Abstract
Imaging neurons, glia and vasculature in the living brain has become an important experimental tool for understanding how the brain works. Here we describe in detail a protocol for imaging cortical structures at high optical resolution through a thinned-skull cranial window in live mice using two-photon laser scanning microscopy (TPLSM). Surgery can be performed within 30–45 min and images can be acquired immediately thereafter. The procedure can be repeated multiple times allowing longitudinal imaging of the cortex over intervals ranging from days to years. Imaging through a thinned-skull cranial window avoids exposure of the meninges and the cortex, thus providing a minimally invasive approach for studying structural and functional changes of cells under normal and pathological conditions in the living brain.
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Acknowledgements
This work was supported by grants from the National Institutes of Health to W.-B.G. and J.G. as well as an Ellison Medical Foundation/AFAR Postdoctoral Fellowship to G.Y.
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Contributions
All authors contributed to the development/improvement of the thinned-skull imaging technique and the manuscript preparation. G.Y. wrote the initial draft and made the figures. F.P. obtained the movie of imaging through a thinned-skull preparation. C.P. conducted microglia-imaging experiment. J.G. and W.G. were responsible for the initial development of the thinned-skull imaging approach. G.Y. and F.P. improved the technique.
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Supplementary information
Supplementary Figure 1 | Home made head immobilization device.
Head immobilization device includes a custom built plate and a skull holder. The custom built plate was made of a 14×10×0.1 cm aluminum plate, two 18×18×18 mm aluminum blocks, two ¼ inch screws and two spacers. The skull holder was made of 3 to 4 double-edge shaving blades. All experiments using animals were carried out under institutional and national guidelines. (PDF 228 kb)
Supplementary Figure 2 | The dendrites and spines can be clearly visualized on a conventional fluorescence microscope after proper thinning of the skull.
(a-c) Images taken from a CCD camera equipped on a fluorescence microscope. Arrows indicate the spines located in the focal plane of the camera. Structures that were out of focus appear blurred in these images. Scale bar, 10 µm. All experiments using animals were carried out under institutional and national guidelines. (PDF 99 kb)
Supplementary Movie 1
Neuronal structures imaged at high resolution through a thinned-skull window in 1-month old YFP-H mice. The whole movie is an image stack of optical sections that span the thinned skull (∼20 µm in thickness), meningeal layers and neuronal structures within the first 100 µm under the pial surface. The imaged region is 66.6 µm × 66.6 µm and step size is 0.75 µm. The movie is played at a speed of 8 frames (6 µm) per second. Note there is a ∼20 µm space between the skull and the pial surface representing the subarachnoid space. This space is not well visualized when the skull is depressed during the thinning process. All experiments using animals were carried out under institutional and national guidelines. (MOV 3998 kb)
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Yang, G., Pan, F., Parkhurst, C. et al. Thinned-skull cranial window technique for long-term imaging of the cortex in live mice. Nat Protoc 5, 201–208 (2010). https://doi.org/10.1038/nprot.2009.222
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DOI: https://doi.org/10.1038/nprot.2009.222
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