High-performance probes for light and electron microscopy.
We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Sarada
S
Viswanathan
Department of Neurobiology &Anatomy, University of Utah, Salt Lake City, Utah, USA.
Megan E
ME
Williams
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Erik B
EB
Bloss
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Timothy J
TJ
Stasevich
Howard Hughes Medical Institute, Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.
Colenso M
CM
Speer
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Aljoscha
A
Nern
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Barret D
BD
Pfeiffer
1] Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. [2] Laboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, Maryland, USA.
Bryan M
BM
Hooks
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Wei-Ping
WP
Li
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Brian P
BP
English
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Teresa
T
Tian
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Gilbert L
GL
Henry
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
John J
JJ
Macklin
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Ronak
R
Patel
Laboratory of Systems Neuroscience, National Institute of Mental Health, Bethesda, Maryland, USA.
Charles R
CR
Gerfen
1] Howard Hughes Medical Institute, Department of Chemistry &Chemical Biology, Harvard University, Cambridge, Massachusetts, USA. [2] Howard Hughes Medical Institute, Department of Physics, Harvard University, Cambridge, Massachusetts, USA.
Xiaowei
X
Zhuang
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Yalin
Y
Wang
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Gerald M
GM
Rubin
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA.
Loren L
LL
Looger
D000818:Animals; D000941:Antigens; D001931:Brain Mapping; D004330:Drosophila; D008164:Luminescent Proteins / Q000737:chemistry*; D051379:Mice; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009474:Neurons; D011487:Protein Conformation
D000941:Antigens; D008164:Luminescent Proteins