Photo-switchable chromophores are fluorescent inorganic dyes or proteins characterized by the tunability of their emission depending on the properties of the exciting radiation such as wavelength or intensity. The opportunities o ered by this kind of markers in the field of fluorescence microscopy, range from super-resolution imaging to molecular intracellular sensing of pH, calcium or second messengers. In this thesis I have studied the properties of two photo switchable GFP Mut2 mutants (E222Q and Mut2GQ) and their possible applications to biological fluorescence pump and probe imaging. From a strictly biological point of view the choice of a biological marker such as GFPs is very convenient since the uorescent label can be engineered to be naturally expressed by the cell under study. The risk of compromising or altering the cell cycle is reduced and, at the same time, labeling a ect the parent as well the daughter cells. These two mutants o er the possibility to investigate the role of the proton network around the GFP chromophore (in the case of the E222Q mutant) and the external protonation (in the case of the Mut2GQ mutant) of the GFP chromophore on the photo-switching. From the physical point of view, GFP Mut2 mutants, when excited by a less energetic pump wavelength, are characterized by long-lived non-fluorescent states whose depopulation can be triggered by irradiation with a more energetic probe beam. The first effect of the photo-induced depopulation of the long-lived state is the enhancement of the fluorescence signal due to the fact that the molecules are no more prevented to be excited to the high quantum yield singlet excited state. I have been able not only to characterize the fluorescence enhancement ratio of the GFP-Mut2 mutants, but also the characteristics relaxation times of the population and depopulation processes of the dark state. These parameters have a dependence on physical properties of their surroundings such as pH and viscosity, that could be exploited in imaging and sensing applications. Basing on this characterization I reasoned that the fluorescence enhancement (called in the thesis Enhancement Ratio) and the related relaxation time (Photoswitching Time) characteristic of these GFP mutants and other photoswitchable proteins, could be the basis for a new functional imaging technique. With this new protocol for image acquisition (called Beating Mode) it is possible to acquire images in which the contrast agent is not only the fluorescence intensity, but also the Enhancement Ratio or the Photoswitching Times. This imaging protocol can be easily extended to other photo-switchable proteins and by further characterizing their photo-switching properties as a function of pH or viscosity, it will offer the possibility to map biological functions in the samples. Here I outline and test the algorithms to perform what I called Intensity Modulation Beating Mode Imaging. However it must be noted that in order to reach the ultimate application of this technique, namely to perform intra-cellular sensing experiments via the detection of the photoswithcing parameters, the GFP mutants should be further engineered to allow cell transfection or membrane adhesion. This molecular biology step goes beyond the aim of my thesis. In this work, after the first introductory theoretical Chapters, I present a theoretical introduction to photo switchable GFP mutants in terms of physical and conformational properties (Chapter 3). In Chapter 4 we present the characterization of the behavior of the photo switching properties of the two mutants under study as a function of physical characteristics of the surroundings such as pH and viscosity. In the same Chapter we also present a joined theoretical-simulative model in order to analyze, in terms of FCS analysis, the coupling of the uorescence properties with the long lived dark state conformational dynamics. In Chapter 5, we describe the new image acquisition protocol developed in this work and called Beating Mode, both in terms of its potentialities and limitations, with a particular interest in the rationalization of the best operative condition and the applicability to biological sample. In Chapter 6 the applications of the Beating Mode protocol are finally outlined.

(2012). GFP photo switchable mutants for intensity modulation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).

GFP photo switchable mutants for intensity modulation

DAGLIO, STEFANO CARLO
2012

Abstract

Photo-switchable chromophores are fluorescent inorganic dyes or proteins characterized by the tunability of their emission depending on the properties of the exciting radiation such as wavelength or intensity. The opportunities o ered by this kind of markers in the field of fluorescence microscopy, range from super-resolution imaging to molecular intracellular sensing of pH, calcium or second messengers. In this thesis I have studied the properties of two photo switchable GFP Mut2 mutants (E222Q and Mut2GQ) and their possible applications to biological fluorescence pump and probe imaging. From a strictly biological point of view the choice of a biological marker such as GFPs is very convenient since the uorescent label can be engineered to be naturally expressed by the cell under study. The risk of compromising or altering the cell cycle is reduced and, at the same time, labeling a ect the parent as well the daughter cells. These two mutants o er the possibility to investigate the role of the proton network around the GFP chromophore (in the case of the E222Q mutant) and the external protonation (in the case of the Mut2GQ mutant) of the GFP chromophore on the photo-switching. From the physical point of view, GFP Mut2 mutants, when excited by a less energetic pump wavelength, are characterized by long-lived non-fluorescent states whose depopulation can be triggered by irradiation with a more energetic probe beam. The first effect of the photo-induced depopulation of the long-lived state is the enhancement of the fluorescence signal due to the fact that the molecules are no more prevented to be excited to the high quantum yield singlet excited state. I have been able not only to characterize the fluorescence enhancement ratio of the GFP-Mut2 mutants, but also the characteristics relaxation times of the population and depopulation processes of the dark state. These parameters have a dependence on physical properties of their surroundings such as pH and viscosity, that could be exploited in imaging and sensing applications. Basing on this characterization I reasoned that the fluorescence enhancement (called in the thesis Enhancement Ratio) and the related relaxation time (Photoswitching Time) characteristic of these GFP mutants and other photoswitchable proteins, could be the basis for a new functional imaging technique. With this new protocol for image acquisition (called Beating Mode) it is possible to acquire images in which the contrast agent is not only the fluorescence intensity, but also the Enhancement Ratio or the Photoswitching Times. This imaging protocol can be easily extended to other photo-switchable proteins and by further characterizing their photo-switching properties as a function of pH or viscosity, it will offer the possibility to map biological functions in the samples. Here I outline and test the algorithms to perform what I called Intensity Modulation Beating Mode Imaging. However it must be noted that in order to reach the ultimate application of this technique, namely to perform intra-cellular sensing experiments via the detection of the photoswithcing parameters, the GFP mutants should be further engineered to allow cell transfection or membrane adhesion. This molecular biology step goes beyond the aim of my thesis. In this work, after the first introductory theoretical Chapters, I present a theoretical introduction to photo switchable GFP mutants in terms of physical and conformational properties (Chapter 3). In Chapter 4 we present the characterization of the behavior of the photo switching properties of the two mutants under study as a function of physical characteristics of the surroundings such as pH and viscosity. In the same Chapter we also present a joined theoretical-simulative model in order to analyze, in terms of FCS analysis, the coupling of the uorescence properties with the long lived dark state conformational dynamics. In Chapter 5, we describe the new image acquisition protocol developed in this work and called Beating Mode, both in terms of its potentialities and limitations, with a particular interest in the rationalization of the best operative condition and the applicability to biological sample. In Chapter 6 the applications of the Beating Mode protocol are finally outlined.
CHIRICO, GIUSEPPE
GFP, Imaging Methods, FCS, Beating Mode, fluorescence, microscopy, imaging
FIS/07 - FISICA APPLICATA (A BENI CULTURALI, AMBIENTALI, BIOLOGIA E MEDICINA)
English
13-giu-2012
Scuola di dottorato di Scienze
FISICA E ASTRONOMIA - 30R
24
2010/2011
open
(2012). GFP photo switchable mutants for intensity modulation. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2012).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/31053
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