Novel Applications of Super-Resolution Microscopy in Molecular Biology and Medical Diagnostics

by William Zhang
Doctoral thesis
Date of Examination:2015-11-18
Date of issue:2016-03-31
Advisor:Prof. Dr. Silvio Rizzoli
Referee:Prof. Dr. Tiago Fleming Outeiro
Referee:Prof. Dr. Gerhard Braus
Persistent Address: http://dx.doi.org/10.53846/goediss-5589

 

 

Files in this item

Name:PhD-thesis_v4.pdf
Size:2.03Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Despite recent advances in microscopy techniques, the underlying labeling assays remained mostly unchanged. However, super-resolution techniques require other staining conditions than conventional light microscopy, like more dense labeling, which is not always trivial to achieve. In this work, I present two novel applications of stimulated emission depletion (STED) microscopy to the fields of molecular biology and medical diagnostics. For one, I established a fluorescent in situ hybridization (FISH) protocol to investigate mRNAs of the three synaptic proteins synaptophysin, synaptobrevin, and synaptotagmin in primary hippocampal neurons. This achieved more precise information regarding the mRNA numbers and organization than conventional confocal microscopy. Secondly, I applied STED microscopy for the development of a novel diagnosis method for Alzheimer’s disease (AD). The increased resolution was sufficiently high to discriminate low and high molecular weight β-amyloid (Aβ) aggregates produced in vitro. Analysis of cerebrospinal fluid (CSF) samples from 36 AD patients, 11 patients with mild cognitive impairment (MCI), and 21 controls allowed a separation of AD patients from controls with ~87% specificity and ~79% sensitivity. In conclusion, this work illustrates the need for optimization of long established methods depending on the imaging technique and sample to obtain more accurate data.
Keywords: super-resolution; Alzheimer's disease; fluorescence in situ hybridization
 
Statistik