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Abstract: Autofluorescence of aldehyde-fixed tissues greatly hinders fluorescence microscopy. In particular, lipofuscin, an autofluorescent component of aged brain tissue, complicates fluorescence imaging of tissue in neurodegenerative diseases. Background and lipofuscin [...]

Abstract. In light sheet microscopy, optical sectioning by selective fluorescence excitation with a sheet of light is combined with fast full-frame acquisition. This illumination scheme provides minimal photobleaching and phototoxicity. [...]

Podocytes are critical in the maintenance of a healthy glomerular filter; however, they have been difficult to study in the intact kidney because of technical limitations. Here we report the [...]

The transient receptor potential channel TRPC3 is exclusively expressed in the apical membrane of principal cells of the collecting duct (CD) both in vivo and in the mouse CD cell line IMCD-3. Previous studies revealed that ATP-induced apical-to-basolateral transepithelial [Ca.sup.2+] flux across IMCD-3 monolayers is increased by overexpression of TRPC3 and attenuated by a dominant negative TRPC3 construct, suggesting that [Ca.sup.2+] entry across the apical membrane occurs via TRPC3 channels. To test this hypothesis, we selectively measured the [Ca.sup.2+] permeability of the apical membrane of fura-2-1oaded IMCD-3 cells using the [Mn.sup.2+] quench technique. [Mn.sup.2+] influx across the apical membrane was increased 12- to 16-fold by apical ATP and was blocked by the pyrazole derivative BTP2, a known inhibitor of TRPC3 channels, with an [IC.sub.50] value calcium channels; transient receptor potential channel; fluorescence microscopy; fura 2; manganese quench; inner medullary collecting duct cell line IMCD-3 doi: 10.1152/ajprenal.00670.2009.

To define the molecular architecture of the kinetochore in vertebrate cells, we measured the copy number of eight kinetochore proteins that link kinetochore microtubules (MTs [kMTs]) to centromeric DNA. We used a fluorescence ratio method and chicken DT40 cell lines in which endogenous loci encoding the analyzed proteins were deleted and complemented using integrated green fluorescent protein fusion transgenes. For a mean of 4.3 kMTs at metaphase, the protein copy number per kMT is between seven and nine for members of the MT-binding KNL-1/Mis12 complex/Ndc80 complex network. It was between six and nine for four members of the constitutive centromere-associated network: centromere protein C (CENP-C), CENP-H, CENP-I, and CENP-T. The similarity in copy number per kMT for all of these proteins suggests that each MT end is linked to DNA by six to nine fibrous unit attachment modules in vertebrate cells, a conclusion that indicates architectural conservation between multiple MT-binding vertebrate and single MT-binding budding yeast kinetochores. doi/10.1083/jcb.200912022

Total internal reflection fluorescence microscopy (TIRFM) images the plasma membrane--cytosol interface and has allowed insights into the behavior of individual secretory granules before and during exocytosis. Much less is known about the dynamics of the other partner in exocytosis, the plasma membrane. In this study, we report the implementation of a TIRFM-based polarization technique to detect rapid submicrometer changes in plasma membrane topology as a result of exocytosis. A theoretical analysis of the technique is presented together with image simulations of predicted topologies of the post-fusion granule membrane--plasma membrane complex. Experiments on dil-stained bovine adrenal chromaffin cells using polarized TIRFM demonstrate rapid and varied submicrometer changes in plasma membrane topology at sites of exocytosis that occur immediately upon fusion. We provide direct evidence for a persistent curvature in the exocytotic region that is altered by inhibition of dynamin guanosine triphosphatase activity and is temporally distinct from endocytosis measured by VMAT2-pHluorin. doi/10.1083/jcb.200908010

We present a method to separate clustered nuclei from fluorescence microscopy cellular images, using shape markers and marking function in a watershed-like algorithm. Shape markers are extracted using an adaptive H-minima transform. A marking function based on the outer distance transform is introduced to accurately separate clustered nuclei. With synthetic images, we quantitatively demonstrate the performance of our method and provide comparisons with existing approaches. On mouse neuronal and Drosophila cellular images, we achieved 6%-7% improvement of segmentation accuracies over earlier methods. Index Terms--Active contours, cell segmentation, cellular imaging, fluorescence microscopy, watershed segmentation.

