Correlative light- and electron microscopy with chemical tags.
Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Mario
M
Perkovic
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Michael
M
Kunz
Goethe University Frankfurt, Institute for Physical and Theoretical Chemistry, Max-von-Laue Str. 13, 60438 Frankfurt am Main, Germany.
Ulrike
U
Endesfelder
Max-Planck-Institute for Brain Research, Max-von-Laue Str. 4, 60438 Frankfurt am Main, Germany.
Stefanie
S
Bunse
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Christoph
C
Wigge
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Zhou
Z
Yu
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Victor-Valentin
VV
Hodirnau
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Margot P
MP
Scheffer
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany.
Anja
A
Seybert
Goethe University Frankfurt, Institute for Physical and Theoretical Chemistry, Max-von-Laue Str. 13, 60438 Frankfurt am Main, Germany.
Sebastian
S
Malkusch
Max-Planck-Institute for Brain Research, Max-von-Laue Str. 4, 60438 Frankfurt am Main, Germany.
Erin M
EM
Schuman
Goethe University Frankfurt, Institute for Physical and Theoretical Chemistry, Max-von-Laue Str. 13, 60438 Frankfurt am Main, Germany.
Mike
M
Heilemann
Goethe University Frankfurt, Buchmann Institute for Molecular Life Sciences and Institute for Biophysics, Max-von-Laue Str. 15, 60438 Frankfurt am Main, Germany. Electronic address: achilleas.frangakis@biophysik.org.
Achilleas S
AS
Frangakis
D022005:Adherens Junctions / Q000648:ultrastructure; D000818:Animals; D015820:Cadherins / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism / Q000648:ultrastructure; D005456:Fluorescent Dyes; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D009942:Organometallic Compounds; D013194:Staining and Labeling / Q000379:methods*
D015820:Cadherins; D005456:Fluorescent Dyes; D009942:Organometallic Compounds; C005460:uranyl acetate
Adherens junctions; Correlative electron and light microscopy; Electron tomography