Optics Team

We are applying tools of physics, optics, and computer science to produce super-resolution multi-color 3D imaging of nanoscale structures inside living cells.

Principal Investigator

Peter Kner

Peter Kner

  • Associate Professor of Engineering
  • The University of Georgia

Peter focuses on the development of techniques of super-resolution microscopy and adpative optics for biological research and biomedical applications.

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Participant

Abhijit Marar

Abhijit Marar

  • Graduate Student
  • The University of Georgia

"I was born in Mumbai, India. I was raised around the world — in Kazakhstan, Algeria, England, India..."

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Research Goals

  • The overall goal of QSTORM-AO is to develop a wavefront-shaping, light-sheet microscope, using photoswitchable quantum dot fluorophores, that will enable 3D single-molecule localization 100um below the surface of a whole organism.
  • The Optics Team is currently designing and building hardware and software components of the system. The new instrument will incorporate Adaptive Optics (AO) and Light Sheet Excitation as well as a novel holographic approach to 3D Single Molecule Localization (SML).

Optics Team Posts

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Background

STORM: a type of Super-Resolution Microscopy

STORM stands for STochastic Optical Reconstruction Microscopy, a localization-based super-resolution imaging technique, first developed in 2006. It was designed to penetrate the 250 nanometer (nm) resolution limit of classical optical microscopy. That limit exists because 250 nm is approximately half the wavelength of visible light.  Any point of light smaller in diameter than 250 nm will still appear to be at least 250 nm in diameter.  And because of that "diffraction limit," any light-emitting tags (fluorophores) spaced any closer than 250 nm, will be indistinguishable from one another. To address this fundamental physical limitation, STORM, and similar techniques (PALM, FSTORM) rely on “photo-switchable fluorophores.” These are photon-emitting molecules that can be controlled so that only a small scattered fraction of them are “on” at any given time. Each one that is “on” and at least 250 nm from its nearest neighbor can then be accurately localized and plotted without interference. The point source of the light is assumed to be at the very center of the circular diffraction pattern it produces.

 

A computer collects the point source localization data for each subset of fluorophores as they randomly switch on and off, at something like 10-50,000 images every 30 milliseconds. (Contrast this to film, which runs at 30 frames per second, and you understand how STORM might eventually be able to capture essentially video of molecular scale activity within living cells.)   Carefully designed algorithms process the multiple images to create one composite "reconstructed" image, yielding all the illuminated structures in “super resolution." In this way, STORM tricks nature into yielding up optical images of intricate biological structures that would be invisible to human eyes.

The video below, by Ricardo Henriques, provides a terrific analogy for STORM, applying a similar process of spot detection and image reconstruction to the blinking lights on a familiar structure.

The brighter and longer-lasting the photon-emitters, the stronger the signal, especially against the "pollution" of diffraction from other light sources. The brighter the fluorophore, the higher the signal to noise ratio, and the more precise the point source localization.  This is particularly important when imaging in live cell or tissue samples that are thick with features.

Most STORM microscopists use organic dyes as their light-emitting agents; the goal of the QSTORM team, however, is to substitute photo-switchable quantum dots (QDots) for the dye molecules. Quantum dots are small (10-20 nm) semiconductor crystals that absorb light and emit different colors of light depending on their diameter. QDots are brighter and longer lasting than dye molecules, and these characteristics should help to produce much higher resolution STORM images of molecular dynamics within cells.  The trick is that QDots have to be made switchable for use in the random localization strategy essential to STORM's success. That's the challenge the QSTORM QDot team is currently addressing.