The yeeK gene of Bacillus subtilis is predicted to encode a protein of 145 amino acids composed of 28% glycine, 23% histidine, and 12% tyrosine residues. Previous studies were unable to detect YeeK in wild-type spores; however, the 18-kDa YeeK polypeptide has been identified in yabG mutant spores. In this study, we analyze the expression and localization of YeeK to explore the relationship between YeeK and YabG. Northern hybridization analysis of wild-type RNA indicated that transcription of the yeeK gene, which was initiated 5 h after the onset of sporulation, was dependent on a SigK-containing RNA polymerase and the GerE protein. Genetic disruption of yeeK did not impair vegetative growth, development of resistant spores, or germination. Fluorescent microscopy of in-frame fusions of YeeK with green fluorescent protein (YeeK-GFP) and red fluorescent protein (YeeK-RFP) confirmed that YeeK assembles into the spore integument. CotE, SafA, and SpoVID were required for the proper localization of YeeK-GFP. Comparative analysis of YeeK-RFP and an in-frame GFP fusion of YabG indicated that YeeK colocalized with YabG in the spore coat. This is the first use of fluorescent proteins to show localization to different layers of the spore coat. Immunoblotting with anti-GFP antiserum indicated that YeeK-GFP was primarily synthesized as a 44-kDa molecule, which was then digested into a 29-kDa fragment that corresponded to the molecular size of GFP in wild-type spores. In contrast, a minimal amount of 44-kDa YeeK-GFP was digested in yabG mutant spores. Our findings demonstrate that YeeK is guided into the spore coat by CotE, SafA, and SpoVID. We conclude that YabG is directly or indirectly involved in the digestion of YeeK.

Tetraspanins regulate cell migration, sperm-egg fusion, and viral infection. Through interactions with one another and other cell surface proteins, tetraspanins form a network of molecular interactions called the tetraspanin web. In this study, we use single-molecule fluorescence microscopy to dissect dynamics and partitioning of the tetraspanin CD9. We show that lateral mobility of CD9 in the plasma membrane is regulated by at least two modes of interaction that each exhibit specific dynamics. The majority of CD9 molecules display Brownian behavior but can be transiently confined to an interaction platform that is in permanent exchange with the rest of the membrane. These platforms, which are enriched in CD9 and its binding partners, are constant in shape and localization. Two CD9 molecules undergoing Brownian trajectories can also codiffuse, revealing extra platform interactions. CD9 mobility and partitioning are both dependent on its palmitoylation and plasma membrane cholesterol. Our data show the high dynamic of interactions in the tetraspanin web and further indicate that the tetraspanin web is distinct from raft microdomains.

Decreased renal blood flow following an ischemic insult contributes to a reduction in glomerular filtration. However, little is known about the underlying cellular or subcellular mechanisms mediating reduced renal blood flow in human ischemic acute kidney injury (AKI) or acute renal failure (ARF). To examine renal vascular injury following ischemia, intraoperative graft biopsies were performed after reperfusion in 21 cadaveric renal allografts. Confocal fluorescence microscopy was utilized to examine vascular smooth muscle and endothelial cell integrity as well as peritubular interstitial pericytes in the biopsies. The reperfused, transplanted kidneys exhibited postischemic injury to the renal vasculature, as demonstrated by disorganization/disarray of the actin cytoskeleton in vascular smooth muscle cells and disappearance of yon Willebrand factor from vascular endothelial cells. Damage to peritubular capillary endothelial cells was more severe in subjects destined to have sustained ARF than in those with rapid recovery of their graft function. In addition, peritubular pericytes/myofibroblasts were more pronounced in recipients destined to recover than those with sustained ARF. Taken together, these data suggest damage to the renal vasculature occurs after ischemia-reperfusion in human kidneys. Preservation of peritubular capillary endothelial integrity and increasing pericytes may be critical to recovery from postischemic AKI. acute renal failure; confocal fluorescence microscopy; vascular smooth muscle cell; vasculature

