Single Molecule Cryo-Fluorescence Microscopy
by Weixing Li
Date of Examination:2016-10-26
Date of issue:2016-11-07
Advisor:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Holger Stark
Referee:Prof. Dr. Kai Tittmann
Persistent Address:
http://dx.doi.org/10.53846/goediss-5968
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Abstract
English
This work presents a newly developed cryo-fluorescence microscopy system and several of its applications. The thesis is divided into three parts. Part I introduces the design of the cryo-fluorescence microscope, and demonstrates its superior cooling efficiency, mechanical stability, and imaging quality. The high performance of this system allows for continuous single molecule imaging for up to six hours with minor post-correctable drift at 89 K. This system also provides the possibility to transfer vitrified samples at cryogenic temperatures, which enables correlative measurements with cryo-electron microscopy. Part II explores the properties of commonly used organic dyes at 89 K. The photostability is drastically enhanced at liquid nitrogen temperatures, resulting in an average photon yield (detected) of several millions per molecule. The blinking of Alexa647, Atto647N, and Cy5 dyes is slowed down by a factor of ten at 89 K. Atto488 and Alexa488 dyes enter prolonged dark states in the cryostat due to the lack of oxygen (sample located in vacuum in the cryostat), which makes single molecule localization microscopy directly applicable at 89 K. Part III demonstrates single molecule localization with exceptional sub-nanometer precision exploiting the large photon yield at 89 K, and suggests approaches to colocalize two fluorescent molecules with a similar precision.
Keywords: Microscopy+Super-resolution+Cryo-microscopy+Cryostat