Cellular Imaging Case Study

Obtaining first-of-a-kind images of micronuclear structure

Errors in mitosis that cause chromosomes to lag behind during cell division cause the formation of micronuclei. These extra-nuclear bodies containing damaged chromosomes, or chromosome fragments, are often seen in cancer cells. While structurally similar to the primary nucleus, micronuclei are much more prone to membrane rupture. And when a micronucleus ruptures, its DNA can shatter, causing genome instability and tumorigenesis.

Postdoctoral research fellow Dr. Anna Mammel of Fred Hutchinson Cancer Research Center and her mentor, faculty member Dr. Emily Hatch, wanted to learn what causes micronuclei to become unstable — information that would be critical in devising a possible strategy to prevent rupture and cancer genome instability.

The Challenge

To do so, Mammel and Hatch needed to image and quantify nanoscale features of micronuclei. But they didn’t know which imaging approach and microscope would work best for their needs. In addition, they wanted the highest possible resolution for 3D imaging of fixed, adherent mammalian cells, which would require super-resolution imaging — but neither had much experience with the method.

The Approach

The team turned to the experts in Fred Hutch Shared Resources for help. Mammel and Hatch met to discuss the project’s challenges and goals with Cellular Imaging core Director Dr. Peng Guo and two of the core’s imaging experts, Dr. Lena Schroeder and Dave McDonald, and with image analysis expert Dr. Julien Dubrulle of the Genomics & Bioinformatics shared resource. Over several conversations, the group mapped out an appropriate course of action to get Mammel and Hatch the high-resolution images they needed and to develop a pipeline for image analysis and quantification.

Schroeder and Guo held development sessions in which Mammel tried several microscopes and compared images of her samples acquired with two state-of-the-art, super-resolution imaging approaches: instant structured illumination microscopy, or iSIM, and stimulated emission depletion, or STED. Because each sample and each research project is unique, it would have been impossible to know which technology was the best fit for the team’s needs without this valuable testing process. Mammel also benefited from the core’s expert staff members to refine sample labeling and identify the optimal imaging parameters.

Primary nucleus and two micronuclei.
Primary nucleus and two micronuclei containing one chromosome each. The bottom micronucleus (filled arrowhead) is ruptured and has a porous nuclear lamina structure. The top micronucleus (open arrowhead) contains chromosome 1, is intact and has a uniform nuclear lamina structure. In the left image, the centromere is shown in black, chromosome 1 is shown in red, the intact marker is shown in green, DNA is shown in blue and the lamina is shown in pink. Sample made by and imaged by Dr. Anna Mammel, Hatch Lab, Fred Hutch

The Outcome

After the development sessions, Mammel and Hatch agreed that the STED images best fit the needs of their project. Schroeder trained Mammel to be a proficient, independent user of the core’s Leica SP8 STED microscope.

Thanks to the first-of-a-kind STED and iSIM images they obtained, Mammel and Hatch deepened their understanding of micronuclear biology. These technologies allowed the researchers to observe defects in the micronuclei lamina they could not have detected with a lower-resolution confocal microscope. They believe the large gaps in the lamina meshwork they’ve seen likely compromise membrane stability and may provide an explanation for why micronuclei rupture. They plan to include in an upcoming manuscript their four-color confocal plus one-color STED channel images, plus the quantification of the abnormal lamina obtained with Dubrulle’s help.

Read More

Learn more about the Hatch Lab’s research.

“I have been highly impressed with everyone in the imaging core facility and their dedication to making this project successful. Through this collaborative effort, we were able to obtain, for the first time, high-resolution images of micronuclear lamina structure.”

– Dr. Anna Mammel, postdoctoral fellow, Hatch Lab, Fred Hutch