Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.

/pdf/lipid-rafts-as-a-membrane-organizing-principle-3mxewaemed.pdf

Lipid Rafts As a Membrane-Organizing Principle

Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

/pdf/nonlinear-magic-multiphoton-microscopy-in-the-biosciences-3fszv5aq8z.pdf

Nonlinear magic: multiphoton microscopy in the biosciences

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

Ultra-High Resolution Imaging by Fluorescence Photoactivation Localization Microscopy

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has provided a myriad of applications for biological systems Over the last several years, mutagenesis studies have improved folding properties of GFP (refs 1,2) However, slow maturation is still a big obstacle to the use of GFP variants for visualization These problems are exacerbated when GFP variants are expressed at 37 degrees C and/or targeted to certain organelles Thus, obtaining GFP variants that mature more efficiently is crucial for the development of expanded research applications Among Aequorea GFP variants, yellow fluorescent proteins (YFPs) are relatively acid-sensitive, and uniquely quenched by chloride ion (Cl-) For YFP to be fully and stably fluorescent, mutations that decrease the sensitivity to both pH and Cl- are desired Here we describe the development of an improved version of YFP named "Venus" Venus contains a novel mutation, F46L, which at 37 degrees C greatly accelerates oxidation of the chromophore, the rate-limiting step of maturation As a result of other mutations, F64L/M153T/V163A/S175G, Venus folds well and is relatively tolerant of exposure to acidosis and Cl- We succeeded in efficiently targeting a neuropeptide Y-Venus fusion protein to the dense-core granules of PC12 cells Its secretion was readily monitored by measuring release of fluorescence into the medium The use of Venus as an acceptor allowed early detection of reliable signals of fluorescence resonance energy transfer (FRET) for Ca2+ measurements in brain slices With the improved speed and efficiency of maturation and the increased resistance to environment, Venus will enable fluorescent labelings that were not possible before

A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications

In 1873, Ernst Abbe discovered what was to become a well-known paradigm: the inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. However, for its most popular imaging mode, fluorescence microscopy, the diffraction barrier is crumbling. Here, I discuss the physical concepts that have pushed fluorescence microscopy to the nanoscale, once the prerogative of electron and scanning probe microscopes. Initial applications indicate that emergent far-field optical nanoscopy will have a strong impact in the life sciences and in other areas benefiting from nanoscale visualization.

/pdf/far-field-optical-nanoscopy-3qevt2g1wp.pdf

Far-Field Optical Nanoscopy

Elongated Membrane Zones Boost Interactions of Diffusing Proteins

Quantification of Mitochondrial Membrane Curvature by Three-Dimensional Localization Microscopy

Design strategies and structure-property relationships for two-photon absorption in conjugated molecules are described on the basis of correlated quantum-chemical calculations. We first focus on stilbene derivatives with centrosymmetric structures. We found that derivatization of the conjugated molecule with electroactive groups in a quadrupolarlike arrangement leads to a large increase in the two-photon absorption cross section, δ. Quantum-chemical description provides rich insight into the mechanisms for the two-photon absorption phenomenon.

Theoretical Design of Organic Chromophores with Large Two-Photon Absorption Cross-Sections

The pattern of substitution mutation in different nearest-neighbor environments of the human genome☆

Fluorescence microscopy is an essential and flexible tool for the study of biology, chemistry, and physics. It can provide information on a wide range of spatial and temporal scales. However, since the inception of light microscopy, diffraction has limited the size of the smallest details that could be imaged in any sample using light. Because much of biology occurs on molecular length scales, interest in circumventing the diffraction limit has been high for many years. Recently, several techniques have been introduced that can bend or break the diffraction limit. Localization-based methods introduced in 2006 have reached this goal and are now rapidly growing in popularity.

/pdf/localization-based-super-resolution-light-microscopy-5ay4nkck6d.pdf

Samuel T. Hess

Papers

Elongated Membrane Zones Boost Interactions of Diffusing Proteins

Quantification of Mitochondrial Membrane Curvature by Three-Dimensional Localization Microscopy

Theoretical Design of Organic Chromophores with Large Two-Photon Absorption Cross-Sections

The pattern of substitution mutation in different nearest-neighbor environments of the human genome☆

Localization-Based Super-Resolution Light Microscopy