An effective signal amplifying strategy for copper (II) sensing by using in situ fluorescent proteins as energy donor of FRET

2018 ◽  
Vol 259 ◽  
pp. 633-641 ◽  
Author(s):  
Qianchen Zhang ◽  
Duoduo Zhao ◽  
Chengwu Zhang ◽  
Jinhua Liu ◽  
Zhongfu An ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Energy Donor ◽  
Effective Signal

scholarly journals Using Scuba for In Situ Determination of Chlorophyll Distributions in Corals by Underwater Near Infrared Fluorescence Imaging

2020 ◽  
Vol 8 (1) ◽  
pp. 53
Author(s):  
Thomas Oh ◽  
Jittiwat Sermsripong ◽  
Barry W. Hicks
Keyword(s):  
Fluorescence Imaging ◽  
Large Scale ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Fluorescence Emission ◽  
Florida Keys ◽  
Nir Fluorescence ◽  
Novel Method ◽  
Near Infrared Fluorescence Imaging

Studies reporting quantitation and imaging of chlorophyll in corals using visible fluorescent emission in the red near 680 nm can suffer from competing emission from other red-emitting pigments. Here, we report a novel method of selectively imaging chlorophyll distributions in coral in situ using only the near infrared (NIR) fluorescence emission from chlorophyll. Commercially available equipment was assembled that allowed the sequential imaging of visible, visible-fluorescent, and NIR-fluorescent pigments on the same corals. The relative distributions of chlorophyll and fluorescent proteins (GFPs) were examined in numerous corals in the Caribbean Sea, the Egyptian Red Sea, the Indonesian Dampier Strait, and the Florida Keys. Below 2 m depth, solar induced NIR chlorophyll fluorescence can be imaged in daylight without external lighting, thus, it is much easier to do than visible fluorescence imaging done at night. The distributions of chlorophyll and GFPs are unique in every species examined, and while there are some tissues where both fluorophores are co-resident, often tissues are selectively enriched in only one of these fluorescent pigments. Although laboratory studies have clearly shown that GFPs can be photo-protective, their inability to prevent large scale bleaching events in situ may be due to their limited tissue distribution.

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

scholarly journals PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergey Gorelick ◽  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Travis K Johnson ◽  
Ava Handley ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Fluorescent Proteins ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Ion Beam ◽  
Correlative Microscopy ◽  
New Approach ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

scholarly journals Role of Ubiquitin and Proteasomes in Phagosome Maturation

2005 ◽  
Vol 16 (4) ◽  
pp. 2077-2090 ◽  
Author(s):  
Warren L. Lee ◽  
Moo-Kyung Kim ◽  
Alan D. Schreiber ◽  
Sergio Grinstein
Keyword(s):  
Flow Cytometry ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Continuous Distribution ◽  
Endocytic Pathway ◽  
Phagosome Maturation ◽  
Fcγ Receptors ◽  
Acidic Compartment

Phagosomes undergo multiple rounds of fusion with compartments of the endocytic pathway during the course of maturation. Despite the insertion of vast amounts of additional membrane, the phagosomal surface area remains approximately constant, implying active ongoing fission. To investigate the mechanisms underlying phagosomal fission we monitored the fate of Fcγ receptors (FcγR), which are known to be cleared from the phagosome during maturation. FcγR, which show a continuous distribution throughout the membrane of nascent phagosomes were found at later times to cluster into punctate, vesicular structures, before disappearing. In situ photoactivation of receptors tagged with a light-sensitive fluorescent protein revealed that some of these vesicles detach, whereas others remain associated with the phagosome. By fusing FcγR to pH-sensitive fluorescent proteins, we observed that the cytoplasmic domain of the receptors enters an acidic compartment, indicative of inward budding and formation of multivesicular structures. The topology of the receptor was confirmed by flow cytometry of purified phagosomes. Phagosomal proteins are ubiquitylated, and ubiquitylation was found to be required for formation of acidic multivesicular structures. Remarkably, proteasomal function is also involved in the vesiculation process. Preventing the generation of multivesicular structures did not impair the acquisition of late endosomal and lysosomal markers, indicating that phagosomal fusion and fission are controlled separately.

