Contributions of fluorescence techniques to understanding G protein-coupled receptor dimerisation

G protein-coupled receptors (GPCRs) are the largest class of eukaryotic cell-surface receptors and, over the last decade, it has become clear that they are capable of dimerisation. Whilst many biochemical and biophysical approaches have been used to study dimerisation, fluorescence techniques, including Förster resonance energy transfer and single molecule fluorescence, have been key players. Here we review recent contributions of fluorescence techniques to investigate GPCR dimers, including dimerisation in cell membranes and native tissues, the effect of ligand binding on dimerisation and the kinetics of dimer formation and dissociation. The challenges of studying multicomponent membrane protein systems have led to the development and refinement of many fluorescence assays, allowing the functional consequences of receptor dimerisation to be investigated and individual protein molecules to be imaged in the membranes of living cells. It is likely that the fluorescence techniques described here will be of use for investigating many other multicomponent membrane protein systems.     

Fluorescence molecular imaging of small animal tumor models

In vivo imaging of molecular events in small animals has great potential to impact basic science and drug development. For this reason, several imaging technologies have been adapted to small animal research, including X-ray, magnetic resonance, and radioisotope imaging. Despite this plethora of visualization techniques, fluorescence imaging is emerging as an important alternative because of its operational simplicity, safety, and cost-effectiveness. Fluorescence imaging has recently become particularly interesting because of advances in fluorescent probe technology, including targeted fluorochromes as well as fluorescent "switches" sensitive to specific biochemical events. While past biological investigations using fluorescence have focused on microscopic examination of ex vivo, in vitro, or intravital specimens, techniques for macroscopic fluorescence imaging are now emerging for in vivo molecular imaging applications. This review illuminates fluorescence imaging technologies that hold promise for small animal imaging. In particular we focus on planar illumination techniques, also known as Fluorescence Reflectance Imaging (FRI), and discuss its performance and current use. We then discuss fluorescence molecular tomography (FMT), an evolving technique for quantitative three-dimensional imaging of fluorescence in vivo. This technique offers the promise of non-invasively quantifying and visualizing specific molecular activity in living subjects in three dimensions.     

Advances in Image Correlation Spectroscopy: Measuring Number Densities,Aggregation States,and Dynamics of Fluorescently labeled Macromolecules in Cells

A brief historical outline of fluorescence fluctuation correlation techniques is presented, followed by an in-depth review of the theory and development of image correlation techniques, including: image correlation spectroscopy (ICS), temporal ICS (TICS), image cross-correlation spectroscopy (ICCS), spatiotemporal ICS (STICS), k-space ICS (kICS), raster ICS (RICS), and particle ICS (PICS). These techniques can be applied to analyze image series acquired on commercially available laser scanning or total internal reflection fluorescence microscopes, and are used to determine the number density, aggregation state, diffusion coefficient, velocity, and interaction fraction of fluorescently labeled molecules or particles. A comprehensive review of the application of ICS techniques to a number of systems, including cell adhesion, membrane receptor aggregation and dynamics, virus particle fusion, and fluorophore photophysics, is presented.     

Optical microscopy in photosynthesis

Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.     

Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy

The spatiotemporal localization of neuronal signaling is important for triggering neuronal responses in specific locations at precise times. Fluorescence resonance energy transfer imaging enables measurement of spatiotemporal dynamics of signaling activity in live neurons. Although the usefulness of fluorescence resonance energy transfer is well recognized, there are many difficulties in applying it, particularly when imaging in neuronal micro-compartments in light-scattering brain tissue. Fluorescence resonance energy transfer has been imaged using several techniques including intensity-based methods, fluorescence lifetime imaging and fluorescence anisotropy imaging. These methods have different advantages and disadvantages, and thus are suitable in different applications.     

Computer processing of microscopic images of bacteria: morphometrY and fluorimetry

Several techniques that use computer analysis of microscopic images have been developed to study the complicated microbial flora in the human intestine, including measuring the shape and fluorescence intensity of bacteria. These techniques allow rapid assessment of changes in the intestinal flora and could apply equally to other complex microbial ecosystems.     

Fluorescence imaging in vivo: recent advances

In vivo fluorescence imaging uses a sensitive camera to detect fluorescence emission from fluorophores in whole-body living small animals. To overcome the photon attenuation in living tissue, fluorophores with long emission at the near-infrared (NIR) region are generally preferred, including widely used small indocarbocyanine dyes. The list of NIR probes continues to grow with the recent addition of fluorescent organic, inorganic and biological nanoparticles. Recent advances in imaging strategies and reporter techniques for in vivo fluorescence imaging include novel approaches to improve the specificity and affinity of the probes and to modulate and amplify the signal at target sites for enhanced sensitivity. Further emerging developments are aiming to achieve high-resolution, multimodality and lifetime-based in vivo fluorescence imaging.     

Toward an understanding of the structural basis of translation

The recently solved X-ray crystal structures of the ribosome have provided opportunities for studying the molecular basis of translation with a variety of methods including cryo-electron microscopy - where maps give the first glimpses of ribosomal evolution - and fluorescence spectroscopy techniques.     

