Localization-Based Super-Resolution Light Microscopy
the use of relatively non-invasive labeling procedures (that is, transfection), and high specificity due to genetic encoding of PA-FP-protein chimeras. Time-lapse imaging. Temporally-resolved super-resolu-
tion imaging is useful for live or dynamic specimens. Time resolution depends on desired resolution: 50-nm resolution requires a nearest-neighbor distance of 25–50 nm, or approxi- mately 70–280 molecules per diffraction-limited area, which requires (for one rendered image) at least 70–280 frames of acquisition, or 0.2–0.7 seconds per acquisition at 400 Hz. Such frame rates are achievable using a number of commer- cially available EMCCD cameras, such as the Andor iXon+ DU-897. Time resolution of 25 nm requires nearest-neighbor distance of 12.5–25 nm, or 280–1150 frames, requiring 0.7–3 seconds per rendered image. Intensity should be adjusted to bleach molecules within approximately one frame. Note that the structure of interest should not rearrange significantly during this time period, although the individual molecules can move within the structure as long as their motion samples or is meaningfully representative of the structure overall. For example, the dynamics of actin structures at the cell leading edge (motion toward the cell periphery at 0.1 to 2 μm/min) [31] is compatible with live-cell FPALM. Te dynamics of membrane protein clusters is also well-suited to time-lapse imaging [26]. Of course, control experiments must always be performed to confirm that addition of labels to the sample does not perturb structure or function. Figures 4 and 5 show examples of two-color live-cell and fixed-cell FPALM, respectively. Molecular trajectories. Imaging conditions can be adjusted
to allow probe molecules to remain fluorescent for several frames before photobleaching. Tus, several consecutive images of the same molecule can be analyzed to construct a short trajectory, which can be equivalent to a few milliseconds or as much as a few seconds. A trade-off results between trajectory length and localization precision per step from the limited photon budget per molecule: for a thousand photons detected from a single molecule before bleaching, 10 steps with 100 photons per frame results in a localization precision of ~20–30 nm at best, whereas 40 steps would yield 50–60 nm localization precision. On the other hand, trajectories may be obtained for hundreds of thousands of molecules, allowing higher density sampling of structures [32]. Clearly, such methods are not well-suited to situations where long-term tracking of the same molecule(s) is important. However, for many applications, such as tracking of membrane proteins and lipids within small clusters, or measurement of the local velocity of cytoskeletal structures, trajectory imaging can be quite useful. Probes that are particularly resistant to photobleaching, allowing detection of >103 photons, are highly desirable. Tree-dimensional
imaging.
Because most biological struc- tures are three-dimensional (3D),
2011 July •
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Figure 4: Multicolor live-cell FPALM. (A) Pseudocolor image of individual PA-FP molecules (30 ms exposure with Andor iXon+ EMCCD) illustrating FPALM with simultaneous two-channel detection. (B) Two-color FPALM image of Dendra2-HA (green) and PAmCherry actin (red) in a live HAb2 fibroblast cell at 37° C (8000 frames total). (C) Dendra2-HA only. (D) PAmCherry actin only. Credit: M. Gudheti, M. Siyath Gunewardene, T. Gould, and S. Hess.
methods that can image beyond the diffraction limit in the axial direction (z) are essential for advancement of our understanding of biological systems. However, the diffraction-limited resolution in z is typically worse than the lateral resolution. Advances in 3D localization micro- scopy have been demonstrated in several incarnations, including biplane detection [21], optical astigmatism [22], interferometry [23], and double-helix point-spread function [24].
Figure 5: Multicolor FPALM image showing colocalization of PAmCherry-caveolin-1 (red) with Dendra2-interferon receptor molecules (green) in fixed ZFL cells. Credit: K. Gabor, T. Gould, C. Kim, and S. Hess.
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