Imaging Intracellular Fluorescent Proteins at Nanometer Resolution

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Science  15 Sep 2006:
Vol. 313, Issue 5793, pp. 1642-1645
DOI: 10.1126/science.1127344
  • Fig. 1.

    The principle behind PALM. A sparse subset of PA-FP molecules that are attached to proteins of interest and then fixed within a cell are activated (A and B) with a brief laser pulse at λact = 405 mm andthenimagedat λexc = 561 mm until most are bleached (C). This process is repeated many times (C and D) until the population of inactivated, unbleached molecules is depleted. Summing the molecular images across all frames results in a diffraction-limited image (E and F). However, if the location of each molecule is first determined by fitting the expected molecular image given by the PSF of the microscope [(G), center] to the actual molecular image [(G), left], the molecule can be plotted [(G), right] as a Gaussian that has a standard deviation equal to the uncertainty σx,y in the fitted position. Repeating with all molecules across all frames (A′ through D′) and summing the results yields a superresolution image (E′ and F′) in which resolution is dictated by the uncertainties σx,y as well as by the density of localized molecules. Scale: 1 × 1 μm in (F) and (F′), 4 × 4 μm elsewhere.

  • Fig. 2.

    Comparative summed-molecule TIRF (A) and PALM (B) images of the same region within a cryo-prepared thin section from a COS-7 cell expressing the lysosomal transmembrane protein CD63 tagged with the PA-FP Kaede. The larger boxed region in (B), when viewed at higher magnification (C) reveals smaller associated membranes that may represent interacting lysosomes or late endosomes that are not resolvable by TIRF. In a region where the section is nearly orthogonal to the lysosomal membrane, the most highly localized molecules fall on a line of width ∼10 nm (inset). In an obliquely cut region [(D), from the smaller boxed region in (B)], the distribution of CD63 within the membrane plane can be discerned.

  • Fig. 3.

    Comparative summed-molecule TIRF (A), PALM (B), TEM (C), and PALM/TEM overlay (D) images of mitochondria in a cryo-prepared thin section from a COS-7 cell expressing dEosFP-tagged cytochrome-C oxidase import sequence. Higher magnification PALM (E), TEM (F), and overlay (G) images within the box in (B) reveal that these matrix reporter molecules extend up to, but not into, the ∼20-nm outer mitochondrial membrane. The molecular distribution across two mitochondria along lines 1 and 2 in PALM image (E) are compared in (H) to the TEM signal along lines 3 and 4 in (F) across the same mitochondria. Scale bars: 1.0 μm in (A) to (D); 0.2 μm in (E) to (G).

  • Fig. 4.

    Examples of PALM imaging near the surfaces of whole, fixed cells. (A) A summed-molecule TIRF image of focal adhesions for a FoLu cell expressing dEos-tagged vinculin. (B) A magnified PALM view of the structure within a single adhesion over the region indicated by the box in (A), including apparent assembly of vinculin in a partial network (arrows). (C) A summed-molecule TIRF image near the periphery of a FoLu cell expressing tdEos-tagged actin. (D) A magnified PALM view of the actin distribution within the portion of the lamellipodium outlined by the box in (C). Inset, a further magnified view near the leading edge over the region indicated by the smaller box. (E and F) Summed-molecule TIRF and PALM images, respectively, of a COS-7 cell expressing the retroviral protein Gag tagged with dEos. The PALM image highlights voids (arrows labeled V), a higher density region (arrow R), and probable condensation at several points (arrows labeled P) into VLPs of ∼100- to 150-nm size (inset).

Additional Files

  • Imaging Intracellular Fluorescent Proteins at Nanometer Resolution
    Eric Betzig, George H. Patterson, Rachid Sougrat, O. Wolf Lindwasser, Scott Olenych, Juan S. Bonifacino, Michael W. Davidson, Jennifer Lippincott-Schwartz, Harald F. Hess

    Supporting Online Material

    This supplement contains:
    Materials and Methods
    Figs. S1 to S10
    Table S1
    Movie S1

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