CONFOCAL MICROSCOPY STUDIES OF LIVING FUNGAL HYPHAE AND CONIDIA USING RHENIUM (I) TRICARBONYL COMPLEXES AS FLUORESCENT DYES.

Polymeric microcapsules with the fungal laccase from Pycnoporus sanguineus CS43 may represent an attractive avenue for the removal or degradation of dyes from wastewaters. Microcapsules of alginate/chitosan (9.23 ± 0.12 µm) and poly(styrenesulfonate) (PSS) (9.25 ± 0.35 µm) were synthesized and subsequently tested for catalytic activity in the decolorization of the diazo dye Congo Red. Successful encapsulation into the materials was verified via confocal microscopy of labeled enzyme molecules. Laccase activity was measured as a function of time and the initial reaction rates were recovered for each preparation, showing up to sevenfold increase with respect to free laccase. The ability of substrates to diffuse through the pores of the microcapsules was evaluated with the aid of fluorescent dyes and confocal microscopy. pH and thermal stability were also measured for encapsulates, showing catalytic activity for pH values as low as 4 and temperatures of about 80 °C. Scanning electron microscope (SEM) analyses demonstrated the ability of PSS capsules to avoid accumulation of byproducts and, therefore, superior catalytic performance. This was corroborated by the direct observation of substrates diffusing in and out of the materials. Compared with our PSS preparation, alginate/chitosan microcapsules studied by others degrade 2.6 times more dye, albeit with a 135-fold increase in units of enzyme per mg of dye. Similarly, poly(vinyl) alcohol microcapsules from degrade 1.7 times more dye, despite an eightfold increase in units of enzyme per mg of dye. This could be potentially beneficial from the economic viewpoint as a significantly lower amount of enzyme might be needed for the same decolorization level achieved with similar encapsulated systems.

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A Rapid Method for Combined Laser Scanning Confocal Microscopic and Electron Microscopic Visualization of Biocytin or Neurobiotin-labeled Neurons

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549804600216 ◽

1998 ◽

Vol 46 (2) ◽

pp. 263-273 ◽

Cited By ~ 10

Author(s):

Xue J. Sun ◽

Leslie P. Tolbert ◽

John G. Hildebrand ◽

Ian A. Meinertzhagen

Keyword(s):

Confocal Microscopy ◽

Laser Scanning ◽

Fluorescent Dyes ◽

Laser Scanning Confocal Microscopy ◽

Single Step ◽

Three Dimensions ◽

Fluorescent Label ◽

Electron Microscopic ◽

Scanning Confocal Microscopy ◽

Long Term Storage

Intracellular recording and dye filling are widely used to correlate the morphology of a neuron with its physiology. With laser scanning confocal microscopy, the complex shapes of labeled neurons in three dimensions can be reconstructed rapidly, but this requires fluorescent dyes. These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a technique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is filled with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable electron-dense material by a single-step conversion using a commercially available ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin–peroxidase conjugate, is both faster and less labor intensive than photo-oxidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate using anti-serotonin as an example.

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DNA Staining for Fluorescence and Laser Confocal Microscopy

Journal of Histochemistry & Cytochemistry ◽

10.1177/002215549704500107 ◽

1997 ◽

Vol 45 (1) ◽

pp. 49-53 ◽

Cited By ~ 218

Author(s):

Takeshi Suzuki ◽

Keiko Fujikura ◽

Tetsuya Higashiyama ◽

Kuniaki Takata

Keyword(s):

Confocal Microscopy ◽

Nuclear Dna ◽

Fluorescent Dyes ◽

Sybr Green ◽

Sybr Green I ◽

Nuclear Staining ◽

Nucleic Acid Binding ◽

Laser Confocal Microscopy ◽

Dna Staining ◽

Cytoplasmic Rna

We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

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New Lipophilic Fluorescent Dyes for Exosome Labeling: Monitoring of Cellular Uptake of Exosomes

10.1101/2020.02.02.931295 ◽

2020 ◽

Cited By ~ 1

Author(s):

Takashi Shimomura ◽

Ryo Seino ◽

Kaori Umezaki ◽

Asako Shimoda ◽

Takatoshi Ezoe ◽

...

