Autofluorescence and Apoptosis in Flow Cytometry: Correlative Methods for Fluorescence and Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 618-619
Author(s):  
R. W. Smith
Keyword(s):  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Fluorescent Protein ◽  
Statistical Evaluation ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Apoptotic Cells ◽  
Chemical Probes ◽  
Clear Separation ◽  
Dying Cells

Flow cytometry offers several advantages in evaluating cellular fluorescence and separating values from chemical probes or transfected fluorescent proteins from autofluorescence. Autofluorescence can introduce artificial values that cannot be separated by examination of the fluorescence histogram. Often a comparison of fluorescence in two detectors can separate autofluorescence from positive fluorescence. A region can be defined around the cells of interest for further statistical evaluation or for subsequent sorting.Autofluorescence is also correlated with apoptosis in that apoptotic cells often increase autofluorescence values. A clear separation of living cells from dying cells can be achieved by the introduction of a DNA stain, such as 7AAD, which will penetrate cells with disrupted or damaged cellular membranes. Intact living cells will not show the 7AAD fluorescence.This separation is important in transfected fluorescent protein studies, as the transfection process is often toxic to cells.

Illuminating subcellular structures and dynamics in plants: a fluorescent protein toolboxThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 515-522 ◽  
Author(s):  
Preetinder K. Dhanoa ◽  
Alison M. Sinclair ◽  
Robert T. Mullen ◽  
Jaideep Mathur
Keyword(s):  
Plant Cell ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Live Imaging ◽  
Living Cells ◽  
Special Issue ◽  
Exciting Possibility ◽  
Selection Of

The discovery and development of multicoloured fluorescent proteins has led to the exciting possibility of observing a remarkable array of subcellular structures and dynamics in living cells. This minireview highlights a number of the more common fluorescent protein probes in plants and is a testimonial to the fact that the plant cell has not lagged behind during the live-imaging revolution and is ready for even more in-depth exploration.

scholarly journals Caspase Activity Is Required for Engulfment of Apoptotic Cells

2013 ◽  
Vol 33 (16) ◽  
pp. 3191-3201 ◽  
Author(s):  
Boris Shklyar ◽  
Flonia Levy-Adam ◽  
Ketty Mishnaevski ◽  
Estee Kurant
Keyword(s):  
Central Nervous System ◽  
Normal Development ◽  
Function Analysis ◽  
Living Cells ◽  
Apoptotic Cells ◽  
Caspase Activity ◽  
Internal Inhibition ◽  
Content Type ◽  
Multicellular Organisms ◽  
Dying Cells

Clearance of apoptotic cells by phagocytic neighbors is crucial for normal development of multicellular organisms. However, how phagocytes discriminate between healthy and dying cells remains poorly understood. We focus on glial phagocytosis of apoptotic neurons during development of theDrosophilacentral nervous system. We identified phosphatidylserine (PS) as a ligand on apoptotic cells for the phagocytic receptor Six Microns Under (SIMU) and report that PS alone is not sufficient for engulfment. Our data reveal that, additionally to PS exposure, caspase activity is required for clearance of apoptotic cells by phagocytes. Here we demonstrate that SIMU recognizes and binds PS on apoptotic cells through its N-terminal EMILIN (EMI), Nimrod 1 (NIM1), and NIM2 repeats, whereas the C-terminal NIM3 and NIM4 repeats control SIMU affinity to PS. Based on the structure-function analysis of SIMU, we discovered a novel mechanism of internal inhibition responsible for differential affinities of SIMU to its ligand which might prevent elimination of living cells exposing PS on their surfaces.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

1997 ◽  
Vol 3 (S2) ◽  
pp. 133-134 ◽  
Author(s):  
Ammasi Periasamy ◽  
Richard N. Day
Keyword(s):  
Transcription Factors ◽  
Living Cell ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Protein Localization ◽  
Resonance Energy ◽  
Living Cells ◽  
Specific Gene ◽  
Green Fluorescent ◽  
Prl Gene

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

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.

scholarly journals Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells

2008 ◽  
Vol 105 (46) ◽  
pp. 17789-17794 ◽  
Author(s):  
Gerard Marriott ◽  
Shu Mao ◽  
Tomoyo Sakata ◽  
Jing Ran ◽  
David K. Jackson ◽  
...  
Keyword(s):  
Optical Switching ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Optical Switch ◽  
Fluorescence Emission ◽  
Optical Probe ◽  
Living Cells ◽  
Cross Correlation Analysis ◽  
Total Fluorescence ◽  
Lock In

One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed “optical switches.” This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.

scholarly journals An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17896 ◽  
Author(s):  
Michele L. Markwardt ◽  
Gert-Jan Kremers ◽  
Catherine A. Kraft ◽  
Krishanu Ray ◽  
Paula J. C. Cranfill ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Site Directed Mutagenesis ◽  
Quantum Yields

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells

2009 ◽  
Vol 2009 (12) ◽  
pp. pdb.top64-pdb.top64 ◽  
Author(s):  
M. A. Rizzo ◽  
M. W. Davidson ◽  
D. W. Piston
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Protein Tracking

scholarly journals Fusion of a fluorescent protein to the pUL25 minor capsid protein of pseudorabies virus allows live-cell capsid imaging with negligible impact on infection

2012 ◽  
Vol 93 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Kevin P. Bohannon ◽  
Patricia J. Sollars ◽  
Gary E. Pickard ◽  
Gregory A. Smith
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Nuclear Dynamics ◽  
Infection Models ◽  
Simplex Virus

In order to resolve the location and activity of submicroscopic viruses in living cells, viral proteins are often fused to fluorescent proteins (FPs) and visualized by microscopy. In this study, we describe the fusion of FPs to three proteins of pseudorabies virus (PRV) that allowed imaging of capsids in living cells. Included in this study are the first recombinant PRV strains expressing FP–pUL25 fusions based on a design applied to herpes simplex virus type 1 by Homa and colleagues. The properties of each reporter virus were compared in both in vitro and in vivo infection models. PRV strains expressing FP–pUL25 and FP–pUL36 preserved wild-type properties better than traditional FP–pUL35 isolates in assays of plaque size and virulence in mice. The utility of these strains in studies of axon transport, nuclear dynamics and viral particle composition are documented.

scholarly journals Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

2003 ◽  
Vol 69 (11) ◽  
pp. 6848-6855 ◽  
Author(s):  
Yutao Fu ◽  
Charles O'Kelly ◽  
Michael Sieracki ◽  
Daniel L. Distel
Keyword(s):  
Flow Cytometry ◽  
Prey Species ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Heterotrophic Flagellate ◽  
Broad Host Range ◽  
Bacterial Cells ◽  
Protein Markers ◽  
Significant Difference ◽  
Paraphysomonas Imperforata

ABSTRACT Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.

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