Resolution Fluorescence Microscopy Research Articles

Application of aptamers and autofluorescent proteins for RNA visualization

The repertoire of RNAs transcribed and processed within living cells is of extraordinary complexity. With new types of RNA being identified, the need for tools to investigate the spatio-temporal aspects of processing and trafficking of these molecules has become more evident. To visualize RNA in living cells, autofluorescent proteins (AFPs) appear as a promising alternative to synthetic fluorescent compound based labels. While current fluorescent protein-based RNA labelings have provided many new insights into the biology of RNA regulation, further improvements and adaptations are desirable to make AFP labels as valuable in the RNA world as they have proven to be for protein tagging. This article reviews the achievements and existing challenges in engineering AFPs as efficient RNA tags for high resolution fluorescence microscopy in living cells.

Photoreactions and dynamics of the green fluorescent protein

The wild type green fluorescent protein (GFP) from Aequorea victoria has been extensively investigated with a strong focus on the photochemistry and structural dynamics that are linked with its diverse activities. GFP combines a number of remarkable, and some unique, features that are still intensely researched both experimentally and theoretically. The protein environment effectively inhibits deactivation pathways that are dominant in the isolated chromophore and is therefore responsible for the bright fluorescence. Its p-hydroxybenzylidene-imidazolidinone chromophore acts as a photoacid, and optical excitation triggers ultrafast proton transfer reactions in the active site. The microscopic details of the proton transfer mechanism through a hydrogen bonding network are discussed in this critical review. This property of the wild type GFP has provided the opportunity to characterise the role of the specific protein environment in the proton transfer reactions in comparison to photoacid reactions in the condensed phase. In addition, GFP displays a photochromic side reaction that is uniquely caused by electron transfer from a buried anionic glutamic acid to the optically excited chromophore. This phototransformation property has also been exploited in high resolution fluorescence microscopy techniques. The discussion in this review is extended to include vibrational spectroscopy and structural dynamics (106 references).

In situ formation, manipulation, and imaging of droplet-encapsulated fibrin networks

The protein fibrin plays a principal role in blood clotting and forms robust three dimensional networks. Here, microfluidic devices have been tailored to strategically generate and study these bionetworks by confinement in nanoliter volumes. The required protein components are initially encapsulated in separate droplets, which are subsequently merged by electrocoalescence. Next, distinct droplet microenvironments are created as the merged droplets experience one of two conditions: either they traverse a microfluidic pathway continuously, or they "park" to fully evolve an isotropic network before experiencing controlled deformations. High resolution fluorescence microscopy is used to image the fibrin networks in the microchannels. Aggregation (i.e."clotting") is significantly affected by the complicated flow fields in moving droplets. In stopped-flow conditions, an isotropic droplet-spanning network forms after a suitable ripening time. Subsequent network deformation, induced by the geometric structure of the microfluidic channel, is found to be elastic at low rates of deformation. A shape transition is identified for droplets experiencing rates of deformation higher than an identified threshold value. In this condition, significant densification of protein within the droplet due to hydrodynamic forces is observed. These results demonstrate that flow fields considerably affect fibrin in different circumstances exquisitely controlled using microfluidic tools.

Abstract 1779: Autophagy is an Important Survival Response to Bnip3-Mediated Mitochondrial Injury

