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?

The finding of nonlinear nanometric-sized probes is of key importance for the development of nonlinear microscopy in nanosciences and biology. We isolate nonlinear KTiOPO4 nanocrystals with remarkable photostability in second-harmonic generation under femtosecond infrared laser light excitation. Their size distribution is determined using dynamic light scattering and atomic force microscopy. With both polarization analysis and defocused imaging of the emitted second-harmonic field, we also extract the Euler angles of the crystalline axes of a single nanocrystal. These sub-wavelength particles can find application as near-field vectorial probes.

/pdf/photostable-single-ktiopo-4-nanocrystals-for-second-harmonic-1rk81704wu.pdf

Photostable Single KTiOPO 4 Nanocrystals for Second-Harmonic Generation Microscopy

Engineering of new nonlinear materials, structures and devices with enhanced figures of merit has acted over the two last decades as a major driving force in optics: electrooptic polymers have emerged in this context as a sound basis for a currently maturing integrated optics technology with a variety of industrial applications at stake. We report here on a major turning point in this field whereby nonlinear optical phenomena, while remaining a major functional end-goal, are being furthermore implemented in the elaboration process itself. Coherent multiphoton processes appear indeed as unique tools to pattern linear and nonlinear structures to a sophistication level out of the reach of earlier technologies. We will review the basic physical phenomena involved which mainly pertain to quantum interferences between multiphoton excitation pathways in photosensitive media. Multipolar nonlinear patterns reflecting the multipolar symmetry of controllable polarization states of the coherent writing beams can be permanently or imprinted on a variety of substrates from films to waveguides, or gratings.

Nonlinear Photonic Engineering: Physics and Applications

Amyloid fibrils are proteins misfolding structures that involve a beta-sheet structure and are associated with the pathologies of various neurodegenerative diseases. We show that Thioflavine-T and Congo Red, two major dyes used to image fibrils by fluorescence assays, can provide deep structural information when probed by means of polarization resolved fluorescence microscopy [1]. This technique allows indeed to retrieve simultaneously both mean orientation and orientation dispersion of the dye, used here as a reporter of the fibril structure [2].

Sophie Brasselet

Papers

Photostable Single KTiOPO 4 Nanocrystals for Second-Harmonic Generation Microscopy

Nonlinear Photonic Engineering: Physics and Applications

Polymorphism of amyloid fibrils monitored by polarization resolved microscopy

Probing molecular order of lipid reporters di-8-ANEPQ in cell plasma membrane by fluorescence polarimetry imaging

Multimodal polarimetric nonlinear microscopy in tissues