velikost textu

Optical microscopy to study the role of cytoskeleton in cell locomotion and virus trafficking

Upozornění: Informace získané z popisných dat či souborů uložených v Repozitáři závěrečných prací nemohou být použity k výdělečným účelům nebo vydávány za studijní, vědeckou nebo jinou tvůrčí činnost jiné osoby než autora.
Název:
Optical microscopy to study the role of cytoskeleton in cell locomotion and virus trafficking
Typ:
Disertační práce
Autor:
Ing. Francesco Difato, Ph.D.
Školitel:
doc. RNDr. Jitka Forstová, CSc.
Oponenti:
prof. RNDr. Pavel Hozák, DrSc.
prof. RNDr. Jaromír Plášek, CSc.
Id práce:
112812
Fakulta:
Přírodovědecká fakulta (PřF)
Pracoviště:
Katedra genetiky a mikrobiologie (31-140)
Program studia:
Molekulární a buněčná biologie, genetika a virologie (P1519)
Obor studia:
-
Přidělovaný titul:
Ph.D.
Datum obhajoby:
11. 12. 2008
Výsledek obhajoby:
Prospěl/a
Jazyk práce:
Angličtina
Abstrakt:
3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1. The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF permited to better quantify the 3D morphological properties of the sample and remove noise from the image as shown in my experimental results. Successively, I’ve focused on the the time resolution of optical microscopy. , the available resolution permits following the evolution of biological processes which cannot be understood through structural organization of the living organism. For example, by live imaging we followed the infectious pathway of Polyomavirus and we were able to observe the active role of actin polymerization in virion transport, while immunofluorescence assays indicated cortical actin as a merely steric barrier during first steps of infections. Additionally, I’ve explored the potentialities of FRAP technique to study reaction kinetics in living sample51. With the four dimensions of space and time and available resolution with optical microsopy, other measurable variables can be analyzed. The wavelength λ dimention enables for example multi labeling and discrimination of different entities in the sample. Spectral properties of fluorochromes such as the lifetime of the excited state, give information on the environmental condition of the molecule. Changes in the intracellular environment, related to the cell metabolism, could influence the structure of the fluorescent molecules and its optical properties. Therefore, cell states and physiology of living sample could be studied at molecular level. This is the so called lifetime dimension. Futhermore, the Second Harmonic Generation, occuring when an intense laser beam passes through a polarizable material with non-centrosymmetric molecular organization, gives another possible dimension for optical microscopy measurements206. This unique multidimensionality of data acquired by innovative optical microscopy methods has to face the limited spatial resolution in comparison with other kind of imaging methods. An important effort was dedicated in the last decade to improve the resolution of optical microscopy by developing alternative methodologies. For example fluorescent techniques like FRET represent a distance ruler at nanometer resolution which could be used to break the resolution limit in combination with scanning probe microscopy techniques67. Improvements in resolution are also achieved with new optical configurations. Total Internal Reflection Fluorescence Microscopy can follow processes in an illuminated thin layer (on the order of 100 nm) of the sample near the coverslip interface207. Structured light excitation permit to expand the spatial frequency bandwidth of the optical system. The interference of light shift the Fourier transform of the sample image in the bandwidth of the microscope and it allow to acquires a larger frequency range than the support of the system28,208. Techniques as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) apply a fitting between the PSF of the system and the point like features of the image to obtain the centre of mass of the labelled molecule at 20nm resolution209. Stimulated emission depletion (STED) exploits the optical properties of dyes to obtain nanometre resolution. Stimulated emission of photons from molecules inside a ring shaped region permits to observe the fluorescence from the centre hole. The dimension of such hole can be modulated through laser intensity for stimulated emission, and can 151 reach nanometre dimension13,14,210. This technique in combination with another ones called 4pi, (which utilize interference of two counter propagating short pulse infrared laser beams) reaches 100 nm resolution in the z axis and 10 nm resolution in x,y211. The classical optical resolution of microscopes is well suited for biology studies on tissues: two photon microscopy improved the deep penetration of optical systems while SPIM (Selective Plane Illumination Microscopy) technique permits to obtain 3D imaging of entire organisms212. With the evolution of new optical systems, the optical resolution go well beyond the diffraction limit and opens the perspective to follow biological process of living samples at molecular resolution. The last aspects treated in my work were optical tweezers tools, given the high potentialities