Myocardial ischemia and reperfusion (I/R) is associated with increased autophagy and cell death. We previously found that the mitochondrial death protein Bnip3 contributes to I/R injury and induces autophagy in cardiac cells. However, the role of Bnip3-mediated autophagy is not clear and has been reported to be a protective, as well as a detrimental process. In this study, we investigated the functional role of Bnip3-mediated autophagy by utilizing adenoviral infections of cells coupled with fluorescence deconvolution microscopy for the analysis of autophagy and Bnip3 function. Imaging of cells infected with adenoviruses expressing GFP-LC3 and Bnip3 or beta-galactosidase (beta-gal) allowed us to monitor the formation of autophagosomes in live cells. Cell death was measured by assessing LDH activity in the media. Monitoring of GFP-LC3 incorporation into autophagosomes confirmed that overexpression of Bnip3 induced upregulation of autophagy in HL-1 myocytes and neonatal myocytes. We previously found that Bnip3 mediates cell death via Bax/Bak and that mouse embryonic fibroblasts (MEFs) lacking Bax and Bak are resistant to Bnip3-mediated cell death. Bnip3 still caused significant upregulation of autophagy in Bax/Bak−/− MEFs and imaging using high resolution fluorescence microscopy showed that a majority of the cells mitochondria were sequestered in autophagosomes. These results were confirmed by electron microscopy which also revealed the presence of a number of giant mitochondria in the cytosol, suggesting stimulation of mitochondrial biogenesis. Although Bnip3 alone did not cause cell death in Bax/Bak−/− MEFs, addition of 3-methyladenine (3-MA), an inhibitor of autophagy induced significant cell death in cells infected with Bnip3 but had no effect on cells overexpressing beta-gal. Overexpression of Bnip3 caused significant cell death in wild type MEFs, which was enhanced when inhibiting autophagy with 3-MA. These results suggest that Bnip3 stimulates autophagy to remove damaged mitochondria and mitochondrial biogenesis to synthesize new functional mitochondria. Since Bnip3 is upregulated in heart failure, stimulation of autophagy and mitochondrial biogenesis might represent potential therapeutic targets to treat heart disease. This research has received full or partial funding support from the American Heart Association, AHA National Center.

Three-dimensional sub–100 nm resolution fluorescence microscopy of thick samples

Imaging volumes as thick as whole cells at three-dimensional (3D) super-resolution is required to reveal unknown features of cellular organization. We report a light microscope that generates images with translationally invariant 30 x 30 x 75 nm resolution over a depth of several micrometers. This method, named biplane (BP) FPALM, combines a double-plane detection scheme with fluorescence photoactivation localization microscopy (FPALM) enabling 3D sub-diffraction resolution without compromising speed or sensitivity.

Nanoscale resolution fluorescence microscopy using electromagnetically induced transparency

We suggest a type of scanning fluorescence microscope that is capable of resolving nanometer-size objects in the far field. The key idea is to use the spatial sensitivity of the dark state of electromagnetically induced transparency and localize an atomic excitation to a spot much smaller than the wavelength of light.

Micrometer-Sized Rings Self-assembled from Thiacyanine Dye Molecules and Their Waveguiding Properties

To fabricate ring-shaped waveguides on a micrometer scale, chloroform solutions of thiacyanine (TC) dyes having alkyl groups of different chain lengths ranging from −C2H5 to −C8H17 were drop-cast on a glass substrate and were allowed to evaporate under different conditions. It was found that the TC dyes with −C8H17 groups (TC8) self-assembled into well-defined rings with diameters of 5−30 μm on evaporating the solution under highly humid conditions (relative humidity: ∼90%). The morphological and optical properties of the individual rings were investigated by microscopy techniques. The polarized fluorescence microscope image of the rings showed pronounced polarization effects, indicating that the molecules inside the rings are highly ordered. The mechanism of ring formation was discussed on the basis of the molecular arrangement within the rings. Furthermore, the waveguiding properties of the rings were investigated by spatially resolved fluorescence microscopy. It was found that the TC8 rings guided their fluorescence along the rings.

3-Dimensional reconstruction of fluorescent structures in tardigrades

Tardigrades are microscopic animals, thus brightfield microscopy is a well established method for tardigrade observation. Modern techniques in functional genetics like fluorescence in situ hybridisation or fluorescently labelled expression markers demand high resolution fluorescence microscopy. Nevertheless tardigrades are still considered to be difficult objects for fluorescence techniques as they are covered by an opaque and diffracting cuticle. We show a modern technique of structured light illumination that enables us to acquire thin optical sections and consequently to reconstruct 3-dimensional structures in tardigrades with a high spatial resolution in all 3 dimensions. This technique is evaluated on taxonomically valuable internal as well as external structures of eutardigrades: the bucco-pharyngeal apparatus and the claws. The 3-dimensional reconstructions allow the measurement of distances in all 3 dimensions.

