Quantitative Fluorescence Microscopy of Protein Dynamics in Living Cells

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 induced fluorescence fluctuations due to transitions between the dark and excitable state of EGFP in bulk samples. We have found that the average lifetime of the long-lived dark state of the chromophore is about 2.3 s irrespective of the excitation intensity whereas the average on-time is dependent on the intensity used.