of their use for biological studies. Optical Tweezers open the field of single molecule manipulation and its biophysical caracterization70,100. Calibrated optical tweezers, the so called photonic force microscope, add another controlled101,103 or measurable102 variable to optical microscopy: the force. In this work, we used optical tweezers to measure force exerted by cellular structure, and to study the molecular system involved in cell motility. Such measurements give the possibility to analyze quantitatively motor planning of the cell in response to different stimuli and study its mechanotrasduction process. With perspective of advancing from microscopy to nanoscopy, I think that usual artificial microsphere manipulation and visualization as presented in my studies will be replaced in the near future by bio-particles213. In the experimental result with Polyoma-virus, I developed an experimental protocol to study endocytic routes and cytoskeleton organization during intracellular trafficking. Better understanding of virus infection is necessary to define possible application of virus like particle as bio-probe but this can evolve sinergically with their employment and characterization as nano-devices213. Optical tweezers were already used to manipulate single virion70; mPyV empty caspid can be used as a trafficking cargo model, to measure PSF inside cells, as probe to perform intracellular force measurements, to apply intracellular stresses, or target specific molecule in cellular compartments214 through their optical manipulation70,183,192. A complementary system to light manipulation tools that is fast evolving is laser dissection. By laser scissors it was possible to cut living biological samples with subcellular resolution: the cytoskeleton organization could be disturbed while leaving the cells unaffected in any other respect215,216. Optical tweezers and laser dissection tools were also used to control the location and timing of optical uncaging in chemical stimulation experiments217, or for optoporating the cell membrane218. These opportunities encourage the use of optical tools in probing and quantifying biophysical cellular parameters as alternative descriptive markers, and open the new field of nanosurgery216. 152
Abstract v angličtině:
3. General conclusions 150 The interest in optical microscopy is constanly growing, mainly because of its unique features in examining biological systems in four dimensions (x-y-z-t)1. The work presented here was focused on biological applications of optical microscopy by exploring and improving the spatial and temporal resolution performances and by futher developing optical tools for manipulating biological samples. First, I studied the resolution performances of the system in the three dimensional space and I contributed in improving the experimental spatial resolution of microscope by applying deconvolution. In this respect, theoretical modelling can characterize the image formation process of the microscope, but only experimental measurement of the PSF can quantify the limitations of the real system. Indeed, experimental PSF presents shape assymetry due to spherical aberrations introduced by optical elements, while theoretical PSF is symmetric and account only for the resolution limits of an ideal imaging system. The disadvantage of experimental PSF is that could be corrupted by noise, otherwise deconvolution with the theoretical PSF offer only a qualitative improvement of the image, because the introduced artefacts cannot be quantified. Deconvolution of the acquired data with experimental PSF permited to better quantify the 3D morphological properties of the sample and remove noise from the image as shown in my experimental results. Successively, I’ve focused on the the time resolution of optical microscopy. , the available resolution permits following the evolution of biological processes which cannot be understood through structural organization of the living organism. For example, by live imaging we followed the infectious pathway of Polyomavirus and we were able to observe the active role of actin polymerization in virion transport, while immunofluorescence assays indicated cortical actin as a merely steric barrier during first steps of infections. Additionally, I’ve explored the potentialities of FRAP technique to study reaction kinetics in living sample51. With the four dimensions of space and time and available resolution with optical microsopy, other measurable variables can be analyzed. The wavelength λ dimention enables for example multi labeling and discrimination of different entities in the sample. Spectral properties of fluorochromes such as the lifetime of the excited state, give information on the environmental condition of the molecule. Changes in the intracellular environment, related to the cell metabolism, could influence the structure of the fluorescent molecules and its optical properties. Therefore, cell states and physiology of living sample could be studied at molecular level. This is the so called lifetime dimension. Futhermore, the Second Harmonic Generation, occuring when an intense laser beam passes through a polarizable material with non-centrosymmetric molecular