TY - JOUR
T1 - Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes
AU - Shroff, Hari
AU - Galbraith, Catherine G.
AU - Galbraith, James A.
AU - White, Helen
AU - Gillette, Jennifer
AU - Olenych, Scott
AU - Davidson, Michael W.
AU - Betzig, Eric
PY - 2007/12/18
Y1 - 2007/12/18
N2 - 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.
AB - 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.
KW - Fluorescent proteins
KW - Multi-label
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UR - http://www.scopus.com/inward/citedby.url?scp=38049110263&partnerID=8YFLogxK
U2 - 10.1073/pnas.0710517105
DO - 10.1073/pnas.0710517105
M3 - Article
C2 - 18077327
AN - SCOPUS:38049110263
SN - 0027-8424
VL - 104
SP - 20308
EP - 20313
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 51
ER -