Sublethal Trauma Model With Systemic Endotoxemia for the Study of Microcirculatory Disorders After the Second Hit

Experimental models often cannot simulate the clinical situation in traumatized patients where the septic second hit finally leads to multiple organ dysfunction syndrome and death. We present an experimental animal model combining initial standardized soft tissue trauma to the left hindlimb of rats with subsequent sublethal systemic endotoxemia. This study characterizes the influence of trauma and systemic endotoxemia on nutritive blood flow, inflammatory cell-cell interaction and tissue cell integrity in the traumatized region. At 24 h after local tissue contusion, in vivo analysis of the skeletal muscle microcirculation by means of high resolution fluorescence microscopy revealed intravascular leukocyte accumulation and impairment of nutritive perfusion with tissue hypoxia. Moreover, muscle tissue damage was characterized by myocyte cell apoptosis. Additional systemic exposure of animals to E. coli lipopolysaccharide at 6 h after soft tissue contusion caused a drop in arterial blood pressure as well as coagulatory disorders, as given by marked thrombocytopenia and reduced thromboplastin times. In double-hit exposed animals, skeletal muscle microcirculation presented with an aggravation of inflammation, perfusion failure, and apoptotic cell death after 24 h. This model more closely resembles the scenario of polytraumatized patients with the risk of secondary infections and, thus, is suited to characterize anti-inflammatory drugs with respect to their potential to interfere with microcirculatory disorders and elaboration of disseminated intravascular coagulation after trauma.

Waveguiding Properties of Fiber-Shaped Aggregates Self-Assembled from Thiacyanine Dye Molecules

The waveguiding properties of the fiber-shaped aggregates self-assembled from the thiacyanine (TC) dye in solution were investigated by spatially resolved fluorescence microscopy. The TC fibers transferred onto a glass substrate were excited by a focused laser beam. The spectra of the outcoupled light from the fiber end were recorded as a function of the distance between the laser spot and the fiber end. The spectral images in a false-color intensity scale constructed from the outcoupled light spectra exhibited the waveguiding properties of the fibers well. The optical losses for the wavelength range over the entire emission band (470−620 nm) were quantitatively determined. It was found that the fibers have a cutoff wavelength at ∼480 nm, below which the light is not guided, and these fibers act as low-loss waveguides for the wavelength range of 530−600 nm. The spectral images also revealed that the TC fiber functions as an optical cavity (Fabry−Perot cavity) by the recurrence of the guided light due to t...

Localizations of endogenous APP/APP-Proteolytic products are consistent with microtubular transport

Dementia of the Alzheimer type (DAT) is associated with the accumulation of beta-amyloid (A beta) peptides derived from beta-amyloid precursor protein (APP). Goldstein and coworkers have suggested that APP acts as a cargo receptor connecting post-Golgi vesicles and motor proteins. Sisodia and colleagues have suggested that APP is a passive passenger within the vesicles. Both views predict that one should be able to visualize colocalizations of APP with microtubules, the object of the present investigation. To avoid possible artifacts created by APP overexpression, we studied endogenous expression in a human neuroblastoma cell line (SK-N-SH). Using high resolution fluorescence microscopy and antibodies specific for the amino termini of APP and A beta sequences, we found that endogenous APP and A beta peptide immunoreactivities colocalized with microtubules in interphase cells. Disruption of microtubules, followed by fixation at various time points during repolymerization, allowed us to observe the sequence and timing of these colocalizations in interphase cells. In addition, to our surprise, we found that A beta immunoreactivities colocalize with the mitotic spindle, a bundle of specialized microtubules. Because of the condensed cytoplasm found in neurons, we suggest that SK-N-SH cells might be a more convenient experimental system for exploring the mechanisms that underlie these protein localizations and the pathology that might result from altered APP protein structure and function.

