2012 Dec 11
Visualizing Cell Structure and Function With Point-Localization Superresolution Imaging
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Visualizing Cell Structure and Function With Point-Localization Superresolution Imaging
Fundamental to the success of cell and developmental biology is the ability to tease apart molecular organization in cells and tissues by localizing specific proteins with respect to one another in a native cellular context. However, many key cellular structures (from mitochondrial cristae to nuclear pores) lie below the diffraction limit of visible light, precluding analysis of their organization by conventional approaches. Point-localization superresolution microscopy techniques, such as PALM and STORM, are poised to resolve, with unprecedented clarity, the organizational principles of macromolecular complexes within cells, thus leading to deeper insights into cellular function in both health and disease.
Copyright © 2012 Elsevier Inc. All rights reserved.
Principle of photoactivation/switching based pointillist superresolution microscopy. (A) The probe molecules are in the dark state and are invisible at the start of the experiment. A sparse subset of molecules is photoactivated and imaged as spatially separate fluorescence spots. The activated fluorescent molecules are photobleached, and a new set of molecules is activated to continue the identification and localization of single molecules. (B) Discrete fluorescence spots of photoactivated molecules in each frame are individually fit to localize the molecules with high spatial resolution. The bleaching, activation and imaging steps are repeated thousands of times, and a superresolution image is finally generated by combining the localization information from all the frames.
Working principle of bleaching/blinking based superresolution microscopy. Subtracting consecutive image frames from each other isolates single fluorescent spots, corresponding to single molecules that have bleached or blinked during the image acquisition process. The single molecule spots in the difference images are mathematically fit to estimate the center of molecules with tens of nanometer spatial resolution. By combining the single molecule localization information from all the difference images, a superresolution image is reconstructed with structural details unresolvable in diffraction-limited image.
Exhibition of the macromolecular details reconstructed using point-localization microscopy methods. (A) Class averaged dSTORM reconstruction of components of the nuclear pore complex (NPC) from isolated nuclear lamina of a
Xenopus laevis oocyte. The integral membrane component gp210 (left) labeled with antibody highlights the eight-fold symmetry of the NPC, while wheat-germ agglutinin labels nucleoporin central channel (middle). The two averaged structures are docked into one another to reveal a double ring structure of the NPC (right). Scale bars, 100 nm. (Adapted from reference Z). (B) Three-dimensional reconstructions of the focal adhesion integrin receptor (α v) and overlying actin stress fiber bundles using interferometric-PALM (iPALM) and the photoactivatable fluorescent protein variants of EOS. Top view of cell (top images) and side view projection of region within white box (bottom images: Scale bars, 500nm). Distribution of localized molecules along the optical axis (bottom right). (Adapted from reference X). (C) Correlative three-dimensional focused-ion beam scanning electron microscopy (FIB-SEM) and iPALM imaging of mitochondrial membranes and the associated mitochondrial nucleoid DNA. The mitochondrial nucleoid (red isosurface) is labeled with the photoactivatable fluorescent protein mEOS2 and is overlaid with an SEM slice (grayscale). Three-dimensinal representation of mitochondrial inner membrane cristae (yellow isosurface) is shown overlaid with nuceloid-associated mEOS2. Scale bars 400nm. (Adapted from reference Y).
(A–B) Pair-correlation PALM (PC-PALM) analysis enables the estimation of physical parameters of molecular clusters in single molecule super-resolution images. (A) Super-resolution PALM image of Lyn kinase tagged with photoactivatable GFP (PAGFP) expressed in COS-7 cells. Autocorrelation analysis is performed on a sub-section (red box) of the cell to estimate the size, protein number and density in clusters of Lyn-PAGFP. By mathematical fitting (red line) of the measured correlation function (g(r), black circles), the clustering contribution from multiple appearances of same fluorescent molecule (blue spots) and the actual correlation function of protein clustering (green circles) is evaluated. (B) PC-PALM analysis showing the nanoscale organization of PAGFP labeled GPI anchored protein (PAGFP-GPI) and photoactivatable mCherry1 tagged actin (PAmCh-actin). The two proteins are scattered across the membrane at steady state (I), with no detectable cross-correlation. However, the PALM image and cross-correlation curve shows significant co-clustering of PAGFP-GPI and PAmCh-actin following antibody crosslinking of PAGFP-GPI. The cross-correlation curve provides an estimate of the spatial scales of colocalization between the two proteins. (C-D) Superresolution imaging with fast acquisition time has been used to characterize the biogenesis and maturation of focal adhesion complexes and the endocytic clathrin coated pits. (C) PALM time-series images of tdEos-paxillin expressed in CHO cells. The image shows the incorporation of tdEos-Paxillin in a maturing focal adhesion complex. Scale bars, 500 nm (D) Three-dimensional STORM image series showing temporal evolution of labeled transferrin cluster in live cell. Scale bars, 50 nm.
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