Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure - PubMed (original) (raw)

. 2009 Mar 3;106(9):3125-30.

doi: 10.1073/pnas.0813131106. Epub 2009 Feb 6.

James A Galbraith, Catherine G Galbraith, Jennifer Lippincott-Schwartz, Jennifer M Gillette, Suliana Manley, Rachid Sougrat, Clare M Waterman, Pakorn Kanchanawong, Michael W Davidson, Richard D Fetter, Harald F Hess

Affiliations

Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure

Gleb Shtengel et al. Proc Natl Acad Sci U S A. 2009.

Abstract

Understanding molecular-scale architecture of cells requires determination of 3D locations of specific proteins with accuracy matching their nanometer-length scale. Existing electron and light microscopy techniques are limited either in molecular specificity or resolution. Here, we introduce interferometric photoactivated localization microscopy (iPALM), the combination of photoactivated localization microscopy with single-photon, simultaneous multiphase interferometry that provides sub-20-nm 3D protein localization with optimal molecular specificity. We demonstrate measurement of the 25-nm microtubule diameter, resolve the dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors within endoplasmic reticulum and adhesion complexes, 3D protein organization previously resolved only by electron microscopy. iPALM thus closes the gap between electron tomography and light microscopy, enabling both molecular specification and resolution of cellular nanoarchitecture.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Schematics and operating principle of multiphase interferometric microscope illustrating how Z-position is resolved. (A and B) Schematic of the single-photon multiphase fluorescence interferometer. A point source with z-position δ emits a single photon both upwards and downwards. These 2 beams (color coded as red and green in B) interfere in a special 3-way beam splitter. (C) The self-interfered photon propagates to the 3 color-coded CCD cameras with amplitudes that oscillate 120° out of phase as indicated.

Fig. 2.

Fig. 2.

X, Y, Z resolution of iPALM and its dependence on source brightness, illustrating iPALM's sub-20-nm 3D resolution with endogenous FP labels. (A) A histogram of experimentally determined positions from repeatedly sampling (25,000 frames) a source where ≈1,200 photons are detected per frame from ≈1,500 photons emitted into a 4π solid angle. (B) Axial (solid red circles) and lateral (solid blue squares) resolution of iPALM determined from FWHM of localization of Au beads of different brightness. Note that the positional FWHM number is 2.4 times larger than σ the variance that is also used to characterize resolution. Axial (empty red circles) and lateral (empty blue squares) resolution of the defocusing method determined from FWHM of localized position of Au beads of different brightness. Large ovals indicate approximately the published results for axial (red) and lateral (blue) resolutions of 3D STORM (2) and BP PALM (3). The typical photon output of fluorescent protein tags and synthetic fluorophores are depicted as pink and green gradients. Also shown (horizontal dashed lines) are addition uncertainties resulting from the displacement between the target protein and the fluorescent probes for different imaging methods.

Fig. 3.

Fig. 3.

Superresolution iPALM image of microtubules in a PtK1 cell expressing human α-tubulin fused to _m_-KikGR, rendered with z axis color-coding. (A) Large area overview. (B and C) Zoom-in of the area bound by the white box in X–Y (B) and Z–Y (C) projections (_z_-scale is magnified 5×). (D) Histogram of _z_-positions of molecules in the boxed region. Each microtubule has a FWHM of 30 nm, and the separation distance of 70 nm between the cyan and purple microtubules is easily resolved.

Fig. 4.

Fig. 4.

Superresolution iPALM image of COS7 cell expressing the membrane protein VSVG fused to td-EosFP, rendered with z axis color-coding. (A) Large area overview. (B and C) Area outlined in white is enlarged in B and shown in C as a z cross-section. Shown in the Inset is the histogram of vertical distribution of fluorescent molecules in the area limited by the red rectangle.

Fig. 5.

Fig. 5.

U2OS cell expressing td-EosFP-αv-integrin. (A–C) Widefield (A) and PALM (B) images, and z color-coded iPALM image (C). The coverslip surface (CS), focal adhesion (FA), cell plasma membrane (PM), and endoplasmic reticulum (ER) can be identified. (D) _Z_-position histogram of area limited by the green box in C with peaks corresponding to CS and FA. (E and F) X–Y (E) and X–Z (F) projections of the area bound by the white box in C. Z-scale is magnified by 4 in F.

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