Fundamental limits to superresolution fluorescence microscopy

Alex Small

doi:10.1117/12.2003035

22 February 2013 Fundamental limits to superresolution fluorescence microscopy

Alex Small

Proceedings Volume 8590, Single Molecule Spectroscopy and Superresolution Imaging VI; 859010 (2013) https://doi.org/10.1117/12.2003035
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

Superresolution fluorescence microscopy techniques such as PALM, STORM, STED, and Structured Illumination Microscopy (SIM) enable imaging of live cells at nanometer resolution. The common theme in all of these techniques is that the diffraction limit is circumvented by controlling the states of fluorescent molecules. Although the samples are labeled very densely (i.e. with spacing much smaller than the Airy distance), not all of the molecules are emitting at the same time. Consequently, one does not encounter overlapping blurs. In the deterministic techniques (STED, SIM) the achievable resolution scales as the wavelength of light divided by the square root of the intensity of a beam used to control the fluorescent state. In the stochastic techniques (PALM, STORM), the achievable resolution scales as the wavelength of light divided by the square root of the number of photons collected. Although these limits arise from very different mechanisms (parabolic beam profiles for STED and SIM, statistics for PALM and STORM), in all cases the resolution scales inversely with the square root of a measure of the number of photons used in the experiment. We have developed a proof that this relationship between resolution and photon count is universal to techniques that control the states of fluorophores using classical light. Our proof encompasses linear and nonlinear optics, as well as computational post-processing techniques for extracting information beyond the diffraction limit. If there are techniques that can achieve a more efficient relationship between resolution and photon count, those techniques will require light exhibiting non-classical correlations.

Citation Download Citation

Alex Small "Fundamental limits to superresolution fluorescence microscopy", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 859010 (22 February 2013); https://doi.org/10.1117/12.2003035

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KEYWORDS

Photons

Signal detection

Microscopy

Luminescence

Stimulated emission depletion microscopy

Super resolution

Molecules

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