Single-molecule spectroscopic methods

Being praised for the mere fact of enabling the detection of individual fluorophores a dozen years ago, single-molecule techniques nowadays represent standard methods for the elucidation of the structural rearrangements of biologically relevant macromolecules. Single-molecule-sensitive techniques, such as fluorescence correlation spectroscopy, allow real-time access to a multitude of molecular parameters (e.g. diffusion coefficients, concentration and molecular interactions). As a result of various recent advances, this technique shows promise even for intracellular applications. Fluorescence imaging can reveal the spatial localization of fluorophores on nanometer length scales, whereas fluorescence resonance energy transfer supports a wide range of different applications, including real-time monitoring of conformational rearrangements (as in protein folding). Still in their infancy, single-molecule spectroscopic methods thus provide unprecedented insights into basic molecular mechanisms.     

蛋白质相互作用实验技术的最新进展

蛋白质相互作用的研究, 是揭示生物体正常生长发育及其应对各种生物或(和)非生物胁迫的分子机制及其调控网络的重要途径。文章综述了近年来发展起来的研究蛋白质相互作用的常用实验性方法, 如酵母双杂交系统、串联亲和纯化、免疫共沉淀、GST Pull-down、双分子荧光互补、荧光共振能量转移、表面等离子共振分析, 介绍了其原理、发展进程, 并分析了其优缺点。     

Pore-forming protein structure analysis in membranes using multiple independent fluorescence techniques

A large number of transmembrane proteins form aqueous pores or channels in the phospholipid bilayer, but the structural bases of pore formation and assembly have been determined experimentally for only a few of the proteins and protein complexes. The polypeptide segments that form the transmembrane pore and the secondary structure that creates the aqueous-lipid interface can be identified using multiple independent fluorescence techniques (MIFT). The information obtained from several different, but complementary, fluorescence analyses, including measurements of emission intensity, fluorescence lifetime, accessibility to aqueous and to lipophilic quenching agents, and fluorescence resonance energy transfer (FRET) can be combined to characterize the nature of the protein-membrane interaction directly and unambiguously. The assembly pathway can also be determined by measuring the kinetics of the spectral changes that occur upon pore formation. The MIFT approach therefore allows one to obtain structural information that cannot be obtained easily using alternative techniques such as crystallography. This review briefly outlines how MIFT can reveal the identity, location, conformation, and topography of the polypeptide sequences that interact with the membrane.     

Advances in laboratory evolution of enzymes

We address recent developments in the area of laboratory, or directed evolution, with a focus on enzymes and on new methodologies of generic potential. We survey three main areas: (i) library making techniques, including the application of computational and rational methods for library design; (ii) screening and selection techniques, including recent applications of enzyme screening by FACS (fluorescence activated cell sorter); (iii) new approaches for performing directed evolution, and in particular, the application of 'neutral drifts' (libraries generated by rounds of mutation and selection for the enzyme's original function) and of consensus mutations to generate highly evolvable starting points for directed evolution.     

FRET imaging

F?rster (or Fluorescence) Resonance Energy Transfer (FRET) is unique in generating fluorescence signals sensitive to molecular conformation, association, and separation in the 1-10 nm range. We introduce a revised photophysical framework for the phenomenon and provide a systematic catalog of FRET techniques adapted to imaging systems, including new approaches proposed as suitable prospects for implementation. Applications extending from a single molecule to live cells will benefit from multidimensional microscopy techniques, particularly those adapted for optical sectioning and incorporating new algorithms for resolving the component contributions to images of complex molecular systems.     

Tag–probe labeling methods for live-cell imaging of membrane proteins

Instead of using reconstituted proteoliposomes, in situ investigations of membrane proteins in living cell membranes are important because the heterogeneous and dynamic nature of biomembranes significantly affects their behavior. Protein-specific labeling is a key technique for the detection of a target protein by fluorescence measurements, particularly fluorescence microscopy. However, conventional genetic fusion with fluorescent proteins has several shortcomings. Post-translational labeling methods using a genetically encodable tag and synthetic probes targeting to the tag can overcome these limitations. This review summarizes emerging tag–probe techniques for labeling specific membrane proteins and their applications, including endocytotic internalization, partitioning to specific membrane domains, interprotein interactions, and conformational changes.     

DNA-induced distamycin A fluorescence

The fluorescent properties of the antibiotic distamycin A were investigated in a range of materials including Trypanosoma cruzi epimastigotes, chicken erythrocytes, calf thymus DNA and synthetic polynucleotides using both microscopic and spectroscopic techniques. A bright blue-white fluorescence was observed from kinetoplast DNA and chromatin after treatment with distamycin A under ultraviolet (365 nm) excitation. Considerable enhancement of distamycin A fluorescence (emission peak at 455 nm under 320-340 nm excitation) was found in the presence of DNA and poly(dA-dT).poly(dA-dT). We discuss a possible explanation for this unexpected fluorescent emission, as well as its implications for microscopic and fluorimetric studies.     