Membrane potential governs lateral segregation of plasma membrane proteins and lipids in yeast

The plasma membrane potential is mainly considered as the driving force for ion and nutrient translocation. Using the yeast Saccharomyces cerevisiae as a model organism, we have discovered a novel role of the membrane potential in the organization of the plasma membrane. Within the yeast plasma membrane, two non-overlapping sub-compartments can be visualized. The first one, represented by a network-like structure, is occupied by the proton ATPase, Pma1, and the second one, forming 300-nm patches, houses a number of proton symporters (Can1, Fur4, Tat2 and HUP1) and Sur7, a component of the recently described eisosomes. Evidence is presented that sterols, the main lipid constituent of the plasma membrane, also accumulate within the patchy compartment. It is documented that this compartmentation is highly dependent on the energization of the membrane. Plasma membrane depolarization causes reversible dispersion of the H(+)-symporters, not however of the Sur7 protein. Mitochondrial mutants, affected in plasma membrane energization, show a significantly lower degree of membrane protein segregation. In accordance with these observations, depolarized membranes also considerably change their physical properties (detergent sensitivity).

Single-molecule mountains yield nanoscale cell images

Methods to simultaneously localize the positions of multiple single fluorophores by precisely determining their individual positions are now yielding impressive gains in fluorescence microscopy resolution.

Fine Structural Analysis of Brefeldin A-Induced Compartment Formation After High-Pressure Freeze Fixation of Maize Root Epidermis

Formation of large perinuclear brefeldin A (BFA)-induced compartments is a characteristic feature of root apex cells, but it does not occur in shoot apex cells. BFA-induced compartments have been studied mostly using low resolution fluorescence microscopy techniques. Here, we have employed a high-resolution ultrastructural method based on ultra rapid freeze fixation of samples in order to study the formation of BFA-induced compartment in intact maize root epidermis cells in detail. This approach reveals five novel findings. Firstly, plant TGN/PGN elements are not tubular networks, as generally assumed, but rather vesicular compartments. Secondly, TGN/PGN vesicles interact with one another extensively via stalk-like connections and even fuse together via bridge-like structures. Thirdly, BFA-induced compartments are formed via extensive homotypic fusions of the TGN/PGN vesicles. Fourthly, multivesicular bodies (MVBs) are present within the BFA-induced compartments. Fifthly, mitochondria and small vacuoles accummulate abundantly around the large perinuclear BFA-induced compartments.

Photophysics of 9-amino acridine hydrochloride hydrate single microcrystals

Single microcrystals of 9-amino acridine hydrochloride hydrate (9AAHH) have been studied by steady state and time resolved fluorescence microscopy. Crystalline 9AAHH exists in the form of two types of microcrystals viz., stick type and slab type. The stick-shaped microcrystals are found to undergo excimer formation. With increase in size, the excimer fluorescence is masked by the concomitant appearance of self-absorption effect in dimer. The fluorescence lifetimes increase on increasing the microcrystal size. While the bandwidth of fluorescence spectra of slab shaped microcrystals are found to increase, the fluorescence lifetimes decrease with increasing their size. Results are explained on the basis of interplay between the self-absorption effect of the dimer, its conversion efficiency to the excimer, and higher aggregate formation in large microcrystals.

Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins

Fluorescence microscopy is indispensable in many areas of science, but until recently, diffraction has limited the resolution of its lens-based variant. The diffraction barrier has been broken by a saturated depletion of the marker's fluorescent state by stimulated emission, but this approach requires picosecond laser pulses of GW/cm2 intensity. Here, we demonstrate the surpassing of the diffraction barrier in fluorescence microscopy with illumination intensities that are eight orders of magnitude smaller. The subdiffraction resolution results from reversible photoswitching of a marker protein between a fluorescence-activated and a nonactivated state, whereby one of the transitions is accomplished by means of a spatial intensity distribution featuring a zero. After characterizing the switching kinetics of the used marker protein asFP595, we demonstrate the current capability of this RESOLFT (reversible saturable optical fluorescence transitions) type of concept to resolve 50-100 nm in the focal plane. The observed resolution is limited only by the photokinetics of the protein and the perfection of the zero. Our results underscore the potential to finally achieve molecular resolution in fluorescence microscopy by technical optimization.

