Pictured: High resolution 4Pi microscopy vs. traditional confocal microscopy. 3-dimensional 4Pi microscopy visualization of Hela H2AX (green) and gamma-H2AX (red) chromatin clusters at 100 nm resolution (center image) is shown in front of a traditional overview z-projected data stack of HeLa cells stained for H2AX, gamma-H2AX and DAPI taken with a confocal microscope.
HIGH RESOLUTION 4PI MICROSCOPY REACHES THE NUCLEUS
Multidisciplinary collaboration offers unique look at DNA damage and repair
November 13, 2006
WORCESTER, MA- Confocal light microscopy has been an important tool for biomedical scientists as they work to unravel molecular events occurring within human cells. Less than a decade ago, an important advance in microscopy technology was achieved with Dr. Stefan Hell's invention of "4Pi microscopy." Recently developed and commercialized by Leica Microsystems, 4Pi technology offers a significant increase in resolution; however, fundamental optical limitations prevented scientists from visualizing molecules in the cell's nucleus using this new method. Now, through the work of Brian Bennett, PhD, of the University of Massachusetts Medical School (UMMS) Department of Biochemistry & Molecular Pharmacology and Leica Microsystems, UMMS Associate Professor of Biochemistry & Molecular Pharmacology Kendall Knight, PhD, and collaborator Jörg Bewersdorf, PhD, of the Institute for Molecular Biophysics at The Jackson Laboratory, this limitation has been overcome-for the first time high resolution 4Pi microscope images of endogenous nuclear proteins in human cells have been realized. In "H2AX chromatin structures and their response to DNA damage revealed by 4Pi microscopy," published online this week by the Proceedings of the National Academy of Sciences, Drs. Bewersdorf, Bennett and Knight used the Leica TCS 4Pi microscope to examine histones, nuclear proteins that are involved in packaging and protecting DNA as well as in processes that repair damaged DNA. Exposure to environmental carcinogens can lead to a particularly devastating form of DNA damage, DNA double-strand breaks. The occurrence of such DNA breaks invokes a rapid and highly coordinated series of molecular events within the nucleus, and defects in this process correlate with increased risks for cancer, as well as developmental and immunological abnormalities.
Bewersdorf, Bennett and Knight examined a specific histone protein, H2AX. Within seconds after DNA damage has occurred, H2AX undergoes a subtle but functionally significant molecular modification, which in turn plays an important role in coordinating the recruitment of proteins that will repair the break. Prior to these results, researchers had been unable to visualize how H2AX is distributed throughout the nucleus. However, this study provides never-before-seen high resolution 3D images of H2AX, demonstrating that it exists in distinct clusters that are themselves evenly distributed throughout the entire nuclear volume. This finding suggests that H2AX clusters provide a platform from which signaling and repair events can be coordinated. Additionally, high resolution 4Pi images of modified H2AX reveal for the first time the complex three-dimensional character and dynamics of molecular associations that occur at the site of a DNA break.
"We are very excited to have finally overcome the obstacle that had prevented visualization of nuclear proteins," said Bennett, who devised the solution enabling the use of 4Pi microscopy during his PhD thesis work in the Knight lab. "The collaborations between myself, Drs. Knight and Bewersdorf, and those at Leica Microsystems were critical in achieving this first step. We look forward to continuing our investigations with the analysis of other nuclear proteins involved in cancer prevention and the repair of DNA damage."
This work was supported by a Department of Defense Breast Cancer Research Program pre-doctoral fellowship (Bennett) and grants from the National Institutes of Health (Knight). Funding for the 4Pi microscope was provided by the W.M. Keck Foundation and National Science Foundation to The Jackson Laboratory
About The University of Massachusetts Medical School
The University of Massachusetts Medical School is one of the fastest growing medical schools in the country, attracting more than $174 million in research funding annually. A perennial top finisher in the annual U.S.News & World Report ranking of primary care medical schools, UMMS comprises a medical school, graduate school of nursing, graduate school of biomedical sciences and an active research enterprise, and is a leader in health sciences education, research and public service.
About Leica Microsystems
Leica Microsystems is a leading global designer and producer of innovative high-tech precision optics systems for the analysis of microstructures. It is one of the market leaders in each of the fields of Microscopy, Confocal Laser Scanning Microscopy, Imaging Systems, Specimen Preparation and Medical Equipment. For more information on Leica's 4Pi technology, visit www.Leica-Microsystems.com/4Pi .
About The Jackson Laboratory
The Jackson Laboratory is an independent, nonprofit research institution based in Bar Harbor, Maine, with facilities in West Sacramento, Calif. Its research staff of more than 450 investigates the genetic basis of cancers, heart disease, osteoporosis, Alzheimer's disease, glaucoma, diabetes, and many other human diseases and disorders, as well as normal mammalian development and bioinformatics. The Laboratory is also the world's source for nearly 3,000 strains of genetically defined mice, home of the Mouse Genome Database and many other publicly available information resources, and an international hub for scientific courses, conferences, training and education.