The brain would be much easier to see if that pesky skull was not there. Now, using a deformable mirror to correct the light it disperses, researchers led by Meng Cui at the Howard Hughes Medical Institute in Ashburn, Virginia, have come close to making the brain's protective shell vanish. As reported in the July 13 Proceedings of the National Academy of Sciences, their optical prestidigitation revealed writhing microglia and tiny dendritic spines in intact mice.  The optics upgrade can be added to any standard two-photon microscope.

Spines Through the Skull.

After correcting for the light-scattering properties of the intact mouse skull, two-photon imaging brings dendritic spines into view. Scale = 5μm. [Courtesy of Park et al., PNAS 2015.]

Two-photon imaging takes advantage of the long wavelength of infrared light to reduce scattering and produce high-resolution images of living tissue that can't be seen with standard microscopy. However, the mouse skull poses a stubborn barrier, allowing some light to pass but scattering it widely. To see the rodent brain, researchers typically install “cranial windows” or they thin rodent skulls to give them some transparency. However, these invasive procedures can trigger an inflammatory response in the brain that could confound results, especially when researchers are studying microglia, the brain’s immune cells.

To get a look through the skull, first author Jung-Hoon Park and colleagues borrowed an optical technique from astronomy. Used to correct for atmospheric perturbations that make stars twinkle and galaxies look fuzzy, adaptive optics adjust incoming light in real time with a rapidly deformable mirror. The mirror jiggles thousands of times per second to correct the wayward angles of the incoming rays. The researchers fitted a microelectromechanical system (MEMS)-based deformable mirror to a standard two-photon microscope, and programed it to correct for light distortions. Because the shape and thickness of the cranium are fixed entities, the researchers could generate precisely targeted “wavefront corrections” to cancel out interference. Using this set, the researchers could visualize cells in a 300μm2 field of view at submicron resolution.

Park tested the technique on 11- to 12-week-old transgenic mice in which microglia and neurons were labeled with green or yellow fluorescent protein. Shape shifting microglia and dendritic spines came into view (see movie below).

“Considering how sensitive microglia are to their microenvironment, the development of 'minimally' invasive—through a thinned- or opened-skull—two-photon in vivo imaging, combined with electron microscopy and other in situ techniques, was a necessary step to understand their roles in non-pathological conditions,” commented Marie-Eve Tremblay at  Laval University, Quebec. “This advanced wavefront correction technique could take our understanding of microglial physiology a step further.”

Compared with similar imaging through a cranial window, which can probe to a depth of 300μm, the intact skull technique can only visualize small structures such as dendritic spines, Cui said. This limits detailed imaging to the first cortical layer. In some instances removing or thinning the skull may still be the method of choice.

Brian Bacskai of Massachusetts General Hospital in Charlestown called the study an important proof of concept. However, he said that removing the skull—while not quite as elegant as looking through it—is still optimal due to the far-expanded field of view. Bacskai said his standard field of view through a cranial window is 1,000 fold, as described here. This allows Bacskai to view dozens of cells, while the images in Cui’s paper only show parts of cells. The researchers could tile over many cells with the technique, but then they would need to recalibrate the microscope each time, Bacskai said. For these reasons, the adaptive optics technique may not yet be optimal for studying neurodegenerative disease models, said Bacskai. However, he is confident that future advances will bring it closer to reality. “Though we have a long way to go, this is the direction we should be heading.” —Jessica Shugart 



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Primary Papers

  1. . High-resolution in vivo imaging of mouse brain through the intact skull. Proc Natl Acad Sci U S A. 2015 Jul 13; PubMed.