TY - CHAP
T1 - Nanometer-resolution fluorescence electron microscopy (Nano-EM) in cultured cells
AU - Watanabe, Shigeki
AU - Lehmann, Martin
AU - Hujber, Edward
AU - Fetter, Richard D.
AU - Richards, Jackson
AU - Söhl-Kielczynski, Berit
AU - Felies, Annegret
AU - Rosenmund, Christian
AU - Schmoranzer, Jan
AU - Jorgensen, Erik M.
PY - 2014
Y1 - 2014
N2 - Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.
AB - Nano-resolution fluorescence electron microscopy (nano-fEM) pinpoints the location of individual proteins in electron micrographs. Plastic sections are first imaged using a super-resolution fluorescence microscope and then imaged on an electron microscope. The two images are superimposed to correlate the position of labeled proteins relative to subcellular structures. Here, we describe the method in detail and present five technical advancements: the use of uranyl acetate during the freeze-substitution to enhance the contrast of tissues and reduce the loss of fluorescence, the use of ground-state depletion instead of photoactivation for temporal control of fluorescence, the use of organic fluorophores instead of fluorescent proteins to obtain brighter fluorescence signals, the use of tissue culture cells to broaden the utility of the method, and the use of a transmission electron microscope to achieve sharper images of ultrastructure.
KW - Correlative light and electron microscopy
KW - Direct stochastic optical reconstruction microscopy (dSTORM)
KW - Fluorescence electron microscopy
KW - Fluorescence nanoscopy
KW - Ground-state depletion and single-molecule return (GSDIM)
KW - Nano-fEM
KW - Photoactivated localization microscopy (PALM)
KW - Protein localization
KW - Stochastic optical reconstruction microscopy (STORM)
KW - Super-resolution fluorescence microscopy
UR - http://www.scopus.com/inward/record.url?scp=84934444073&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84934444073&partnerID=8YFLogxK
U2 - 10.1007/978-1-62703-776-1_22
DO - 10.1007/978-1-62703-776-1_22
M3 - Chapter
C2 - 24357377
AN - SCOPUS:84934444073
SN - 9781627037754
T3 - Methods in Molecular Biology
SP - 503
EP - 526
BT - Electron Microscopy
PB - Humana Press Inc.
ER -