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Three-dimensional Interferometric Microscopy
This invention combines interferometry and photoactivated localization microscopy (PALM) to create a new light microscopy technique that provides sub 20-nm, three-dimensional localization.
This invention combines interferometry and photoactivated localization microscopy (PALM) to create a new light microscopy technique that provides sub 20-nm, three-dimensional localization. PALM is a widely used, super resolution biological imaging technique that improves the spatial resolution of optical microscopy by at least an order of magnitude and is based on imaging and localizing a large number of single fluorescent molecules sequentially in time and reconstructing a high resolution image based on the molecular coordinates. PALM generates two-dimensional images; obtaining 3D PALM images requires another measurement sensitive to the third dimension. The most sensitive method is to perform multiphase interferometery on the fluorescent light of each molecule and thereby extract its position in the third dimension. The technology described here called iPALM for “interferometric” PALM extends the use of PALM bridging the gap between light and electron microscopy.
The 3D super resolution of this technology has been demonstrated on fixed cells, where it imaged cellular ultrastructure seen previously only with electron microscopy. Using this method it was also possible to resolve the 25-nm diameter of microtubules, resolve dorsal and ventral plasma membranes, and visualize the arrangement of integrin receptors in the endoplasmic reticulum. The nanoscale architecture of various proteins in cell adhesion structure has also been measured. This technology employs an optical system with multiple detectors and a processor. As with 2D PALM, switchable optical labels are used such as the photo activatable fluorescent proteins or switchable dyes.
- Opens a third dimension to photoactivated localization microscopy
- Enables observation of molecules at the same resolution as electron microscopy
- Best vertical resolution for given number of photons
- Proven with photoactivatable fluorescent proteins
- Resolving protein organization within molecular assemblies in cells
- Deciphering molecular scale architecture and interactions that constitute biological structures
- 3D protein trafficking and diffusion in cytosol
- Correlative microscopy
Available designs for Non-Profit Research.
A commercial instrument is in development.
For inquiries, please reference:
Janelia 2006-001 and 2009-006
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