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?

Supplementary document for Chromato-axial memory effect through a forward-scattering slab - 4477056.pdf

Summary form only given. We show that a departure from the low field value of the nonlinear anisotropy can be obtained in strong poling field conditions. Such possibility of controlling the nonlinear anisotropy ratio has been experimentally evidenced for different values of the corona poling field in the case of polycarbonate matrices doped with the DR1 dipolar chromophore and the multipolar dinitro-di-(n-butylamino)-benzene (DNDAB) compound. As the poling field remains in the range of currently used values for electro-optic devices, the possibility to achieve polarization independence by use of mullipoiar molecules becomes a realistic target.

Strong field poling of multipolar structures: fundamentals and device implications

Summary form only given. The main objective of the present work was to evaluate the potential of subphthalocyanine compounds for second-order nonlinear optical (NLO) applications. The investigation of the second-order NLO properties was carried out by electric-field-induced second harmonic generation (EFISH) and hyper-Rayleigh light scattering (HLS) experiments. The measurements were performed in chloroform solution at, respectively, 1.064 and 1.340 /spl mu/m fundamental wavelength.

High second-order optical nonlinearities in subphthalocyanines

Coherent Anti Stokes Raman Scattering (CARS) offers many advantages for nonlinear bio-imaging, thanks to its sub-cellular spatial resolution and unique chemical specificity. Its working principle requires two incident pulsed laser beams with distinct frequencies to be focused in space and time, which focus quality however rapidly deteriorates when propagating at large depths in biological tissues. The depth limits of CARS and the capability of wavefront correction to overcome these limits are currently unknown. In this work we exploit the spectral correlation properties of the transmission matrix of a scattering medium in a pulsed regime, to recover coherent focusing for two distant incident CARS wavelengths which propagation is initially uncorrelated. Using wavefront shaping with a single spatial light modulator, we recover CARS generation through thick mice spinal cord tissues where initially no signal is measurable due to scattering, and demonstrate point scanning over large field of views of tens of micrometers.

Coherent anti-Stokes Raman scattering through thick biological tissues by single wavefront shaping

We demonstrate the association of two-photon nonlinear microscopy with balanced homodyne detection for investigating second harmonic radiation properties at nanoscale dimensions. Variation of the relative phase between second-harmonic and fundamental beam is retrieved, as a function of the absolute orientation of the nonlinear emitters. Sensitivity of ≈ 1.6 photon per second, in the spatio-temporal mode of the local oscillator, is obtained. This value is high enough to efficiently detect the second-harmonic emission from a single KTiOPO 4 crystal of sub-wavelength size, embedded in a thin polymer film.

Sophie Brasselet

Papers

Supplementary document for Chromato-axial memory effect through a forward-scattering slab - 4477056.pdf

Strong field poling of multipolar structures: fundamentals and device implications

High second-order optical nonlinearities in subphthalocyanines

Coherent anti-Stokes Raman scattering through thick biological tissues by single wavefront shaping

Coherent detection of second harmonic generation in nonlinear optics