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?

Myelin around axons is currently widely studied by structural analyses and large scale imaging techniques, with the goal to
decipher its critical role in neuronal protection. While there is strong evidence that in myelin, lipid composition and lipid
membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet
to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in
vivo observations. In this work, we study and quantify molecular order of lipids in myelin at sub-diffraction scales, using labelfree
polarization resolved Coherent Anti Stokes Raman (PR-CARS), which exploits CARS sensitivity to coupling between
light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters
in the sample such as lipid type, orientational organization and composition. We show that lipid molecular order of myelin
in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis
(EAE), a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits
to unravel molecular-scale perturbations of lipid layers at early stage of the demyelination progression, while the membrane
architecture at the mesoscopic scale (here about 100 nm) seems much less affected. Such information cannot be brought
by pure morphological observation and opens new prospectives towards molecular-scale understanding of neurodegenerative
diseases.

/pdf/lipid-order-degradation-in-autoimmune-demyelination-probed-1txyl39fi9.pdf

Lipid order degradation in autoimmune demyelination probed by polarization resolved coherent Raman microscopy

The title compd. has a C-H...O H bond-mediated trigonal network structure that leads to octupolar NLO behavior in the solid state.

Sophie Brasselet

Papers

Lipid order degradation in autoimmune demyelination probed by polarization resolved coherent Raman microscopy

Engineering of an octupolar nonlinear optical crystal: tribenzyl isocyanurate.

Investigation of new push–pull pyridine-1-oxide derivatives as 1D NLO chromophores with vanishing dipole moment

Probing MHC Class I Protein and Lipid Order in Cell Membranes by Fluorescence Polarization - resolved imaging

Photoinduced multipolar symmetry breaking in multipolar nonlinear media: from molecular to photonic engineering