scholarly journals Estimating numbers of intracellular molecules through analysing fluctuations in photobleaching

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Wide Field ◽  
Order Of Magnitude ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact ◽  
Quantitative Fluorescence

Abstract The impact of fluorescence microscopy has been limited by the difficulties of expressing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we propose an estimator to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. Performing experiments in budding yeast, we show that our estimates of numbers agree, within an order of magnitude, with published biochemical measurements, for all six proteins tested. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

scholarly journals Estimating numbers of fluorescent molecules in single cells by analysing fluctuations in photobleaching

2018 ◽  
Author(s):  
Elco Bakker ◽  
Peter S. Swain
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Measurement Noise ◽  
Wide Field ◽  
Stochastic Fluctuations ◽  
Biochemical Measurements ◽  
Green Fluorescent ◽  
The Impact

The impact of fluorescence microscopy has been limited by the difficulties of express-ing measurements of fluorescent proteins in numbers of molecules. Absolute numbers enable the integration of results from different laboratories, empower mathematical modelling, and are the bedrock for a quantitative, predictive biology. Here we develop a general algorithm to infer numbers of molecules from fluctuations in the photobleaching of proteins tagged with Green Fluorescent Protein. To untangle measurement noise from stochastic fluctuations, we use the linear noise approximation and Kalman filtering within a framework of Bayesian inference. Not only do our results agree with biochemical measurements for multiple proteins in budding yeast, but we also provide a statistically verified model of measurement noise for fluorescence microscopes. The experiments we require are straightforward and use only a wide-field fluorescence microscope. As such, our approach has the potential to become standard for those practising quantitative fluorescence microscopy.

scholarly journals Influence of nanobody binding on fluorescence emission, mobility and organization of GFP-tagged proteins

2020 ◽  
Author(s):  
Falk Schneider ◽  
Christian Eggeling ◽  
Erdinc Sezgin
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Fluorescence Emission ◽  
Fluorescence Properties ◽  
Great Care ◽  
Target Molecules ◽  
Green Fluorescent

SummaryAdvanced fluorescence microscopy studies require specific and monovalent molecular labelling with bright and photostable fluorophores. This necessity led to the widespread use of fluorescently labelled nanobodies against commonly employed fluorescent proteins. However, very little is known how these nanobodies influence their target molecules. Here, we observed clear changes of the fluorescence properties, mobility and organisation of green fluorescent protein (GFP) tagged proteins after labelling with an anti-GFP nanobody. Intriguingly, we did not observe any co-diffusion of fluorescently-labelled nanobodies with the GFP-labelled proteins. Our results suggest significant binding of the nanobodies to a non-emissive, oligomerized form of the fluorescent proteins, promoting disassembly into more monomeric forms after binding. Our findings show that great care must be taken when using nanobodies for studying dynamic and quantitative protein organisation.

scholarly journals Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy

1997 ◽  
Vol 73 (5) ◽  
pp. 2782-2790 ◽  
Author(s):  
G.H. Patterson ◽  
S.M. Knobel ◽  
W.D. Sharif ◽  
S.R. Kain ◽  
D.W. Piston
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
Quantitative Fluorescence

scholarly journals Phagocytosed Bordetella pertussis Fails To Survive in Human Neutrophils

2000 ◽  
Vol 68 (2) ◽  
pp. 956-959 ◽  
Author(s):  
Derrick H. Lenz ◽  
Christine L. Weingart ◽  
Alison A. Weiss
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Bordetella Pertussis ◽  
Fluorescent Protein ◽  
Survival Rates ◽  
Polymyxin B ◽  
Immune Serum ◽  
Human Neutrophils ◽  
Green Fluorescent

ABSTRACT Previous studies have reported that phagocytosed Bordetella pertussis survives in human neutrophils. This issue has been reexamined. Opsonized or unopsonized bacteria expressing green fluorescent protein (GFP) were incubated with adherent human neutrophils. Phagocytosis was quantified by fluorescence microscopy, and the viability of phagocytosed bacteria was determined by colony counts following treatment with polymyxin B to kill extracellular bacteria. Only 1 to 2% of the phagocytosed bacteria remained viable. Opsonization with heat-inactivated immune serum reduced the amount of attachment and phagocytosis of the bacteria but did not alter survival rates. In contrast to previous reports, these data suggest that phagocytosed B. pertussis bacteria are killed by human neutrophils.

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

ACS Nano ◽  
2015 ◽  
Vol 9 (10) ◽  
pp. 9528-9541 ◽  
Author(s):  
Sam Duwé ◽  
Elke De Zitter ◽  
Vincent Gielen ◽  
Benjamien Moeyaert ◽  
Wim Vandenberg ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

scholarly journals Constitutive and Inducible Expression of Green Fluorescent Protein in Brucella suis

1999 ◽  
Vol 67 (12) ◽  
pp. 6695-6697 ◽  
Author(s):  
Stephan Köhler ◽  
Safia Ouahrani-Bettache ◽  
Marion Layssac ◽  
Jacques Teyssier ◽  
Jean-Pierre Liautard
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Fluorescence Microscopy ◽  
Gene Fusion ◽  
Fluorescent Protein ◽  
Fusion System ◽  
Chromosomal Dna ◽  
Brucella Suis ◽  
Green Fluorescent ◽  
Inducible Genes

ABSTRACT A gene fusion system based on plasmid pBBR1MCS and the expression of green fluorescent protein was developed for Brucella suis, allowing isolation of constitutive and inducible genes. Bacteria containing promoter fusions of chromosomal DNA togfp were visualized by fluorescence microscopy and examined by flow cytometry. Twelve clones containing gene fragments induced inside J774 murine macrophages were isolated and further characterized.

scholarly journals Relationship between Acropora millepora juvenile fluorescence and composition of newly established Symbiodinium assemblage

2018 ◽  
Author(s):  
KM Quigley ◽  
ME Strader ◽  
MV Matz
Keyword(s):  
Life Cycle ◽  
Green Fluorescent Protein ◽  
Coral Reefs ◽  
Deep Sequencing ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Biological Interaction ◽  
Acropora Millepora ◽  
Continuous Trait ◽  
Green Fluorescent

AbstractCoral-dinoflagellate symbiosis is the key biological interaction enabling existence of modern-type coral reefs, but the mechanisms regulating initial host–symbiont attraction, recognition and symbiont proliferation thus far remain largely unclear. A common reef-building coral, Acropora millepora, displays conspicuous fluorescent polymorphism during all phases of its life cycle, due to the differential expression of fluorescent proteins (FPs) of the green fluorescent protein family. In this study, we examine whether fluorescent variation in young coral juveniles exposed to natural sediments is associated with the uptake of disparate Symbiodinium assemblages determined using ITS-2 deep sequencing. We found that Symbiodinium assemblages varied significantly when redness values varied, specifically in regards to abundances of clades A and C. Whether fluorescence was quantified as a categorical or continuous trait, clade A was found at higher abundances in redder juveniles. These preliminary results suggest juvenile fluorescence may be associated with Symbiodinium uptake, potentially acting as either as an attractant to ecologically specific types or as a mechanism to modulate the internal light environment to control Symbiodinium physiology within the host.

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