Imaging pancreatic β-cells in the intact pancreas

2006 ◽  
Vol 290 (5) ◽  
pp. E1041-E1047 ◽  
Author(s):  
Manami Hara ◽  
Restituto F. Dizon ◽  
Benjamin S. Glick ◽  
Catherine S. Lee ◽  
Klaus H. Kaestner ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Confocal Imaging ◽  
Β Cells ◽  
Β Cell ◽  
Disease States ◽  
Adult Mice ◽  
And Function

We have developed a method to visualize fluorescent protein-labeled β-cells in the intact pancreas through combined reflection and confocal imaging. This method provides a 3-D view of the β-cells in situ. Imaging of the pancreas from mouse insulin I promoter (MIP)-green (GFP) and red fluorescent protein (RFP) transgenic mice shows that islets, β-cell clusters, and single β-cells are not evenly distributed but are aligned along the large blood vessels. We also observe the solitary β-cells in both fetal and adult mice and along the pancreatic and common bile ducts. We have imaged the developing endocrine cells in the embryos using neurogenin-3 (Ngn3)-GFP mice crossed with MIP-RFP mice. The dual-color-coded pancreas from embryos (E15.5) shows a large number of green Ngn3-expressing proendocrine cells with a smaller number of red β-cells. The imaging technique that we have developed, coupled with the transgenic mice in which β-cells and β-cell progenitors are labeled with different fluorescent proteins, will be useful for studying pancreatic development and function in normal and disease states.

scholarly journals Preparation of long single-strand DNA concatemers for high-level fluorescence in situ hybridization

2021 ◽  
Author(s):  
Dongjian Cao ◽  
Sa Wu ◽  
Caili Xi ◽  
Dong Li ◽  
Kaiheng Zhu ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Proteins ◽  
Signal To Noise Ratio ◽  
Rolling Circle Amplification ◽  
Simultaneous Detection ◽  
Single Strand ◽  
Detection Methods ◽  
Rolling Circle ◽  
High Level

Abstract Fluorescence in situ hybridization (FISH) is a powerful tool to visualize transcripts in fixed cells and tissues. Despite the recent advances in FISH detection methods, it remains challenging to achieve high-level FISH imaging with a simple workflow. Here, we introduce a method to prepare long single-strand DNA concatemers (lssDNAc) through a controllable rolling-circle amplification (CRCA). Prepared lssDNAcs were used to develop novel AmpFISH workflows. In addition, we present its applications in different scenarios. AmpFISH shows the following advantages: 1) enhanced FISH signal-to-noise ratio (SNR) up to 160-fold compared with single-molecule FISH; 2) simultaneous detection of FISH signals and fluorescent proteins or immunofluorescence (IF) in tissues; 3) simple workflows; and 4) cost-efficiency. In brief, AmpFISH provides convenient and versatile tools for sensitive RNA/DNA detection and to gain useful information on cellular molecules using simple workflows.

scholarly journals Effect of Signal Compounds and Incubation Conditions on the Culturability of Freshwater Bacterioplankton

2003 ◽  
Vol 69 (4) ◽  
pp. 1980-1989 ◽  
Author(s):  
Alke Bruns ◽  
Ulrich Nübel ◽  
Heribert Cypionka ◽  
Jörg Overmann
Keyword(s):  
In Situ Hybridization ◽  
Fluorescence In Situ Hybridization ◽  
Eutrophic Lake ◽  
Homoserine Lactone ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Probable Number ◽  
Culture Independent ◽  
Effective Signal

ABSTRACT The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months. Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 μM) of the signal compounds N-(oxohexanoyl)-dl-homoserine lactone, N-(butyryl)-dl-homoserine lactone, cyclic AMP (cAMP), or ATP. cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts. Microautoradiography with [2,8-3H]cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells. The bacterial cAMP uptake systems had a very low Km value of ≤1 nM. Analysis of the cultured bacteria by 16S rRNA gene fingerprinting showed that different bacterial phylotypes were recovered in the presence and in the absence of cAMP. Consequently, the addition of cAMP caused a stimulation of otherwise nonculturable bacteria. Phylogenetically different bacteria were also recovered at different temperatures and oxygen partial pressures. Throughout the study period, mainly members of the β-subclass of the Proteobacteria were cultivated. In addition, some members of the Actinomycetales were enriched. Quantification by culture-independent fluorescence in situ hybridization demonstrated that β-Proteobacteria and Actinomycetales also dominated the natural bacterioplankton assemblage. Sequence comparison revealed that two members of the Actinomycetales which reached high numbers in the natural bacterioplankton assemblage could actually be enriched by our cultivation approach.

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