Keyword(s):

Confocal Microscopy ◽

Zeta Potential ◽

Cellular Uptake ◽

Fluorescent Probes ◽

Fluorescent Dyes ◽

Live Cells ◽

Nanoparticle Tracking Analysis ◽

Excellent Performance ◽

Potential Measurement ◽

Highly Sensitive

ABSTRACTWe have developed three types of exosomal membrane binding fluorescent probes, Mem Dye-Green, Mem Dye-Red and Mem Dye-Deep Red, to monitor exosome uptake into cells. The dyes contain a cyanine group as a fluorescent scaffold, which allows for highly sensitive fluorescence imaging of the exosome. These dyes can also be used to observe the dynamics of exosomes in live cells. The use of PKH dyes (Figure 1), which are currently the most widely-used fluorescent probes for exosome labeling, has some limitations. For example, PKH dyes tend to aggregate to form exosome-like nanoparticles, and these nanoparticles are uptaken by cells. Moreover, Mehdi suggested that the use of PKH dyes triggers an enlargement of the exosome size owing to membrane fusion or intercalation. To overcome the limitations of PKH dyes, we introduce amphiphilic moieties to the cyanine. To investigate the characteristics of the Mem Dyes as exosome labeling probes, we perform nanoparticle tracking analysis (NTA), zeta potential measurement and confocal microscopy. The Mem Dyes show excellent performance for exosome labeling (no aggregation and less size shift).

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Understanding the role of seawater vacuolisation in the biomineralisation of planktonic foraminifera using confocal microscopy

10.5194/egusphere-egu2020-18090 ◽

2020 ◽

Author(s):

David Evans ◽

Jonathan Erez ◽

Wolfgang Müller

Keyword(s):

Confocal Microscopy ◽

Fluorescent Dyes ◽

Dominant Role ◽

Planktonic Foraminifera ◽

Future Climate Change ◽

Geochemical Data ◽

Globigerinoides Ruber ◽

Seawater Chemistry ◽

Surface Ocean

The calcite shells of planktonic foraminfera are a key archive for palaeoceanic reconstruction and represent one of the largest sinks of carbon from the surface ocean. Therefore, understanding the biomineralisation process of these organisms, and how responsive it is to ocean acidification, is an important part of accurately predicting the carbon cycle response to past and future climate change events. To date, the majority of the direct observational evidence on which foraminifera biomineralisation models are based comes from shallow-dwelling benthic species. Whilst this has provided a large amount of important information, it is not known how applicable these models are to the low-Mg planktonic foraminifera. In particular, key questions regarding the relative importance of seawater vacuolisation (SWV) versus calcium transmembrane transport (TMT) remain unresolved. We present the results of fluorescent labelling experiments on intact, decalcified planktonic foraminifera (Globigerinoides ruber and Globigerinella siphonifera) using the cell-impermeable dyes calcein, FITC-dextran, and SNARF-dextran, enabling direct observation of seawater vacuoles within the cell via confocal microscopy. Our results indicate that seawater endocytosis plays a dominant role in the calcification process. Seawater vacuoles can make up a large proportion of the intracellular volume, with a residence time on the order of hours. Moreover, we show that the skeleton is labelled with fluorescent dyes such that seawater derived from these vacuoles must be present at the calcification site. Along with inferences based on geochemical data [Evans et al., 2018], our results strongly argue that biomineralisation models centred on seawater endocytosis are applicable to the planktonic foraminifera.Evans, D., Erez, J., M&#252;ller, W. [2018] Assessing foraminifera biomineralisation models through trace element data of cultures under variable seawater chemistry. GCA 236:198.

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A Multiscale Approach to Multi-Component Wetting

Fluids Engineering ◽

10.1115/imece2004-61219 ◽

2004 ◽

Author(s):

Anne M. Grillet ◽

Benjamin J. Ash ◽

Carlton F. Brooks ◽

John A. Emerson

Keyword(s):

Confocal Microscopy ◽

Laser Scanning ◽

Fluorescent Dyes ◽

Laser Scanning Confocal Microscopy ◽

Multiscale Approach ◽

Local Concentration ◽

Proof Of Concept ◽

Scanning Confocal Microscopy ◽

Air Interface ◽

Spectral Separation

Laser scanning confocal microscopy has been applied to study segregation in multi-component wetting. By labeling the two components of a blend with contrasting fluorescent dyes, the approximate local concentration can be determined from the relative fluorescence intensities. As a proof of concept, a coarsely blended mixture was imaged and parameters were adjusted to achieve good spectral separation of the two components. The technique was then applied to a well-blended drop of the two components and one component was observed to segregate to the air interface.

