Fluorescence Deconvolution Microscopy Research Articles

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal andadult cardiomyocytes

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes r esulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments reve aled that in both neonatal andcultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux.We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.

Specificity in intracellular protein aggregation and inclusion body formation.

Protein aggregation is widely considered to be a nonspecific coalescence of misfolded proteins, driven by interactions between solvent-exposed hydrophobic surfaces that are normally buried within a protein's interior. Accordingly, abnormal interactions between misfolded proteins with normal cellular constituents has been proposed to underlie the toxicity associated with protein aggregates in many neurodegenerative disorders. Here we have used fluorescence resonance energy transfer and deconvolution microscopy to investigate the degree to which unrelated misfolded proteins expressed in the same cells coaggregate with one another. Our data reveal that in cells, protein aggregation exhibits exquisite specificity even among extremely hydrophobic substrates expressed at very high levels.

The role of Drosophila CID in kinetochore formation, cell-cycle progression and heterochromatin interactions.

Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.

High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells

High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells

High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells.

High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha1(Cx43), beta1(Cx32), and beta2(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled, and packed normally into functional gap junction channel plaques. Such gap junction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we give a simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts.

High resolution, fluorescence deconvolution microscopy and tagging with the autofluorescent tracers CFP, GFP, and YFP to study the structural composition of gap junctions in living cells

High-resolution, fluorescence deconvolution (DV) microscopy was implemented to obtain a detailed view of the organization and structural composition of gap junctions assembled from one or two different connexin isotypes in live and fixed cells. To visualize gap junctions, the structural protein components of gap junction channels, the connexin polypeptides alpha1(Cx43), beta1(Cx32), and beta2(Cx26), were tagged on their C-termini with the autofluorescent tracers green fluorescent protein (GFP), and its cyan (CFP), and yellow (YFP) color variants. Tagged connexins were expressed in transiently transfected HeLa cells. Comprehensive analysis including dye-transfer analysis demonstrated that the tagged connexins trafficked, assembled, and packed normally into functional gap junction channel plaques. Such gap junction plaques were examined by single, dual, and triple-color DV microscopy. High-resolution images and three-dimensional volume reconstructions of gap junction plaques were obtained by this technique, which revealed several new aspects of gap junction structure. Specifically, the studies demonstrated that the mode of channel distribution strictly depends on the connexin isotypes. Here we present such images, and volume reconstructions in context with images obtained by other light, and electron microscopic techniques, such as laser scanning confocal, conventional wide-field fluorescence, thin section, and freeze-fracture electron microscopy. In addition, we give a simple description of the principal mechanisms of DV microscopy, name advantages and disadvantages, and discuss issues such as dual-color imaging using CFP and YFP, spatial resolution, colocalization, and avoiding imaging artifacts.

Connexin-specific distribution within gap junctions revealed in living cells

To study the organization of gap junctions in living cells, the connexin isotypes alpha(1)(Cx43), beta(1)(Cx32) and beta(2)(Cx26) were tagged with the autofluorescent tracer green fluorescent protein (GFP) and its cyan (CFP) and yellow (YFP) color variants. The cellular fate of the tagged connexins was followed by high-resolution fluorescence deconvolution microscopy and time-lapse imaging. Comprehensive analyses demonstrated that the tagged channels were functional as monitored by dye transfer, even under conditions where the channels were assembled solely from tagged connexins. High-resolution images revealed a detailed structural organization, and volume reconstructions provided a three-dimensional view of entire gap junction plaques. Specifically, deconvolved dual-color images of gap junction plaques assembled from CFP- and YFP-tagged connexins revealed that different connexin isotypes gathered within the same plaques. Connexins either codistributed homogeneously throughout the plaque, or each connexin isotype segregated into well-separated domains. The studies demonstrate that the mode of channel distribution strictly depends on the connexin isotypes. Based on previous studies on the synthesis and assembly of connexins I suggest that channel distribution is regulated by intrinsic connexin isotype specific signals.

Enterococcus faecalis bearing aggregation substance is resistant to killing by human neutrophils despite phagocytosis and neutrophil activation.

