Downey, Mike J. (2012) Extracting fluorescent reporter time courses of cell lineages from high-throughput microscopy at low temporal resolution. PhD thesis, University of Warwick.

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Abstract

Live Cell Imaging and High Throughput Screening are rapidly evolving
techniques and have found many applications in recent years. Modern microscopy enables the visualisation of internal changes in the cell through the
use of
fluorescently tagged proteins which can be targeted to specific cellular
components.
A system is presented here which is designed to track cells at low temporal
resolution within large populations, and to extract
fluorescence data which
allows relative expression rates of tagged proteins to be monitored.
Cell detection and tracking are performed as separate steps, and several
methods are evaluated for suitability using timeseries images of Hoechst-stained
C2C12 mouse mesenchymal stem cells. The use of Hoechst staining ensures
cell nuclei are visible throughout a time-series. Dynamic features, including
a characteristic change in Hoechst
fluorescence intensity during chromosome
condensation, are used to identify cell divisions and resulting daughter cells.
The ability to detect cell division is integrated into the tracking, aiding
lineage construction. To establish the efficiency of the method, synthetic cell
images have been produced and used to evaluate cell detection accuracy. A
validation framework is created which allows the accuracy of the automatic
segmentation and tracking systems to be measured and compared against
existing state of the art software, such as CellProfiler. Basic tracking methods,
including nearest-neighbour and cell-overlap, are provided as a baseline to
evaluate the performance of more sophisticated methods.
The software is demonstrated on a number of biological systems, starting
with a study of different control elements of the Msx1 gene, which regulates
differentiation of mesenchymal stem cells. Expression is followed through
multiple lineages to identify asymmetric divisions which may be due to cell
differentiation.
The lineage construction methods are applied to Schizosaccharomyces pombe
time-series image data, allowing the extraction of generation lengths for individual cells. Finally a study is presented which examines correlations between
the circadian and cell cycles. This makes use of the recently developed FUCCI
cell cycle markers which, when used in conjunction with a circadian indicator
such as Rev-erbα-Venus, allow simultaneous measurements of both cycles.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QH Natural history > QH301 Biology
Library of Congress Subject Headings (LCSH):	Fluorescence microscopy, Cell division -- Microscopy
Official Date:	June 2012
Dates:	Date Event June 2012 Submitted
Institution:	University of Warwick
Theses Department:	Molecular Organisation and Assembly in Cells
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Bretschneider, Till ; Vance, Keith ; Ladds, Graham Robert
Sponsors:	Engineering and Physical Sciences Research Council (EPSRC)
Extent:	vii, 170 leaves : ill., charts
Language:	eng

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