Washington University in St Louis

The Preston M. Green Department of
Electrical & Systems Engineering

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measuring the rotation of single molecules concept art

Since the earliest invention of telescopes, microscopes, and eyeglasses, imaging systems have been designed to help humans visualize the world around us – big and small, near and far. These imaging systems collect the light reflected or emitted from an object and focus it onto our eyes or a camera. The design of these systems dictates that their images only contain two-dimensional (2D) information about an object and that their 2D images are blurred if the object is out of focus. We build imaging systems with new capabilities that surpass these shortcomings.

Super-resolution is a key feature of many of our imaging systems – the ability to overcome the resolution limit of wave physics, termed the diffraction limit, in order to visualize the nanoscale world. Read more about this technology on our resources page.

Three-dimensional imaging


Two examples of rotating PSFs: (left) the corkscrew PSF and (right) the double-helix PSF.

We are investigating new ways of measuring the three-dimensional positions of objects from the two-dimensional images created by imaging systems. One powerful way to implement 3D imaging is through the use of engineered point spread functions (PSFs) – that is, a redesign of the imaging system such that a point emitter of light, like a single molecule, looks dramatically different from a normal focused spot. Rotating PSFs, or images that simply rotate as an emitter moves closer or further away from focus, are an efficient way to implement 3D super-resolution imaging. We are exploring the many possibilities for other PSF behaviors ideal for 3D imaging.

Related papers

  1. M. D. Lew*, S. F. Lee*, J. L. Ptacin, M. K. Lee, R. J. Twieg, L. Shapiro, and W. E. Moerner, “Three-dimensional superresolution colocalization of intracellular protein superstructures and the cell surface in live Caulobacter crescentus,” Proc. Natl. Acad. Sci. USA 108, E1102 (2011). [Journal]
  2. M. D. Lew, S. F. Lee, M. Badieirostami, and W. E. Moerner, “Corkscrew point spread function for far-field three-dimensional nanoscale localization of pointlike objects,” Opt. Lett. 36, 202 (2011). [Journal]

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Measuring the orientation of single molecules


Single fluorescent molecules are exquisitely sensitive probes of their local environment, and their orientations can be used to infer properties like local viscosity or to visualize interactions between biomolecules. They radiate light like nanoscale dipole antennas; their radiation patterns are shaped like donuts around the axis of the dipole. Therefore, we can design an imaging system to measure the orientation of single molecules by modeling how emitted light from each molecule is collected and relayed by the imaging system. The double-helix PSF has the ability to measure simultaneously the 3D position and 2D orientation of single molecules. We are designing other PSFs for measuring orientation.

Related papers

  1. M. P. Backlund, M. D. Lew, A. S. Backer, S. J. Sahl, and W. E. Moerner, “The role of molecular dipole orientation in single-molecule fluorescence microscopy and implications for super-resolution imaging,” ChemPhysChem 15, 587 (2014). [Journal]
  2. M. P. Backlund*, M. D. Lew*, A. S. Backer, S. J. Sahl, G. Grover, A. Agrawal, R. Piestun, and W. E. Moerner, “Simultaneous, accurate measurement of the 3D position and orientation of single molecules,” Proc. Natl. Acad. Sci. USA 109, 19087 (2012). [Highlight in Nat. Methods, Journal]

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