Physics & Astronomy ETDs

Publication Date

Spring 5-11-2018

Abstract

Super-resolution microscopy techniques developed through the past few decades enable us to surpass the classical diffraction limit of light, and thus open new doors to investigate the formerly inaccessible world of nanometer-sized objects. Most importantly, by using super-resolution microscopy, one can visualize sub-cellular structures in the range of 10 to 200 nm. At this range, we can investigate exciting problems in biology and medicine by visualizing protein-protein interactions and spatiotemporal analysis of structures of interest on the surface or inside cells. These techniques (collectively known as nanoscopy) have a high impact on understanding and solving biological questions. This dissertation starts with a brief and general description of current super-resolution techniques and then moves toward a multi-target super-resolution imaging strategy using sequential imaging that has benefits over conventional multi-color imaging methods.

Sequential microscopy takes advantage of the photo-physical properties of the most suitable dye for a particular technique to achieve the optimal and consistent resolution for each of multiple targets of imaging. For example, for dSTORM imaging, this is currently AlexaFluor647.\ Sequential dSTROM has an advantage for multi-target imaging due to having a single imaging channel which avoids dealing with differential aberration-problems between multiple emission paths unlike other multi-color imaging based methods. We show that sequential imaging method can be facilitated using automated imaging.

In this dissertation, a sequential microscope is designed, calibrated, and tested on multiple structures. We show that it can automatically re-find the position of each initially registered cell and can account for sample drift through an entire experiment. The microscope has been used in multiple collaborations with other groups to investigate biological problems of interest.

Two labeling strategies that facilitate sequential imaging are described.\ The first strategy is DNA-strand-displacement , which allows imaging of multiple structures in a controlled and time-efficient binding-unbinding scenario. The second strategy is imaging with the small, actin binding peptide Lifeact.

Finally, future directions and suggestions are made about how we can further improve the microscope. In the Appendix I provide a guide on how to use and troubleshoot the microscope, how to measure the efficiency of the microscope, as well as how to fix and label cells for optimal imaging and how to prepare various imaging buffers.

Degree Name

Physics

Level of Degree

Doctoral

Department Name

Physics & Astronomy

First Committee Member (Chair)

Dr. Keith A. Lidke

Second Committee Member

Dr. Diane S. Lidke

Third Committee Member

Dr. Sudhakar Prasad

Fourth Committee Member

Dr. Aaron Neumann

Language

English

Keywords

Super-Resolution, fluorescence, microscopy

Document Type

Dissertation

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