Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes

Hari Shroff, Catherine Galbraith, James Galbraith, Helen White, Jennifer Gillette, Scott Olenych, Michael W. Davidson, Eric Betzig

Research output: Contribution to journalArticle

369 Citations (Scopus)

Abstract

Accurate determination of the relative positions of proteins within localized regions of the cell is essential for understanding their biological function. Although fluorescent fusion proteins are targeted with molecular precision, the position of these genetically expressed reporters is usually known only to the resolution of conventional optics (≈200 nm). Here, we report the use of twocolor photoactivated localization microscopy (PALM) to determine the ultrastructural relationship between different proteins fused to spectrally distinct photoactivatable fluorescent proteins (PAFPs). The nonperturbative incorporation of these endogenous tags facilitates an imaging resolution in whole, fixed cells of ≈20-30 nm at acquisition times of 5-30 min. We apply the technique to image different pairs of proteins assembled in adhesion complexes, the central attachment points between the cytoskeleton and the substrate in migrating cells. For several pairs, we find that proteins that seem colocalized when viewed by conventional optics are resolved as distinct interlocking nano-aggregates when imaged via PALM. The simplicity, minimal invasiveness, resolution, and speed of the technique all suggest its potential to directly visualize molecular interactions within cellular structures at the nanometer scale.

Original languageEnglish (US)
Pages (from-to)20308-20313
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume104
Issue number51
DOIs
StatePublished - Dec 18 2007
Externally publishedYes

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Color
Proteins
Microscopy
Cellular Structures
Cytoskeleton

Keywords

  • Fluorescent proteins
  • Multi-label

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. / Shroff, Hari; Galbraith, Catherine; Galbraith, James; White, Helen; Gillette, Jennifer; Olenych, Scott; Davidson, Michael W.; Betzig, Eric.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, No. 51, 18.12.2007, p. 20308-20313.

Research output: Contribution to journalArticle

Shroff, Hari ; Galbraith, Catherine ; Galbraith, James ; White, Helen ; Gillette, Jennifer ; Olenych, Scott ; Davidson, Michael W. ; Betzig, Eric. / Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. In: Proceedings of the National Academy of Sciences of the United States of America. 2007 ; Vol. 104, No. 51. pp. 20308-20313.
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