organization, gives another possible dimension for optical microscopy measurements206. This unique multidimensionality of data acquired by innovative optical microscopy methods has to face the limited spatial resolution in comparison with other kind of imaging methods. An important effort was dedicated in the last decade to improve the resolution of optical microscopy by developing alternative methodologies. For example fluorescent techniques like FRET represent a distance ruler at nanometer resolution which could be used to break the resolution limit in combination with scanning probe microscopy techniques67. Improvements in resolution are also achieved with new optical configurations. Total Internal Reflection Fluorescence Microscopy can follow processes in an illuminated thin layer (on the order of 100 nm) of the sample near the coverslip interface207. Structured light excitation permit to expand the spatial frequency bandwidth of the optical system. The interference of light shift the Fourier transform of the sample image in the bandwidth of the microscope and it allow to acquires a larger frequency range than the support of the system28,208. Techniques as photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) apply a fitting between the PSF of the system and the point like features of the image to obtain the centre of mass of the labelled molecule at 20nm resolution209. Stimulated emission depletion (STED) exploits the optical properties of dyes to obtain nanometre resolution. Stimulated emission of photons from molecules inside a ring shaped region permits to observe the fluorescence from the centre hole. The dimension of such hole can be modulated through laser intensity for stimulated emission, and can 151 reach nanometre dimension13,14,210. This technique in combination with another ones called 4pi, (which utilize interference of two counter propagating short pulse infrared laser beams) reaches 100 nm resolution in the z axis and 10 nm resolution in x,y211. The classical optical resolution of microscopes is well suited for biology studies on tissues: two photon microscopy improved the deep penetration of optical systems while SPIM (Selective Plane Illumination Microscopy) technique permits to obtain 3D imaging of entire organisms212. With the evolution of new optical systems, the optical resolution go well beyond the diffraction limit and opens the perspective to follow biological process of living samples at molecular resolution. The last aspects treated in my work were optical tweezers tools, given the high potentialities of their use for biological studies. Optical Tweezers open the field of single molecule manipulation and its biophysical caracterization70,100. Calibrated optical tweezers, the so called photonic force microscope, add another controlled101,103 or measurable102 variable to optical microscopy: the force. In this work, we used optical tweezers to measure force exerted by cellular structure, and to study the molecular system involved in cell motility. Such measurements give the possibility to analyze quantitatively motor planning of the cell in response to different stimuli and study its mechanotrasduction process. With perspective of advancing from microscopy to nanoscopy, I think that usual artificial microsphere manipulation and visualization as presented in my studies will be replaced in the near future by bio-particles213. In the experimental result with Polyoma-virus, I developed an experimental protocol to study endocytic routes and cytoskeleton organization during intracellular trafficking. Better understanding of virus infection is necessary to define possible application of virus like particle as bio-probe but this can evolve sinergically with their employment and characterization as nano-devices213. Optical tweezers were already used to manipulate single virion70; mPyV empty caspid can be used as a trafficking cargo model, to measure PSF inside cells, as probe to perform intracellular force measurements, to apply intracellular stresses, or target specific molecule in cellular compartments214 through their optical manipulation70,183,192. A complementary system to light manipulation tools that is fast evolving is laser dissection. By laser scissors it was possible to cut living biological samples with subcellular resolution: the cytoskeleton organization could be disturbed while leaving the cells unaffected in any other respect215,216. Optical tweezers and laser dissection tools were also used to control the location and timing of optical uncaging in chemical stimulation experiments217, or for optoporating the cell membrane218. These opportunities encourage the use of optical tools in probing and quantifying biophysical cellular parameters as alternative descriptive markers, and open the new field of nanosurgery216. 152
Dokumenty
Stáhnout Dokument Autor Typ Velikost
Stáhnout Text práce Ing. Francesco Difato, Ph.D. 10.07 MB
Stáhnout Abstrakt v českém jazyce Ing. Francesco Difato, Ph.D. 275 kB
Stáhnout Abstrakt anglicky Ing. Francesco Difato, Ph.D. 281 kB
Stáhnout Posudek oponenta prof. RNDr. Pavel Hozák, DrSc. 1.68 MB
Stáhnout Posudek oponenta prof. RNDr. Jaromír Plášek, CSc. 891 kB
Stáhnout Záznam o průběhu obhajoby 703 kB