Membrane trafficking during cytokinesis is not well understood. We used advanced live cell imaging techniques to track exocytosis of single vesicles to determine whether constitutively exocytosed membrane is focally delivered to the cleavage furrow. Ultrasensitive three-dimensional confocal time-lapse imaging of the temperature-sensitive membrane cargo protein vesicular stomatitis virus protein--yellow fluorescent protein revealed that vesicles from both daughter cells traffic out of the Golgi and into the furrow, following curvilinear paths. Immunolocalization and photobleaching experiments indicate that individual vesicles accumulate at the midbody and generate a reserve vesicle pool that is distinct from endosomal and lysosomal compartments. Total internal reflection fluorescence microscopy imaging provided direct evidence that Golgi-derived vesicles from both daughter cells not only traffic to the furrow region but dock and fuse there, supporting a symmetrically polarized exocytic delivery model. In contrast, quantitative analysis of midbody abscission showed inheritance of the midbody remnant by one daughter cell, indicating that cytokinesis is composed of both symmetrical and asymmetrical stages.

Understanding molecular mechanisms of pathophysiology and disease processes requires the development of new methods for studying proteins in animal tissues and organs. Here, we describe a method for adenoviral-mediated gene transfer into tubule or endothelial cells of the rat kidney. The left kidney of an anesthetized rat was exposed and the lumens of superficial proximal tubules or vascular welling points were microinfused, usually for 20 min. The microinfusion solution contained adenovirus with a cDNA construct of either 1) Xenopus laevis actin depolymerizing factor/cofilin [XAC; wt-green fluorescent protein (GFP)], 2) actin-GFP, or 3) GFP. Sudan black-stained castor oil, injected into nearby tubules, allowed us to localize the microinfused structures for subsequent visualization. Two days later, the rat was anesthetized and the kidneys were fixed for tissue imaging or the left kidney was observed in vivo using two-photon microscopy. Expression of GFP and GFP-chimeric proteins was clearly seen in epithelial cells of the injected proximal tubules and the expressed proteins were localized similarly to their endogenously expressed counterparts. Only a minority of the cells in the vitally exposed regions, however, expressed these proteins. Endothelial cells also expressed XAC-GFP after injection of the virus cDNA construct into vascular welling points. An advantage of the proximal tubule and vascular micropuncture approaches is that only minute amounts of virus are required to achieve protein expression in vivo. This micropuncture approach to gene transfer of the virus cDNA construct and intravital two-photon microscopy should be applicable to study of the behavior of any fluorescently tagged protein in the kidney and shows promise in studying renal physiology and pathophysiology. actin; actin depolymerizing factor; adenovirus; green fluorescent protein; intravital microscopy

A technique is described for the visualization of yeast chromosomes in live cells and in real time using a fluorophore labeled protein. Data indicate that yeast cells containing enhanced green fluorescent protein-Dal80p, which is a transcription repressor, show 16 fluorescent foci.

Research points out that lipofuscin-determined population characteristics of Crustacea are more reliable than those inferred from traditional methods. Data show that the yearly pigment accumulation is nearly linear as determined by image analysis of lipofuscin pigment labeled sections of the Antarctic decapod brain.

Results demonstrate that bacterial behavior in biofilms and planktonic bacteria differ as revealed by the analyses of Pseudomonas species genetically tagged with the green and the red fluorescent proteins. Data indicate that initially small microcolonies are formed by cell growth and aggregation that later transforms into compact microcolonies to loose structured colonies.

Results show that flagellar filaments move in varied directions as the bacterial cells alter course and different filaments can change direction at different times. Data indicate that individual filaments exhibit normal, semicoiled, and curly waveforms.

Research was conducted to examine distribution and activity patterns of Pseudomonas fluorescens in the root environment of barley plants. Results show that at normal soil moisture levels the bacteria formed microcolonies around the plant roots with differing viability and activity characteristics.

