Localization of Dense Clusters of Nanoscale Emitters for Super-resolution Microscopy

Barsic, Anthony J.

Graduate Thesis Or Dissertation

Localization of Dense Clusters of Nanoscale Emitters for Super-resolution Microscopy Public Deposited

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Citeable URL: https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/5q47rn958

Abstract

Single-Molecule Localization Microscopy is an optical imaging paradigm which provides computational reconstructions of microscopic objects much smaller than the imaging wavelength. Information regarding physical dimensions of small structures arises from precise localization of individual fluorophores attached to targets within the sample. Complete reconstructions require precise localizations for which neighboring emitters must be resolved beforehand. Control of experimental conditions ensures adjacent emitters are not likely to be activated in the same camera frame. As a result, the gain in spatial resolution occurs at the expense of the acquisition time. Unfortunately, this approach necessitates many images to generate a well-sampled reconstruction. The work presented in this Dissertation relaxes the resolution requirements for emitters in spatiotemporal proximity. The methods described herein use experimental, optical, and computational techniques. Quantum dots are promising for fluorescence experiments due to their brightness and photostability. They exhibit random blinking under constant illumination creating a signal intermittency that is sometimes considered problematic. I present a method for localizing classically unresolved quantum dots by taking advantage of the random, independent nature of the blinking. Experiments confirm the validity of the approach while also showing that nearby quantum dots are indeed independent. Many experiments require three-dimensional information. A microscope can be modified to transmit precise 3D information through a technique called point spread function (PSF) engineering. The gain in axial precision is achieved at the expense of an increased PSF cross-section at focus, thus exacerbating the problem of emitter overlap. The algorithms presented here exploit the mathematical sparsity of localization microscopy data to identify otherwise unresolved PSFs, yielding a tenfold enhancement in allowable fluorophore density. The method is applied to localization microscopy data of dye-labeled microtubules resulting in quantitative measurements of the cellular structure. Localization microscopy is expected to transform biology as the techniques encompassing disparate fields become widespread. This Dissertation also investigates multicolor and 3D super-resolution using original computational and optical tools incorporated into a standard microscope at the BioFrontiers Imaging facility. The system is applied to imaging in the nuclei of virus-infected cells to examine the viral reproductive properties.

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