http://light.ece.illinois.edu/ECE564/Research_Projects_files/fPALM_2006.pdf

Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are ≈25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

/pdf/a-monomeric-red-fluorescent-protein-22nkyufygq.pdf

A monomeric red fluorescent protein

Organic–inorganic hybrid materials do not represent only a creative alternative to design new materials and compounds for academic research, but their improved or unusual features allow the development of innovative industrial applications. Nowadays, most of the hybrid materials that have already entered the market are synthesised and processed by using conventional soft chemistry based routes developed in the eighties. These processes are based on: a) the copolymerisation of functional organosilanes, macromonomers, and metal alkoxides, b) the encapsulation of organic components within sol–gel derived silica or metallic oxides, c) the organic functionalisation of nanofillers, nanoclays or other compounds with lamellar structures, etc. The chemical strategies (self-assembly, nanobuilding block approaches, hybrid MOF (Metal Organic Frameworks), integrative synthesis, coupled processes, bio-inspired strategies, etc.) offered nowadays by academic research allow, through an intelligent tuned coding, the development of a new vectorial chemistry, able to direct the assembling of a large variety of structurally well defined nano-objects into complex hybrid architectures hierarchically organised in terms of structure and functions. Looking to the future, there is no doubt that these new generations of hybrid materials, born from the very fruitful activities in this research field, will open a land of promising applications in many areas: optics, electronics, ionics, mechanics, energy, environment, biology, medicine for example as membranes and separation devices, functional smart coatings, fuel and solar cells, catalysts, sensors, etc.

Applications of hybrid organic–inorganic nanocomposites

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

/pdf/a-practical-guide-to-single-molecule-fret-3blag506ll.pdf

A practical guide to single-molecule FRET

Lipid Rafts: Elusive or Illusive?

Molecular order imaging in cell membranes by polarimetric fluorescence microscopy

The emergence of molecular photonics as a new domain of research at the cross-road of physics, chemistry and device engineering is being triggered by the increasing demands of broadband telecommunication systems which start to challenge the fundamental limits of current inorganic semiconductor based technologies. Increasing to 100 GHz and beyond the bandwidth acceptance of optoelectronic devices, such as modulators and switches, or down-scaling device dimensions into the new frontier of quantum physics have become scientific as well as industrially relevant targets, unlikely to be met by unimaginative extrapolation of current avenues. Facing this formidable challenge, the so-far relatively untapped wealth of molecular structures, and the targeted exploitation of their functional as well as structural flexibility throughout consistent molecular, material and device engineering steps, open-up thoroughly renewed horizons. An important asset is the complementarity and technological compatibility of polymer and inorganic semiconductor structures providing the possibility of smooth and economically viable transitions towards hybrid organic-inorganic technological solutions. Major current and foreseeable impacts appear to be in the realm of nonlinear optics (harmonic generation and carrier frequency shifting, routing, electrooptic modulation and switching, pulse shaping and synchronization, nonlinear refraction) and microlasers which will be exemplified by a selection of recent and ongoing developments in our laboratory at different macroscopic, microscopic and nanoscopic scales.

http://ieeexplore.ieee.org/iel5/10499/33259/01572021.pdf

Advances in polymer based photonics technology: microlasers, electrooptic devices, new concepts

We present a microscopy technique that encodes molecule orientation and orientational fluctuations as well as off-plane displacements into the point spread function, allowing the simultaneous determination of six spatial/directional properties for many molecules simultaneously.

Simultaneous Determination of 3D Orientation and 3D Localization in Single Emitter Microscopy Imaging

We investigate the range of application of single-beam homodyne spectral phase interferometry for direct electric field reconstruction (SPIDER) for multiphoton microscopy. Simulations and experimental studies performed on model spectral phase profiles show that the phase reconstruction technique used in this method makes the phase retrieval quality highly sensitive to the complexity of the profile. In addition, we show that the use of iterative processes is likely to deteriorate the phase retrieval quality, especially for strongly varying phase profiles. These effects are illustrated and quantified for sinusoidal and quadratic spectral phase profiles.

Application of single-beam homodyne SPIDER for the control of complex spectral phase profiles

We measure the quadratic nonlinear optical properties, namely second harmonic generation (SHG) and two-photon excitation fluorescence (TPEF), of nanocrystals of different derivatives of pyrazoline. The derivatives of pirazoline, like for example DCNP are supposed to have high second order nonlinear susceptibility. We use a nonlinear optical microscopy setup, samples were excited with a femtosecond laser operating at 800 nm wavelength, both signals (SHG and TPEF) were measured simultaneously using a dichroic mirror and for SHG a narrow spectral bandwidth filter centered at 400 nm, respectively. The details of the used nonlinear polarimetric microscopy technique can be found in reference. We have produced the nano-size crystals using the precipitation method and estimated the average size of these nanocrystals to be 300 × 600 nm using atomic force microscopy (AFM).

Sophie Brasselet

Papers

Molecular order imaging in cell membranes by polarimetric fluorescence microscopy

Advances in polymer based photonics technology: microlasers, electrooptic devices, new concepts

Simultaneous Determination of 3D Orientation and 3D Localization in Single Emitter Microscopy Imaging

Application of single-beam homodyne SPIDER for the control of complex spectral phase profiles

Second harmonic generation and two-photon excitation fluorescence from individual nanocrystals of pyrazoline derivatives