STED Microscopy: Different Approaches and Applications

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NoTeS 18 Figure 6. STED imaging of activity-dependent postsynaptic morphological plasticity. Nagerl et al. (2008), their Fig. 4C, reprinted with permission. Arrows indicate structural changes in dendritic spines that occurred after plasticity-inducing stimulation using chemical LTP protocol. The first frame was taken before the stimulation. Scale bar: 0.5 µm. light microscopy. STED microscopy, however, can resolve these details without sacrificing the ability to reveal dynamic biological cellular processes inside living tissue, which is a key advantage of light microscopy (Nagerl et al., 2008). To demonstrate the power of STED microscopy for imaging dendritic spines, we used YFP-positive CA1 pyramidal neurons in living organotypic hippocampal slices. Hippocampal slices (300 µm thick) were prepared and embedded in a plasma clot on glass coverslips according to standard procedure (Gähwiler, 1981) and incubated for up to two weeks in a roller incubator at 35°C. For the experiments, cultures were transferred into a heated chamber (35°C), where they were maintained in artificial CSF solution. The chamber was custom-made to fit into the microscope stage. Sample handling and preparation followed standard techniques for fluorescence imaging of living specimens and were not altered specifically for STED microscopy. Figure 5 shows a time-lapse series of STED images (8 out of a total of 20), revealing subtle changes in the shape and position of YFP-labeled dendritic spines. Image series of up to 100 frames could be acquired using a resonant beam scanner without any signs of photodamage (data not shown). To demonstrate the improvement in resolution as compared with confocal microscopy, we measured the full width at half maximum (FWHM) of spine necks. The smallest structures were ~70–80 nm in size, indicating a resolution of better than 70 nm. Because most of the spine necks were broader than 70 nm, the resolution of the microscope was well suited to capture the variability in the width of spine necks. We determined the FWHM of multipixel-line profiles across the narrowest part of the spine necks, which was 129 ± 25 nm in the STED mode and 233 ± 22 nm in the confocal mode. Beyond resolving structural details, it was also possible to directly observe activity-dependent structural changes in the shape and size of dendritic spines. To induce structural changes, we subjected the slices to chemical LTP, which is a standard protocol for globally potentiating synapses by pharmacologically raising neuronal activity in the entire slice. The arrows in Figure 6 indicate changes in spine shape after inducing chemical LTP. These examples show that time-lapse imaging using STED microscopy can reveal details of spines in living tissue that are very difficult, if not impossible, to detect by conventional light microscopy techniques. These can now be rigorously quantified for the first time. References Abbe E (1873) Beiträge zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung. Arch Mikrosk Anat Entwichlungsmech 9:413-468. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, Bonifacino JS, Davidson MW, Lippincott-Schwartz J, Hess HF (2006) Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:1642-1645. Born M, Wolf E (2002) Principles of optics (7th ed). Cambridge, New York, Melbourne, Madrid, Cape Town: Cambridge University Press. Gähwiler BH (1981) Organotypic monolayer cultures of nervous tissue. J Neurosci Methods 4:329-342. © 2009 Willig
Hein B, Willig KI, Hell SW (2008) Stimulated emission depletion (STED) nanoscopy of a fluorescent protein-labeled organelle inside a living cell. Proc Natl Acad Sci USA 105:14271-14276. Hell SW (2009) Microscopy and its focal switch. Nat Methods 6:24-32. Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated-emission–stimulatedemission-depletion fluorescence microscopy. Opt Lett 19:780-782. Hell SW, Jakobs S, Kastrup L (2003) Imaging and writing at the nanoscale with focused visible light through saturable optical transitions. Appl Phys A 77:859-860. Hess ST, Girirajan TPK, Mason MD (2006) Ultra-high resolution imaging by fluorescence photoactivation localization microscopy. Biophys J 91:4258-4272. Nagerl UV, Willig KI, Hein B, Hell SW, Bonhoeffer T (2008) Live-cell imaging of dendritic spines by STED microscopy. Proc Natl Acad Sci USA 105:18982-18987. Rust MJ, Bates M, Zhuang XW (2006) Sub-diffractionlimit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3:793-795. Wildanger D, Rittweger E, Kastrup L, Hell SW (2008) STED microscopy with a supercontinuum laser source. Opt Express 16:9614-9621. Willig KI, Harke B, Medda R, Hell SW (2007) STED microscopy with continuous wave beams. Nat Methods 4:915-918. © 2009 Willig STeD Microscopy: Different Approaches and Applications 19 NoTeS
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