Imaging Chromosome Separation in Mouse Oocytes by Responsive 3D Confocal Timelapse Microscopy
Accurate chromosome segregation is necessary so that genetic material is equally shared among daughter cells. However, maturing mammalian oocytes are particularly prone to chromosome segregation errors, making them a valuable tool for identifying the causes of mis-segregation. Factors such as aging, cohesion loss, DNA damage, and the roles of a plethora of kinetochore and cell cycle-related proteins are involved. To study chromosome segregation in oocytes in a live setting is an imaging challenge that requires advanced techniques. Here we describe a method for examining chromosomes in live oocytes in detail as they undergo maturation. Our method is based on tracking the “center of brightness” of fluorescently labeled chromosomes. Here we describe how to set up our software and run experiments on a Leica TCS SP8 confocal microscope, but the method would be transferable to other microscopes with computer-aided microscopy.
Key wordsImaging Automation Software Microscopy Oocyte
This work was funded by a project grant (mechanisms of DNA damage and repair in mature oocytes) to K.J. from the BBSRC (BB/L006006/1). S.L. is a named postdoctoral researcher on this grant. S.L. wrote the software, S.C. packaged the software for distribution and created the installer, and S.L. drafted the manuscript with input from all authors.