To gain a better understanding of material properties of chromatin and successfully link them to chromatin organization and functions such as transcription, we develop novel methods to actively manipulate a genomic locus inside the nucleus of a living human cell. By targeting iron-containing nanoparticles to a specific genomic locus and applying a controlled magnetic field, we were able to physically move chromatin through nuclear space for the first time. Exertion of near-picoNewton forces led to displacements over microns within minutes. We observe partially reversible stretching of chromatin highlighting its’ viscoelastic nature. We could accurately recapitulate the observed behavior with a Rouse model that included only a weak obstructive effect of the surrounding chromatin and nucleoplasmic material. This challenges the view that interphase chromatin is a gel-like material.
The determination of 3-dimensional arrangement of subcellular assemblies has become a necessary requirement in cellular biology. Unfortunately, the size of most assemblies lies beyond the diffraction limit and therefore they cannot be visualized using conventional fluorescence microscopy techniques. Photoactivation localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) enable the localization of fluorescent molecules with nanometric resolution. We used these microscopy methods together with MicAO 3D-SR – the first adaptive optics device which introduces the three-dimensional imaging capability for PALM/STORM. MicAO also corrects various types of aberrations induced by optical elements inside the microscope and by the biological sample. The correction of these aberrations almost doubles the number of detected photons, increasing the localization precision of PALM/STORM by 40%. At 1000 detected photons the localization precision of our setup is 8 nm in lateral and 16 nm in axial directions. The separate optimization performed for two different colors delivers superb imaging quality, as demonstrated by dual-color 3-dimentional imaging of two centrosomal proteins in HeLa cells.
In fluorescence microscopy, one can distinguish two kinds of imaging approaches, wide field and raster scan
microscopy, differing by their excitation and detection scheme. In both imaging modalities the acquisition is
independent of the information content of the image. Rather, the number of acquisitions N, is imposed by
the Nyquist-Shannon theorem. However, in practice, many biological images are compressible (or, equivalently
here, sparse), meaning that they depend on a number of degrees of freedom K that is smaller that their size N.
Recently, the mathematical theory of compressed sensing (CS) has shown how the sensing modality could take
advantage of the image sparsity to reconstruct images with no loss of information while largely reducing the number M of acquisition. Here we present a novel fluorescence microscope designed along the principles of CS. It uses a spatial light modulator (DMD) to create structured wide field excitation patterns and a sensitive point detector to measure the emitted fluorescence. On sparse fluorescent samples, we could achieve compression ratio N/M of up to 64, meaning that an image can be reconstructed with a number of measurements of only 1.5 % of its pixel number. Furthemore, we extend our CS acquisition scheme to an hyperspectral imaging system.
This paper describes the preparation of bioactive water-soluble fluorescent CdSe/ZnS semi-conductor quantum dots with
a small hydrodynamic diameter of 10 nm. These quantum dots are functionalized with a biotinylated peptide that can be
introduced at different ratios onto the surface of the quantum dots. Their ability to bind to streptavidin in solution is
tested by using gel electrophoresis and fluorescence resonance energy transfer with a fluorescent labeled-streptavidin.
The binding of these quantum dots to Agarose micrometric beads coated with streptavidin is also analyzed by fluorescent
optical microscopy. A synthetic pegylated peptide is successfully used to prevent the non specific adsorption of
streptavidin onto the quantum dots. A specific binding to the streptavidin results in the formation of a very stable
streptavidin-quantum dot complex without any significant aggregation. The average number of streptavidin per quantum
dot is found to be to 4 at the most. Such bioactivate quantum dots can be further conjugated to any biotinylated
biomolecule and used in biological medium.
Colloidal semiconductor quantum dots (QDs) have become common fluorescent probes in biology. Their optical properties not only facilitate spectrally multiplexed detection but also enable single molecule measurements with high signal to noise ratio. This is of particular interest in cell biology since it allows individual QD-tagged biomolecules to be tracked with good spatial and temporal resolution over long durations. Recent measurements on membrane proteins have validated this approach and serve as a basis for more complex experiments in which the motion of different biomolecules, located in various cell compartments (membrane, cytosol, nucleus,...) and tagged with QDs having distinct emission colors, is recorded in real time and with a nanometer resolution. The development of these new imaging methods, equivalent to a molecular positioning system within a single cell, raises many challenges, coming from optics, physical and biological chemistry, as well as image processing.
Conference Committee Involvement (10)
Colloidal Nanoparticles for Biomedical Applications VIII
2 February 2013 | San Francisco, California, United States
Colloidal Nanocrystals for Biomedical Applications VII
21 January 2012 | San Francisco, California, United States
Colloidal Quantum Dots/Nanocrystals for Biomedical Applications VI
22 January 2011 | San Francisco, California, United States
Colloidal Quantum Dots for Biomedical Applications V
23 January 2010 | San Francisco, California, United States
Colloidal Quantum Dots for Biomedical Applications IV
24 January 2009 | San Jose, California, United States
Colloidal Quantum Dots for Biomedical Applications III
19 January 2008 | San Jose, California, United States
Ultrasensitive and Single-Molecule Detection Technologies II
20 January 2007 | San Jose, California, United States
Colloidal Quantum Dots for Biomedical Applications II
20 January 2007 | San Jose, California, United States
Ultrasensitive and Single-Molecule Detection Technologies
21 January 2006 | San Jose, California, United States
Manipulation and Analysis of Biomolecules, Cells and Tissues
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