In vivo optical molecular imaging and analysis in mice using dorsal window chamber models applied to hypoxia, vasculature and fluorescent reporters

Optical techniques for functional imaging in mice have a number of key advantages over other common imaging modalities such as magnetic resonance imaging, positron emission tomography or computed tomography, including high resolution, low cost and an extensive library of available contrast agents and reporter genes. A major challenge to such work is the limited penetration depth imposed by tissue turbidity. We describe a window chamber technique by which these limitations can be avoided. This facilitates the study of a wide range of processes, with potential endpoints including longitudinal gene expression, vascular remodeling and angiogenesis, and tumor growth and invasion. We further describe several quantitative imaging and analysis techniques for characterizing in vivo fluorescence properties and functional endpoints, including vascular morphology and oxygenation. The procedure takes ～2 h to complete, plus up to several weeks for tumor growth and treatment procedures.     

Correlative fluorescence and electron microscopy on ultrathin cryosections: bridging the resolution gap.

Microscopy has become increasingly important for analysis of cells and cell function in recent years. This is due in large part to advances in light microscopy that facilitate quantitative studies and improve imaging of living cells. Analysis of fluorescence signals has often been a key feature in these advances. Such studies involve a number of techniques, including imaging of fluorescently labeled proteins in living cells, single-cell physiological experiments using fluorescent indicator probes, and immunofluorescence localization. The importance of fluorescence microscopy notwithstanding, there are instances in which electron microscopy provides unique information about cell structure and function. Correlative microscopy in which a fluorescence signal is reconciled with a signal from the electron microscope is an additional tool that can provide powerful information for cellular analysis. Here we review two different methodologies for correlative fluorescence and electron microscopy using ultrathin cryosections and the advantages attendant on this approach. (J Histochem Cytochem 49:803-808, 2001)     

Exploring membrane organization and dynamics by the wavelength-selective fluorescence approach

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in fluorescence spectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in a complex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitation shift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation) around an excited state fluorophore which is a function of the motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobility parameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophore merely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the information obtained in such cases will come from the otherwise 'optically silent' water molecules. This makes REES and related techniques extremely useful since hydration plays a crucial modulatory role in a large number of important cellular events, including lipid-protein interactions and ion transport. The interfacial region in membranes, characterized by unique motional and dielectric characteristics, represents an appropriate environment for displaying wavelength-selective fluorescence effects. The application of REES and related techniques (wavelength-selective fluorescence approach) as a powerful tool to monitor the organization and dynamics of probes and peptides bound to membranes, micelles, and reverse micelles is discussed.     

Development of a Plastic Embedding Method for Large-Volume and Fluorescent-Protein-Expressing Tissues

Fluorescent proteins serve as important biomarkers for visualizing both subcellular organelles in living cells and structural and functional details in large-volume tissues or organs. However, current techniques for plastic embedding are limited in their ability to preserve fluorescence while remaining suitable for micro-optical sectioning tomography of large-volume samples. In this study, we quantitatively evaluated the fluorescence preservation and penetration time of several commonly used resins in a Thy1-eYFP-H transgenic whole mouse brain, including glycol methacrylate (GMA), LR White, hydroxypropyl methacrylate (HPMA) and Unicryl. We found that HMPA embedding doubled the eYFP fluorescence intensity but required long durations of incubation for whole brain penetration. GMA, Unicryl and LR White each penetrated the brain rapidly but also led to variable quenching of eYFP fluorescence. Among the fast-penetrating resins, GMA preserved fluorescence better than LR White and Unicryl. We found that we could optimize the GMA formulation by reducing the polymerization temperature, removing 4-methoxyphenol and adjusting the pH of the resin solution to be alkaline. By optimizing the GMA formulation, we increased percentage of eYFP fluorescence preservation in GMA-embedded brains nearly two-fold. These results suggest that modified GMA is suitable for embedding large-volume tissues such as whole mouse brain and provide a novel approach for visualizing brain-wide networks.     

Spatial Covariance Reconstructive (SCORE) Super-Resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy has become a powerful tool to resolve structural information that is not accessible to traditional diffraction-limited imaging techniques such as confocal microscopy. Stochastic optical reconstruction microscopy (STORM) and photoactivation localization microscopy (PALM) are promising super-resolution techniques due to their relative ease of implementation and instrumentation on standard microscopes. However, the application of STORM is critically limited by its long sampling time. Several recent works have been focused on improving the STORM imaging speed by making use of the information from emitters with overlapping point spread functions (PSF). In this work, we present a fast and efficient algorithm that takes into account the blinking statistics of independent fluorescence emitters. We achieve sub-diffraction lateral resolution of 100 nm from 5 to 7 seconds of imaging. Our method is insensitive to background and can be applied to different types of fluorescence sources, including but not limited to the organic dyes and quantum dots that we demonstrate in this work.