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Liposome Sterile Filtration Characterization via X-ray Computed Tomography and Confocal Microscopy

Membranes ◽

10.3390/membranes11110905 ◽

2021 ◽

Vol 11 (11) ◽

pp. 905

Author(s):

Thomas F. Johnson ◽

Kyle Jones ◽

Francesco Iacoviello ◽

Stephen Turner ◽

Nigel B. Jackson ◽

...

Keyword(s):

Computed Tomography ◽

Confocal Microscopy ◽

Fluorescent Dyes ◽

Imaging Techniques ◽

Flow Simulation ◽

Average Diameter ◽

Operating Conditions ◽

X Ray ◽

X Ray Computed ◽

And Performance

Two high resolution, 3D imaging techniques were applied to visualize and characterize sterilizing grade dual-layer filtration of liposomes, enabling membrane structure to be related with function and performance. Two polyethersulfone membranes with nominal retention ratings of 650 nm and 200 nm were used to filter liposomes of an average diameter of 143 nm and a polydispersity index of 0.1. Operating conditions including differential pressure were evaluated. X-ray computed tomography at a pixel size of 63 nm was capable of resolving the internal geometry of each membrane. The respective asymmetry and symmetry of the upstream and downstream membranes could be measured, with pore network modeling used to identify pore sizes as a function of distance through the imaged volume. Reconstructed 3D digital datasets were the basis of tortuous flow simulation through each porous structure. Confocal microscopy visualized liposome retention within each membrane using fluorescent dyes, with bacterial challenges also performed. It was found that increasing pressure drop from 0.07 MPa to 0.21 MPa resulted in differing fluorescent retention profiles in the upstream membrane. These results highlighted the capability for complementary imaging approaches to deepen understanding of liposome sterilizing grade filtration.

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Confocal microscopy analysis reveals that only a small proportion of extracellular vesicles are successfully labelled with commonly utilised staining methods

Scientific Reports ◽

10.1038/s41598-021-04225-4 ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Genevieve E. Melling ◽

Ross Conlon ◽

Paschalia Pantazi ◽

Elizabeth R. Dellar ◽

Priya Samuel ◽

...

Keyword(s):

Confocal Microscopy ◽

Fluorescent Dyes ◽

Extracellular Vesicle ◽

Size Exclusion ◽

Functional Roles ◽

Transmission Electron ◽

Exclusion Chromatography ◽

Staining Methods ◽

Bona Fide ◽

Uptake Studies

AbstractAssessing genuine extracellular vesicle (EV) uptake is crucial for understanding the functional roles of EVs. This study measured the bona fide labelling of EVs utilising two commonly used fluorescent dyes, PKH26 and C5-maleimide-Alexa633. MCF7 EVs tagged with mEmerald-CD81 were isolated from conditioned media by size exclusion chromatography (SEC) and characterised using Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM), MACsPlex immunocapture assay and immunoblots. These fluorescently tagged EVs were subsequently stained with C5-maleimide-Alexa633 or PKH26, according to published protocols. Colocalisation of dual-labelled EVs was assessed by confocal microscopy and quantified using the Rank-Weighted Colocalisation (RWC) algorithm. We observed strikingly poor colocalisation between mEmerald-CD81-tagged EVs and C5-Maleimide-Alexa633 (5.4% ± 1.8) or PKH26 (4.6% ± 1.6), that remained low even when serum was removed from preparations. Our data confirms previous work showing that some dyes form contaminating aggregates. Furthermore, uptake studies showed that maleimide and mEmerald-CD81-tagged EVs can be often located into non-overlapping subcellular locations. By using common methods to isolate and stain EVs we observed that most EVs remained unstained and most dye signal does not appear to be EV associated. Our work shows that there is an urgent need for optimisation and standardisation in how EV researchers use these tools to assess genuine EV signals.

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