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WIREs Nanomed Nanobiotechnol
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Super‐resolution microscopy for nanosensing

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Abstract The recent advances in optical microscopy enable the simultaneous visualization of thousands of structural and signaling molecules as they dynamically rearrange within living cells. Super‐resolution microscopy offers an unprecedented opportunity to define the molecular mechanisms of nanosensing through direct observation of protein movement. This technology provides a real‐time readout of how genetically targeted molecular perturbations affect protein interactions. As we strive to meet the challenge offered by the opportunity to ask questions about the mechanism of cell that we never thought we could answer, we need to be aware that the new technologies are still evolving. The current limitations of each technique need to be considered when matching them to specific biological questions. In this review, we briefly describe the principles of super‐resolution optical microscopy and focus on comparing the characteristics of each technique that are important for their use in studying nanosensing in the cellular microenvironment. WIREs Nanomed Nanobiotechnol 2011 3 247–255 DOI: 10.1002/wnan.130 This article is categorized under: Diagnostic Tools > Biosensing Nanotechnology Approaches to Biology > Cells at the Nanoscale Diagnostic Tools > In Vivo Nanodiagnostics and Imaging

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Super‐resolution microscopy relies on the ability to control the light/dark states of fluorescent molecules. If a fluorescent probe is used that has two distinct states that can be reversibly controlled by photoswitching then the technique is known as RESOLFT11. In one state, the probe is fluorescent while in the other state it is dark because it has either been returned to the ground state [stimulated emission depletion (STED) and saturated structured illumination (SSIM)] or shelved to the meta‐stable triplet state [ground state depletion (GSD)]. Stochastic optical reconstruction microscopy (STORM) is a RESOLFT technique; it toggles a bistable synthetic dye molecule back and forth between states multiple times. However, PALM and fPALM are not RESOLFT techniques when they irreversibly convert from one fluorescent state to the other, but they do share with STORM the requirement to control conversion so that the molecular density is low enough to avoid overlapping molecules. The transition from ground state to excitation takes ∼10−15 seconds; fluorescence emission requires on the order of 10−12 seconds. Molecules can reside in the triplet state variable amounts of time before they give off their phosphorescence and drop to the ground state in ∼10−6 seconds.13

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Molecular organization reveals that proteins that colocalize at the diffraction limit have a distinct molecular organization. (a) DIC image indicating where adhesion complexes are localized on the cell surface. (b–d) Higher magnification views of super‐resolution images indicate that paxillin and vinculin are not colocalized in adhesion molecules. (Reprinted with permission from Ref 44. Copyright 2007 PNAS)

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Live‐cell PALM demonstrates the molecular evolution of molecules within structural and signaling scaffolds. (a) Boxed regions are shown in higher magnification in (b) and (c). (b) Molecules are added to trailing edge of a large adhesion scaffold over the course of time. (c) Small scaffolds located in the central region of a cell show little size variation. (Reprinted with permission from Ref 5. Copyright 2008 Nat Methods)

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Microtubules provide a molecular yardstick for evaluating 3D super‐resolution techniques. Undecorated microtubules have been shown by electron microscopy to have a diameter of ∼22–25 nm and antibody decorated microtubules have a diameter of ∼55 nm.33 (a) 3D STED shows microtubules at 100 nm in diameter. (Reprinted with permission from Ref 34. Copyright 2008 Opt Express) (b) 3D STORM shows 66 nm diameter microtubules. (Reprinted with permission from Ref 25. Copyright 2008 Science) (c) iPALM measures microtubules as 25–30 nm in diameter axially, which is the diameter of the undecorated microtubule plus the 2–3 nm of the PAFP. (Reprinted with permission from Ref 21. Copyright 2009 PNAS)

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Super‐resolution microscopy techniques use either hardware or software to increase resolution. (a) In stimulated emission depletion (STED) microscopy, the excitation point‐spread function (PSF) is combined with the PSF of the STED depletion laser to produce a resultant PSF that is smaller than the diffraction limit of light. (b) In structured illumination microscopy (SIM), the excitation light is patterned with on/off transitions. The high frequency periodicity of the illumination pattern combines with the inherent frequency of the image to permit visualization by the microscope. (c) PALM and STORM techniques turn on subsets of molecules that are separated by distances greater than the diffraction limit of light. Mathematical localization of the PSFs is repeated over many frames that are combined into super‐resolution images.

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Diagnostic Tools > In Vivo Nanodiagnostics and Imaging
Nanotechnology Approaches to Biology > Cells at the Nanoscale
Diagnostic Tools > Biosensing

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