Enterococcus faecalis aggregation substance (AS) mediates efficient bacterium-bacterium contact to facilitate plasmid exchange as part of a bacterial sex pheromone system. We have previously determined that AS promotes direct, opsonin-independent binding of E. faecalis to human neutrophils (PMNs) via complement receptor type 3 and other receptors on the PMN surface. We have now examined the functional consequences of this bacterium-host cell interaction. AS-bearing E. faecalis was phagocytosed and internalized by PMNs, as determined by deconvolution fluorescence microscopy. However, these bacteria were not killed by PMNs, and internalized bacteria excluded propidium iodide, indicating intact bacterial membranes. Resistance to killing occurred despite activation of PMNs, as indicated by an increase in both functional and total surface Mac-1 expression, shedding of L-selectin, and an increase in PMN extracellular superoxide and phagosomal oxidant production. Deconvolution fluorescence microscopy also revealed that phagosomes containing AS-bearing bacteria were markedly larger than phagosomes containing opsonized E. faecalis, suggesting that some modification of phagosomal maturation may be involved in AS-induced resistance to killing. PMN phagosomal pH was significantly higher after ingestion of nonopsonized AS-bearing E. faecalis than after that of opsonized bacteria. The novel ability of AS to promote intracellular survival of E. faecalis inside PMNs suggests that AS may be a virulence factor used by strains of E. faecalis.

An evaluation of two‐photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in xenopus morphogenesis

The ability to visualize cell motility occurring deep in the context of opaque tissues will allow many currently intractable issues in developmental biology and organogenesis to be addressed. In this study, we compare two-photon excitation with laser scanning confocal and conventional digital deconvolution fluorescence microscopy, using the same optical configuration, for their ability to resolve cell shape deep in Xenopus gastrula and neurula tissues. The two-photon microscope offers better depth penetration and less autofluorescence compared to confocal and conventional deconvolution imaging. Both two-photon excitation and confocal microscopy also provide improved rejection of “out-of-focus” noise and better lateral and axial resolution than conventional digital deconvolution microscopy. Deep Xenopus cells are best resolved by applying the digital deconvolution method on the two-photon images. We have also found that the two-photon has better depth penetration without any degradation in the image quality of interior sections compared to the other two techniques. Also, we have demonstrated that the quality of the image changes at different depths for various excitation powers. Microsc. Res. Tech. 47:172–181, 1999. © 1999 Wiley-Liss, Inc.

14] Deconvolution fluorescence microscopy for observation and analysis of membrane biofilm architecture

This chapter provides a detailed description of a unique microscope system that permits both two-dimensional (2D) and three-dimensional (3D) viewing of membrane biofilms from any desired perspective. Several 3D image stacks may be acquired under different imaging (filtration) conditions and combined to produce a color-coded volume that simultaneously maps multiple biofilm features (e.g., dual or triple dye staining). The resulting models may be used to investigate specific biofilm features such as the distribution of specific microorganisms and extracellular polysaccharides, the nature of biofilm fluid channels, or the penetration of biocides or other molecules into the biofilm matrix. The chapter discusses real-time visualization and quantification of bacterial adhesion using microscope flow cells. Microscope flow cells (MFCs) of various design configurations have been developed to allow direct microscopic visualization and quantification of attached biofilm cells in real time under dynamic flow conditions.

The human malaria parasite Plasmodiumfalciparum exports the ATP-binding cassette protein PFGCN20 to membrane structures in the host red blood cell

PFGCN20 is a member of the ATP-binding cassette family of proteins that is closely related to the yeast translational regulator Gcn20p. We have generated a polyclonal antibody against the N-terminal region of PFGCN20 and studied the cellular localization of PFGCN20 throughout the erythrocytic life cycle of Plasmodium falciparum. PFGCN20 was found to be present at all stages and a pronounced export of PFGCN20 into the erythrocyte was observed in the trophozoite and schizont stages. In the indirect immunofluorescence assay, PFGCN20 was found to display significant colocalization with antigens detected by the monoclonal antibody 41E11. In contrast, there was only a minimal overlap of PFGCN20 localization with EMP2 and HRP2. Immunoelectron microscopy demonstrated a pronounced accumulation of PFGCN20 in the lumen of the parasitophorous vacuole and deconvolution fluorescence microscopy showed membrane association with selective regions of a tubovesicular network in the red cell. We also observed a concentration of PFGCN20 in electron-dense plaques just underneath the parasite’s plasma membrane and an association of PFGCN20 with cytoplasmic vesicular structures within the parasite. The observed export of PFGCN20 and its association with the tubovesicular network in host red cells, may be indicative of the fact that PFGCN20 functions as ATP-binding subunit of an unknown multimeric ABC-transporter. The cytoplasmic localization of PFGCN20 in the parasite, however, suggests that the involvement of PFGCN20 in translational regulation or other cytoplasmic biological functions cannot be ruled out.