processing conditions based on the variances of each pixel in the chip of the camera, correcting for any major variations, in order to better fit the point spread function of each fluorophore. Because of this, pixel to pixel variations, like those seen in patterned noise on CMOS cameras becomes a major problem.
If you have a shorter exposure time, you can track faster kinetics. More sensitivity and shorter exposure times with the Prime 95B [Scientific CMOS camera] allow you to image faster and track kinetics better.