Quantitative DNA-paint imaging of AMPA receptors in live neurons

Youn, Yeoan

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https://hdl.handle.net/2142/113962 Copy

Description

Title

Quantitative DNA-paint imaging of AMPA receptors in live neurons

Author(s)

Youn, Yeoan

Issue Date

2021-11-16

Director of Research (if dissertation) or Advisor (if thesis)

Selvin, Paul R

Doctoral Committee Chair(s)

Selvin, Paul R

Committee Member(s)

• Chemla, Yann R
• Chung, Hee Jung
• Zhang, Kai

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Quant Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

DNA-PAINT, single molecule localization microscopy, AMPA receptor, synaptic plasticity

Abstract

DNA-based point accumulation imaging in nanoscale topography (DNA-PAINT) is a single molecule localization microscopy (SMLM) technique that can generate nanometric resolution and quantification of the number of target molecules without knowing the (complex) photo-physics of the fluorophores. DNA-PAINT achieves nanometric resolution by transient binding of a single fluorophore attached to a short single-stranded DNA strand, called an “imager”, to its complementary DNA-target called “docker”, which is attached to the target proteins. Repetition of transient binding and unbinding of imager to the docker achieves nanometric resolution of the image. However, this simple technique has not been used on live cells under physiological condition due to the high salt concentration in the buffer required for fast and specific binding of the imager to the docker. Here, we used a docker that contains multiple binding sequences, from 1-16, for the imager to improve the speed of DNA-PAINT imaging on both fixed and live cells. Also, a monovalent antibody, a single chain variable fragment (scFv), was used to attach the DNA to its protein target; this means that endogenous (native) proteins can be labeled, eliminating overexpression and crosslinking associated with transfection and labeling procedures. Using this technique, we imaged glutamatergic receptor AMPAR (specifically GluA1) in live and fixed neurons. The AMPAR is an important receptor in synaptic transmission and plasticity of excitatory synapses of neurons. After introducing the technique and methods (Chapter 1 and 2), we used DNA-PAINT to image and quantify GluA1 containing AMPARs (GluA1-AMPARs) in fixed neurons (Chapter 3). The position of AMPAR is determined with ~10 nm localization accuracy. The absolute number of the receptors in synapses was quantified using quantitative DNA-PAINT (qPAINT) under basal condition and under the process involved in forming memories, known as chemically induced long term potentiation (cLTP). We found that the average number of GluA1 subunits for fixed neurons goes from 9.2 originally, to 25.2 after 20 minutes of cLTP, or ~170% higher under cLTP condition than that under basal condition. In Chapter 4, we then used the technique to image live neurons, finding both the dynamics of the GluA1-AMPAR and quantifying the amount of AMPARs in physiological buffer solution. We found that the fraction of mobile and immobile AMPARs differs depending on whether their location is inside or outside of synapses. We also found that ~50% of AMPARs in synapses are enriched and immobilized in, generally called nanodomains. Furthermore, we also imaged AMPARs in synapses undergoing cLTP. Due to continuous exchange of fluorescent imager in DNA-PAINT, it is possible to image AMPARs in live neurons over long period of time without losing signal by photobleaching and to observe the change in diffusion of AMPARs as a response to the induction of cLTP. Upon the induction of cLTP, the immobile fraction of AMPARs increased by 10%, resulting in a 40% lower average diffusion coefficient for AMPARs in the synapse. In Chapter 6, we developed a method to correct stage drift in SMLM imaging by using surface pattered fiduciary markers by thermal nanoimprint lithography. Here, we present an improved fiducial micro-pattern prepared by thermal nanoimprint lithography (T-NIL). The new pattern is made of a thermal plastic material with low fluorescence backgrounds across the wide excitation range, particularly in the blue region; robust structural stability under cell culturing condition; and a high biocompatibility in terms of cell viability and adhesion. We demonstrate drift precision to 1.5 nm for lateral (x, y) and 6.1 nm axial (z) axes for 1 min long image acquisition. As a proof of principle, we acquired 4-color wide-field fluorescence images of live mammalian cells; we also acquired SMLM image of fixed and live hippocampal neurons.

Graduation Semester

2021-12

Type of Resource

Thesis

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http://hdl.handle.net/2142/113962

Copyright and License Information

Copyright 2021 Yeoan Youn

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