PUBLICATION
High-density three-dimensional localization microscopy across large volumes
- Authors
- Legant, W.R., Shao, L., Grimm, J.B., Brown, T.A., Milkie, D.E., Avants, B.B., Lavis, L.D., Betzig, E.
- ID
- ZDB-PUB-160308-3
- Date
- 2016
- Source
- Nature Methods 13(4): 359-65 (Journal)
- Registered Authors
- Keywords
- Cell division, Developmental biology, Fluorescence imaging
- MeSH Terms
-
- Animals
- COS Cells
- Cell Membrane/ultrastructure*
- Chlorocebus aethiops
- Embryo, Nonmammalian/ultrastructure*
- Fluorescent Dyes
- Image Processing, Computer-Assisted/methods
- Imaging, Three-Dimensional
- LLC-PK1 Cells
- Microscopy, Electron/methods*
- Microscopy, Fluorescence/methods*
- Mitochondria/ultrastructure*
- Swine
- Zebrafish/embryology
- PubMed
- 26950745 Full text @ Nat. Methods
Citation
Legant, W.R., Shao, L., Grimm, J.B., Brown, T.A., Milkie, D.E., Avants, B.B., Lavis, L.D., Betzig, E. (2016) High-density three-dimensional localization microscopy across large volumes. Nature Methods. 13(4):359-65.
Abstract
Extending three-dimensional (3D) single-molecule localization microscopy away from the coverslip and into thicker specimens will greatly broaden its biological utility. However, because of the limitations of both conventional imaging modalities and conventional labeling techniques, it is a challenge to localize molecules in three dimensions with high precision in such samples while simultaneously achieving the labeling densities required for high resolution of densely crowded structures. Here we combined lattice light-sheet microscopy with newly developed, freely diffusing, cell-permeable chemical probes with targeted affinity for DNA, intracellular membranes or the plasma membrane. We used this combination to perform high-localization precision, ultrahigh-labeling density, multicolor localization microscopy in samples up to 20 μm thick, including dividing cells and the neuromast organ of a zebrafish embryo. We also demonstrate super-resolution correlative imaging with protein-specific photoactivable fluorophores, providing a mutually compatible, single-platform alternative to correlative light-electron microscopy over large volumes.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping