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Table of contents
Remerciements
Introduction
Elements of context
1 3D-super resolution tracking with Stereo Darkfield Interferometry
1.1 Real-time tracking 3D super-resolution: fundamental concepts and challenges
1.2 PSF engineering: a state of the art
1.2.1 High resolution methods
1.2.2 Stereoscopic methods: the advantage of linearity
1.3 Stereo Darkfield Interferometry
1.3.1 Optical setup
1.3.2 Translation of the interferometric pattern and theoretical z-sensitivity
1.3.3 Characterization of the PSF of Stereo Darkfield Interferometry
1.3.4 Localization algorithm
1.3.5 Subnanometer optical precision
1.3.6 Linearity and field dependence
1.3.7 Impact of the size of the imaged particles
1.4 Conclusion
2 High-resolution magnetic tweezers
2.1 Physics of a tethered Brownian bead
2.1.1 Overdamped Langevin dynamics
2.1.2 Stiffness and thermodynamics of a tethered molecule at constant force
2.1.3 Hydrodynamic drag, stiffness and spatio-temporal resolution
2.1.4 Hydrodynamic drag close to a surface
2.1.5 Movement misalignment and post-correction
2.1.6 Discrepancy between this simple model and experimental noise values
2.1.7 Inclusion of hydrodynamic couplings and magnetic anisotropy
2.2 Two biophysical applications
2.2.1 Force spectroscopic measurement of the hybridization of short oligonucleotides with nanometric resolution
2.2.2 Helicase stepping: some preliminary results
2.3 Conclusion
3 Kinetic locking: a method to measure biomolecular interactions involving nucleic acids
3.1 An extremely stable DNA Fluctuating probe
3.1.1 The fluctuations of a 10-bp hairpin in the millisecond range
3.1.2 Correction of the impact of the acquisition frequency for rapid kinetic rates
3.1.3 Temporal stability
3.2 Kinetic locking
3.2.1 Kinetic model
3.2.2 Measurement of the binding kinetics in the case koff ≪k f and kon ≪k f
3.2.3 Binding thermodynamics of very short oligonucleotides
3.2.4 Influence of standard chemical modifiers and comparison with the nearest-neighbour model
3.2.5 Comment on the influence of the force on the thermodynamic measurements
3.3 An application of kinetic locking to the measurement of NA/protein interactions
3.3.1 RecQ detection through the kinetic locking of a fluctuating hairpin
3.3.2 Kinetic locking unveils the stabilization of the replication fork by RecQ
3.4 Conclusion
Conclusion
A Embedded SDI objective
B Brownian simulation of a tethered bead close to a surface
C Material and methods
C.1 Optics
C.2 Single-molecule sample preparation
C.3 Biochemistry
C.4 Single-molecule experiments
C.5 Data treatment
C.6 DNA sequences
C.7 Buffers
D Bibliography
