Complexes macromoléculaires en cellules vivantes
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Fluorescence Microscopy Techniques:
Hetero-FRET by FLIM (Fluorescence Lifetime Imaging Microscopy) and homo-FRET by FAIM (Fluorescence Anisotropy Imaging Microscopy) allow monitoring the spatio-temporal variations of protein-protein interactions and biochemical reactions in living cells.
FLIM is performed by TSCSPC method in wide field microscopy or by fast time resolved-gated CCD method in two-photon excitation confocal microscopy.
FLIM is one of the most reliable methods for hetero-FRET measurement in living cells, since hetero-FRET is determined from the decrease in the fluorescence lifetime of the donor in the presence of the acceptor, a parameter that is insensitive to variations in concentration and optical path length.
TSCSPC-based FLIM method : the temporal and spatial resolutions of the fluorescence decays are less than 100 ps and 500 nm, respectively. The high temporal resolution of the fluorescence decays allows the determination of two discrete fluorescence lifetimes (τD and τFRET) and the proportion of the donor molecules in interaction with the acceptor in the frame of a bi-exponential model.
Time-resolved gated CCD-based FLIM method: Time-lapse hetero-FRET can be performed at relatively fast frame rate, providing the knowledge of fluorescence lifetime values (τD and τFRET) from TSCSP acquisitions.
GFP N-terminal tagged H4 and mCherry-tagged bromodomain protein are coexpressed in living cells (HEK 293) in presence of sodium butyrate. FLIM performed with TSCSPC method gave a value of fluorescence lifetime for unbound GFP-H4: τD = 2.6 ns and and for GFP-H4 bound to mCherry-BromoD; τFRET = 1.6 ns. In the frame of a bi-exponential model of the decay of GFP fluorescence, the proportions of GFP-H4 bound to mCherry-BromoD and unbound are mapped in the cell nucleus.
FAIM method takes advantage of the photoselection of oriented fluorophores by the polarized laser excitation. The measurement of the fluorescence depolarization which takes place during the fluorescence emission can probe homo-FRET between like fluorophore.
Two-photon steady state anisotropy imaging (set up on the TRIMScope) allows to monitor spatio-temporal changes in the oligomerization of GFP-tagged proteins in living cells.
Time-domain FLIM (carried out by the TSCSPC and by the time-resolved gated CCD methods) and FAIM make it possible to perform hetero-FRET and homo-FRET, respectively, at very low excitation light level, 10 to 100 fold lower light intensity than that applied with mercury lamp illumination. This property is crucial to avoid photobleaching, photoconversion and photodynamic reactions in order to perform quantitative imaging in living cell and long period observation.
Photomanipulation is allowed through an external laser set up on the TRIMScope. The possibility to carry out FRAP and FRET measurements on the same cell is under way with the PLEIADES (Picosecond Luminescence Energy-transfer Imaging And Dynamics of Emitting Species) system.
Nano Manipulation by optical tweezers:
Two types of multiple optical tweezers are developped:
- 2D optical tweezers. The multiple traps are confined in the objective focal plane. Two computer-drived acousto-optic deflectors move the laser beam (YAG laser 1064 nm) to make dynamical 2D patterns of optical traps. Different rigidities (and forces) can be associated with each individual trap by changing the residence time of the laser beam on the trap position.
- 3D optical holographic tweezers. A laser beam (IPG, 1064 nm) is expanded on a liquid cristal diffractive optical element (DOE) which works as a light spatial modulator (LSM). Computerized holograms sent to the LSM through video communication generate the 3D patterns of the traps in the microscopic sample. This holographic tweezers are set up on the TRIMScope (with the PLEIADES options), which will allow 3D nano manipulation of living cell and simultaneous fluorescence imaging at high resolution.
Dernière modification 14/03/2011