Cholesterol-dependent Syntaxin-4 and SNAP-23 Clustering Regulates Caveolar Fusion with the Endothelial Plasma Membrane

We determined the organization of target (t) SNARE proteins on the basolateral endothelial plasma membrane (PM) and their role in the mechanism of caveolar fusion. Studies were performed in a cell-free system involving endothelial PM sheets and isolated biotin-labeled caveolae. We monitored the fusion of caveolae with the PM by the detection of biotin-streptavidin complexes using correlative high resolution fluorescence microscopy and gold labeling electron microscopy on ultrathin sections of PM sheets. Imaging of PM sheets demonstrated and biochemical findings showed that the t-SNARE proteins present in endothelial cells (SNAP-23 and syntaxin-4) formed cholesterol-dependent clusters in discrete areas of the PM. Upon fusion of caveolae with the target PM, 50% of the caveolae co-localized with the t-SNARE clusters, indicating that these caveolae were at the peak of the fusion reaction. Fluorescent streptavidin staining of PM sheets correlated with the ultrastructure in the same area. These findings demonstrate that t-SNARE clusters in the endothelial target PM serve as the fusion sites for caveolae during exocytosis.

Concentration sensing based on radiative rate enhancement from a single microcavity

Time resolved fluorescence microscopy has been used to measure the radiative rate of molecules embedded in single spherical microcavities. The value of the measured rate is found to be a function of the diameter of the selected microcavity. The values of radiative rate enhancement (RRE) have been obtained for various cavity sizes and different concentrations. It is shown here for the first time that the concentration dependence of RRE can be used for sensitive detection of changes in the concentration of molecules in microscopic space.

The Spatial Organization of Lipid Synthesis in the Yeast Saccharomyces cerevisiae Derived from Large Scale Green Fluorescent Protein Tagging and High Resolution Microscopy

The localization pattern of proteins involved in lipid metabolism in the yeast Saccharomyces cerevisiae was determined using C-terminal green fluorescent protein tagging and high resolution confocal laser scanning microscopy. A list of 493 candidate proteins ( approximately 9% of the yeast proteome) was assembled based on proteins of known function in lipid metabolism, their interacting proteins, proteins defined by genetic interactions, and regulatory factors acting on selected genes or proteins. Overall 400 (81%) transformants yielded a positive green fluorescent protein signal, and of these, 248 (62% of the 400) displayed a localization pattern that was not cytosolic. Observations for many proteins with known localization patterns were consistent with published data derived from cell fractionation or large scale localization approaches. However, in many cases, high resolution microscopy provided additional information that indicated that proteins distributed to multiple subcellular locations. The majority of tagged enzymes localized to the endoplasmic reticulum (91), but others localized to mitochondria (27), peroxisomes (17), lipid droplets (23), and vesicles (53). We assembled enzyme localization patterns for phospholipid, sterol, and sphingolipid biosynthetic pathways and propose a model, based on enzyme localization, for concerted regulation of sterol and sphingolipid metabolism that involves shuttling of key enzymes between endoplasmic reticulum, lipid droplets, vesicles, and Golgi.

The primacy of affinity over clustering in regulation of adhesiveness of the integrin αLβ2

Dynamic regulation of integrin adhesiveness is required for immune cell–cell interactions and leukocyte migration. Here, we investigate the relationship between cell adhesion and integrin microclustering as measured by fluorescence resonance energy transfer, and macroclustering as measured by high resolution fluorescence microscopy. Stimuli that activate adhesion through leukocyte function–associated molecule-1 (LFA-1) failed to alter clustering of LFA-1 in the absence of ligand. Binding of monomeric intercellular adhesion molecule-1 (ICAM-1) induced profound changes in the conformation of LFA-1 but did not alter clustering, whereas binding of ICAM-1 oligomers induced significant microclustering. Increased diffusivity in the membrane by cytoskeleton-disrupting agents was sufficient to drive adhesion in the absence of affinity modulation and was associated with a greater accumulation of LFA-1 to the zone of adhesion, but redistribution did not precede cell adhesion. Disruption of conformational communication within the extracellular domain of LFA-1 blocked adhesion stimulated by affinity-modulating agents, but not adhesion stimulated by cytoskeleton-disrupting agents. Thus, LFA-1 clustering does not precede ligand binding, and instead functions in adhesion strengthening after binding to multivalent ligands.