Discrete localized fluorescence transients due to openings of a single plasma membrane Ca(super 2+) permeable cation channel were recorded using wide-field digital imaging microscopy with fluo-3 as the Ca(super 2+) indicator. These transients were obtained while simultaneously recording the unitary channel currents using the whole-cell current-recording configuration of the patch-clamp technique. This cation channel in smooth muscle cells is opened by caffeine (Guerrero, A., F.S. Fay, and J.J. Singer. 1994. J. Gen. Physiol. 104:375-394). The localized fluorescence transients appeared to occur at random locations on the cell membrane, with the duration of the rising phase matching the duration of the channel opening. Moreover, these transients were only observed in the presence of sufficient extracellular Ca(super 2+), suggesting that they are due to Ca(super 2+) influx from the bathing solution. The fluorescence transient is characterized by an initial fast rising phase when the channel opens, followed by a slower rising phase during prolonged openings. When the channel closes there is an immediate fast falling phase followed by a slower falling phase. Computer simulations of the underlying events were used to interpret the time course of the transients. The rapid phases are mainly due to the establishment or removal of Ca(super 2+) and Ca(super 2+)-bound fluo-3 gradients near the channel when the channel opens or closes, while the slow phases are due to the diffusion of Ca(super 2+) and Ca(super 2+)-bound fluo-3 into the cytoplasm. Transients due to short channel openings have a 'Ca(super 2+) spark-like' appearance, suggesting that the rising and early falling components of sparks (due to openings of ryanodine receptors) reflect the fast phases of the fluorescence change. The results presented here suggest methods to determine the relationship between the fluorescence transient and the underlying Ca(super 2+) current, to study intracellular localized Ca(super 2+) handling as might occur from single Ca(super 2+) channel openings, and to localize Ca(super 2+) permeable ion channels on the plasma membrane. Keywords: calcium; caffeine-activated channel; nonselective cation channels; fluo-3 fluorescence; smooth muscle

Research was conducted to determine the partial structure of the yeast La protein and compare it with human protein. Structural analysis indicates that the domain of 113-amino acids, involved in the RNA-binding, differs from human La.

A Gram staining technique utilizing fluorescent nucleic acid binding dyes, hexidium iodide (HI) and SYTO 13, was developed for analyzing unfixed organisms in suspension by means of flow cytometry and epifluorescence microscopy. Microscopic analysis found gram-negative strains were stained red-orange by the HI, while gram-positive strains were dyed bright green by the SYTO 13. These results were confirmed by flow cytometric analysis. Quantitative assessments were made in terms of intensity and percentage of staining.

The use of inorganic membrane filters (Anopore) to measure and identify nanoplankton and picoplankton on both epifluorescence and scanning electron microscopes was found to be more effective compared to other filters. Results of this study showed enhanced image clarity and resolution with epifluorescence microscopy due to the flatness and large porosity of the Anopore filters. No charging or darkening of phytoplankton cells was found using scanning electron microscope due to the aluminum oxide composition of thefilter which provided a stable background for microscopy.

The method for the detection of localized calcium ion (Ca2+) concentration by epifluorescence microscopy was improved by modifying the nearest neighbors deblurring scheme toobtain ratiometric fura-2 images from mast cells. It was observed that the restored image is almost the same as that obtained with the nearest neighbors scheme when the in focus image is used in lieu of adjacent adjacent image planes for the deblurring. The improved method allows improved measurements of cytosolic-free Ca2+, which is involved in the regulation of several important biological processes.

The translocation of fluorescently tagged PLC[Gamma] and requirements for this process in cells stimulated with EGF were analyzed using real time fluorescence microscopy applied for the first time to monitor growth factor receptor--effector interactions. The translocation of PLC[Gamma] to the plasma membrane required the functional Src homology 2 domains and was not affected by mutations in the pleckstrin homology domain or inhibition of phosphatidylinositol (PI) 3-kinase. An array of domains specific for PLC[Gamma] isoforms was sufficient for this translocation. The dynamics of translocation to the plasma membrane and redistribution of PLC[Gamma], relative to localization of the EGF receptor and PI 4,5-biphosphate (PI 4,5-[P.sub.2]), were shown. Colocalization with the receptor was observed in the plasma membrane and in membrane ruffles where PI 4,5-[P.sub.2] substrate could also be visualized. At later times, internalization of PLC[Gamma], which could lead to separation from the substrate, was observed. The data support a direct binding of PLC[Gamma] to the receptor as the main site of the plasma membrane recruitment. The presence of PLC[Gamma] in membrane structures and its access to the substrate appear to be transient and are followed by a rapid incorporation into intracellular vesicles, leading to downregulation of the PLC activity. Key words: PLC * EGF receptor * translocation * real time imaging * SH2 and PH domains

Numerous reports have described how RNA polymerase II (RNAPII) enacts transcription of protein-coding genes and how this enzyme is regulated by a variety of cues, but, paradoxically, little is known [...]