Humans and other higher organisms have evolved complex mechanisms for detecting and repairing the chromosome breaks caused by either exposure to genotoxic agents or inherent metabolic processes. This DNA damage signaling and repair involves a highly coordinated series of molecular events, and defects in many of these have been tied to increased risks of cancer as well as to developmental and immunologic abnormalities.Using immunofluorescence methods, researchers have gained significant in-sight into the positioning of signaling and repair proteins and the ways in which the proteins respond to various genomic insults. Still, many of these methods cannot distinguish between fluorescence signals in three-dimensional space when they are within 500 to 800 nm of each other. Increased depth resolution, therefore, could contribute to improved understanding of DNA damage signaling and repair.In the Nov. 28 issue of PNAS, investigators with The Jackson Laboratory in Bar Harbor, Maine, the University of Massachusetts Medical School in Worcester and Leica Microsystems Inc. in Exton, Pa., reported use of Leica’s 4Pi microscope to image proteins with resolution of about 100 nm along the Z-axis. They identified previously undescribed chromatin structures involved in DNA damage signaling and repair, yielding insights into these intricate processes.Using the double-lens 4Pi microscope, researchers have uncovered some of the early events in DNA damage signaling and repair. They have observed the three-dimensional distribution of the histone H2AX, which is involved in the coordination of signaling and repair activities, and tracked the spreading of γ-H2AX following phosphorylation of the histone. The microscope helped them to establish how the boundaries of this spreading are determined. Shown here are 4Pi images of H2AX and γ-H2AX at 15 (a), 45 (b) and 180 minutes (c) after DNA damage has been induced. Images reprinted with permission of PNAS. Developed in the 1990s and commercialized only in the past couple of years, 4Pi microscopy uses two opposing lenses to achieve much higher depth resolution than is typically possible with confocal or two-photon microscopy. Combining the numerical apertures of the two lenses effectively produces a “superlens,” explained Jörg Bewersdorf, a researcher with The Jackson Laboratory and one of the authors of the PNAS paper. “Since the resolution of a microscope depends very much on the numerical aperture,” he said, “it’s obvious that, by doubling the aperture angle, you also increase your resolution dramatically.”The researchers compared 4Pi microscope images (b) with images acquired using conventional confocal microscopy (a). The dotted ellipses in d and c, which represent the same X-Z section from the 4Pi and confocal images, respectively, highlight the enhanced resolution offered by the former.The improved resolution is especially important when imaging 3-D distributions of complex biological structures such as mitochondrial networks. “You don’t have crosstalk between objects of different focal planes hiding fragile details,” Bewersdorf said.He added, however, that 4Pi microscopy as described in the publication is not a fast method. Acquisition of a 3-D dataset takes about half an hour, effectively limiting the technique to imaging of fixed samples, which, he noted, was sufficient for the current research.Previous studies using 4Pi microscopy have shown well-defined images of cellular structures, including microtubules, mitochondria and the Golgi apparatus. But they have not tackled imaging of nuclear proteins, which are essential to understanding DNA damage signaling and repair. The authors now have achieved this goal, through development of new staining methods, as well as optimization of protocols for cell fixation by Brian T. Bennett, currently of Leica Microsystems Inc. and at the time a graduate student in the lab of Kendall L. Knight at the University of Massachusetts Medical School.To demonstrate the improved resolution afforded by this combination of methods, they imaged one of the earliest in the cascade of DNA signaling and repair events: phosphorylation of the histone H2AX to form γ-H2AX. This event is integral to coordinating signaling and repair activities in the wake of double-strand breaks, an especially destructive form of DNA damage. In mammalian cells, H2AX phosphorylation spreads from the DNA break site, but no previous studies have described the distribution of H2AX throughout the nucleus or the mechanisms by which the boundaries of γ-H2AX spreading are established.The investigators began by fixing and staining HeLa cells for H2AX and γ-H2AX and then imaged them with the Leica TCS 4Pi microscope outfitted with a pair of 100/1.35-NA glycerol objectives. A Chameleon laser made by Coherent Laser Group of Santa Clara, Calif., and emitting at 780 nm provided two-photon excitation. They recorded the Alexa Fluor 488 and Rhodamine Red-X fluorescence using the 4Pi system’s photon-counting avalanche photodiodes.They quantitatively analyzed the distribution of H2AX and from there identified special clusters of the histone that seemed to correspond to the known spreading distance of phosphorylation in the case of the double-strand break.The findings showed that H2AX is not distributed randomly throughout the nuclear volume but is instead concentrated in distinct clusters that are uniformly distributed. The researchers subsequently hypothesized that the size and distribution of the clusters determine the boundaries of γ-H2AX spreading. These clusters also may provide a platform for the series of events that initiate within seconds of DNA damage.The investigators are continuing their studies with the 4Pi microscope, applying it to other types of histones to answer further research questions.Contact: Jörg Bewersdorf, The Jackson Laboratory, Bar Harbor, Maine; e-mail: joerg.bewersdorf@jax.org.