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.

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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.

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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

Biological membrane organization mediates numerous cellular functions and has also been connected with an immense number of human diseases. However, until recently, experimental methodologies have been unable to directly visualize the nanoscale details of biological membranes, particularly in intact living cells. Numerous models explaining membrane organization have been proposed, but testing those models has required indirect methods; the desire to directly image proteins and lipids in living cell membranes is a strong motivation for the advancement of technology. The development of super-resolution microscopy has provided powerful tools for quantification of membrane organization at the level of individual proteins and lipids, and many of these tools are compatible with living cells. Previously inaccessible questions are now being addressed, and the field of membrane biology is developing rapidly. This chapter discusses how the development of super-resolution microscopy has led to fundamental advances in the field of biological membrane organization. We summarize the history and some models explaining how proteins are organized in cell membranes, and give an overview of various super-resolution techniques and methods of quantifying super-resolution data. We discuss the application of super-resolution techniques to membrane biology in general, and also with specific reference to the fields of actin and actin-binding proteins, virus infection, mitochondria, immune cell biology, and phosphoinositide signaling. Finally, we present our hopes and expectations for the future of super-resolution microscopy in the field of membrane biology.

Dances with Membranes: Breakthroughs from Super-resolution Imaging.

An optical microscope (101) with heightened resolution and capable of providing three dimensional images is disclosed and described. The microscope (101) can include a sample stage (160) for mounting a sample having a plurality of probe molecules. At least one non-coherent light source (127) can be provided. At least one lens (140a, 140b) can be configured to direct a beam of light from the at least one non-coherent light source (127) toward the sample causing the probe molecules to luminesce. A camera (155) can be configured to detect luminescence from the probe molecules. A light beam path modification module (132, 150) can be configured to alter a path length of the probe molecule luminescence to allow camera luminescence detection at a plurality of object planes.

Non-coherent light microscopy

Quantum dots (QDs) are becoming an increasingly popular fluorescent probe in biological imaging and single molecule applications. With advantages and disadvantages over traditional organic fluorophores, quantitative characterization of the photophysical properties of QDs is a required task for optimizing their performance. For example, maximizing the number of collected photons is essential for high-quality fluorescence imaging and yet is often a limiting factor in biological applications. Using fluorescence correlation spectroscopy (FCS), we compare important photophysical properties (count rates, photobleaching quantum yields, dark state occupancy and dark-state-to-bright-state interconversion rates, among others) of typical commercial CdSe/ZnS QDs against commonly used organic fluorophores relevant to biological applications. Two-photon action cross sections are measured using a novel version of the reference method in a laser-scanning confocal microscope geometry. FCS results for QDs show a correlation between reduced brightness, high fraction of molecules in dark states, and slow interconversion rates between the bright state and dark state(s) consistent with previous work. We confirm large two-photon action cross sections (103−104 GM) and broad two-photon excitation spectra that suggest QDs as advantageous probes for multicolor multiphoton imaging. FCS results show Alexa546 is a particularly bright probe suited for use when probe size is a limitation. While superior in count rate to Alexa555, Alexa546 bleaches faster when used in one-photon excitation.

A Quantitative Comparison of the Photophysical Properties of Selected Quantum Dots and Organic Fluorophores

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.

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Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Biological imaging has been limited by the finite resolution of light microscopy. Recent developments in ultra-high-resolution microscopy methods, many of which are based on fluorescence, are breaking the diffraction barrier; it is becoming possible to image intracellular protein distributions with resolution of tens of nanometers or better. Fluorescence photoactivation localization microscopy (FPALM) is an example of such an ultra-high-resolution method which can image living or fixed cells with demonstrated lateral resolution of better than 20 nm. A detailed description of the methods involved in FPALM imaging of biological samples is presented here, accompanied by comparison with existing methods from the literature.

Samuel T. Hess

Papers

Dances with Membranes: Breakthroughs from Super-resolution Imaging.

Non-coherent light microscopy

A Quantitative Comparison of the Photophysical Properties of Selected Quantum Dots and Organic Fluorophores

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Chapter 12: Nanoscale biological fluorescence imaging: breaking the diffraction barrier.