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Visual analysis of the lysate of single eukaryotic cells by combining µ-fluidics with electron microscopy

Kemmerling, Simon. Visual analysis of the lysate of single eukaryotic cells by combining µ-fluidics with electron microscopy. 2013, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10513

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Abstract

Systems biology aims to understand the emergence of biological functions from underlying interaction networks. The behavior of these networks is stochastic, making the investigation of cell-to-cell variability an important aspect of systems biology. The proteome defines the functional capacity of a cell at a certain point in time. Thus, tools and assays that investigate the concentrations, locations, functions and interactions of proteins at the single cell level will provide new insights in fundamental biology and biomedicine. However, due to the huge diversity of protein species, the dynamic range of protein expression levels, the minute sample volumes, and the lack of amplification techniques, single-cell proteomic studies remain a challenging task, that requires novel and correlative approaches. This is especially true for the analysis of eukaryotic cells, which still challenges existing techniques.
This thesis is part of a project that aims to establish a new approach to proteomic studies on single eukaryotic cells. The project is based on the idea to physically lyse single cells and spread their components onto sample carriers for visual and mass analysis by electron microscopy (EM) and mass spectrometry (MS). The work presented in this thesis focuses on the implementation of some of the basic microfluidic modules that will be part of the envisaged sample preparation pipeline.
The microfluidic pipeline must both prepare the lysate of single cells and deposit it on the sample carriers in an efficient and unbiased manner. A prototype consisting of a versatile conditioning module for in-line sample conditioning and a hand-over module for lossless sample deposition and micro-patterning of EM grids was built. The implemented semiautomatic procedure is capable of processing minute sample volumes and the transferred test samples are suitable for structural analysis by negative stain transmission electron microscopy (TEM). Further results showed that this method can be used to process the total content of cell lysates; lysate components, such as soluble proteins, filaments and membranes were efficiently stained and clearly resolved on the TEM images; stain quality was excellent and highly reproducible. In addition initial tests demonstrated that the modules could be used to prepare sample grids that are suitable for mass determination by scanning TEM (STEM). Further development of the hand-over module focused on its adaption to MS sample carriers. With this modification, the setup was successfully applied to spot sample-matrix mixtures onto MS plates, resulting in homogenous sample spots for matrix-assisted laser desorption/ionization (MALDI) MS analysis.
The next aim was to collect single cell samples. A module allowing single-cell lysis and rapid uptake of the cell lysate was developed to achieve this. Using a freely positionable microcapillary electrode this module is capable of targeting individual adherent cells in situ under light microscopy (LM) observation. It allows fast electrical lysis of single cells followed by rapid aspiration of the cellular components into the microcapillary. The combination with LM enables live cell monitoring and thus facilitates the evaluation of the lysis and aspiration procedure as well as the correlation of LM information with subsequently derived data. The investigation of single cell lysate samples by classical negative stain TEM revealed the presence of several cellular components, such as membrane patches, filaments and other prominent structures with distinctive shapes. This preliminary data indicates that the whole lysate of individual cells can be prepared for a visual investigation by negative stain TEM.
The modules developed during this thesis provide novel and promising methods for the analysis of the protein content of single eukaryotic cells and their combination will result in a versatile sample preparation pipeline that can be applied to biological questions on the single cell level.
Advisors:Stahlberg, Henning
Committee Members:Roderick, Lim
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Structural Biology & Biophysics > Structural Biology (Stahlberg)
Item Type:Thesis
Thesis no:10513
Bibsysno:Link to catalogue
Number of Pages:120 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:53
Deposited On:21 Oct 2013 14:26

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