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Quantitative Fluorescence Microscopy of Protein Dynamics in Living Cells
- Publication date
- 21 December 2006
- Publisher
- The advent of confocal microscopy, fast microcomputers with high storage capacity
and, moreover, the availability of fluorescent proteins of various excitation and
emission properties have made fluorescence microscopy the method of choice in the
study of protein behaviour in living cells. In this thesis we investigated in detail two
important quantitative methods, fluorescence correlation spectroscopy (FCS) and
fluorescence recovery after photobleaching (FRAP). Model systems used in
demonstrating the complementarity of the techniques and their merits included the
nuclear excision repair (NER) system, transcription regulation by the androgen receptor
(AR), and signal transduction by two membrane receptors, the EGF receptor and the
IL2-receptor.
In Chapter 1 an introduction to microscopy is given. A brief history traces the
development of microscopy from the modest lens arrangement of Zacharias Janssen to
modern fluorescence microscopes allowing quantitative investigation of protein
dynamics in living cells. A discussion of fluorescence properties of the GFP is
presented and several quantitative fluorescence microscopy techniques used are
discussed. Also the model systems studied are described.
In Chapter 2 the long-lived dark state of EGFP, the fluorescent tag used in most live
cell studies, is investigated as observed in a set-up similar to a typical fluorescence
recovery after photobleaching (FRAP) experiment. A method is presented to measure
light ind