The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Philippe I H Bastiaens - One of the best experts on this subject based on the ideXlab platform.
-
Imaging in situ protein dna interactions in the cell nucleus using fret flim
Experimental Cell Research, 2005Co-Authors: Frederic Cremazy, Philippe I H Bastiaens, Erik M M Manders, Gertjan Kramer, Gordon L Hager, Erik B Van Munster, Pernette J Verschure, Theodorus W J Gadella, Roel Van DrielAbstract:Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows Imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime Imaging Microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1α and HP1β.
-
improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime Imaging Microscopy data
Journal of Microscopy, 2001Co-Authors: Peter J Verveer, Anthony Squire, Philippe I H BastiaensAbstract:The deconvolution of fluorescence lifetime Imaging Microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.
-
quantitative Imaging of lateral erbb1 receptor signal propagation in the plasma membrane
Science, 2000Co-Authors: Peter J Verveer, Andrew R Reynolds, Fred S. Wouters, Philippe I H BastiaensAbstract:Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)–tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative Imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime Imaging Microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.
-
quantitative Imaging of lateral erbb1 receptor signal propagation in the plasma membrane
Science, 2000Co-Authors: Peter J Verveer, Andrew R Reynolds, Fred S. Wouters, Philippe I H BastiaensAbstract:Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)–tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative Imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime Imaging Microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.
-
global analysis of fluorescence lifetime Imaging Microscopy data
Biophysical Journal, 2000Co-Authors: Peter J Verveer, Anthony Squire, Philippe I H BastiaensAbstract:Global analysis techniques are described for frequency domain fluorescence lifetime Imaging Microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.
Peter J Verveer - One of the best experts on this subject based on the ideXlab platform.
-
improved spatial discrimination of protein reaction states in cells by global analysis and deconvolution of fluorescence lifetime Imaging Microscopy data
Journal of Microscopy, 2001Co-Authors: Peter J Verveer, Anthony Squire, Philippe I H BastiaensAbstract:The deconvolution of fluorescence lifetime Imaging Microscopy (FLIM) data that were processed with global analysis techniques is described. Global analysis of FLIM data enables the determination of relative numbers of molecules in different protein reaction states on a pixel-by-pixel basis in cells. The three-dimensional fluorescence distributions of each protein state can then be calculated and deconvolved. High-resolution maps of the relative concentrations of each state are then obtained from the deconvolved images. We applied these techniques to quantitatively image the phosphorylation state of ErbB1 receptors tagged with green fluorescent protein in MCF7 cells.
-
quantitative Imaging of lateral erbb1 receptor signal propagation in the plasma membrane
Science, 2000Co-Authors: Peter J Verveer, Andrew R Reynolds, Fred S. Wouters, Philippe I H BastiaensAbstract:Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)–tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative Imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime Imaging Microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.
-
quantitative Imaging of lateral erbb1 receptor signal propagation in the plasma membrane
Science, 2000Co-Authors: Peter J Verveer, Andrew R Reynolds, Fred S. Wouters, Philippe I H BastiaensAbstract:Evidence for a new signaling mechanism consisting of ligand-independent lateral propagation of receptor activation in the plasma membrane is presented. We visualized the phosphorylation of green fluorescent protein (GFP)–tagged ErbB1 (ErbB1-GFP) receptors in cells focally stimulated with epidermal growth factor (EGF) covalently attached to beads. This was achieved by quantitative Imaging of protein reaction states in cells by fluorescence resonance energy transfer (FRET) with global analysis of fluorescence lifetime Imaging Microscopy (FLIM) data. The rapid and extensive propagation of receptor phosphorylation over the entire cell after focal stimulation demonstrates a signaling wave at the plasma membrane resulting in full activation of all receptors.
-
global analysis of fluorescence lifetime Imaging Microscopy data
Biophysical Journal, 2000Co-Authors: Peter J Verveer, Anthony Squire, Philippe I H BastiaensAbstract:Global analysis techniques are described for frequency domain fluorescence lifetime Imaging Microscopy (FLIM) data. These algorithms exploit the prior knowledge that only a limited number of fluorescent molecule species whose lifetimes do not vary spatially are present in the sample. Two approaches to implementing the lifetime invariance constraint are described. In the lifetime invariant fit method, each image in the lifetime image sequence is spatially averaged to obtain an improved signal-to-noise ratio. The lifetime estimations from these averaged data are used to recover the fractional contribution to the steady-state fluorescence on a pixel-by-pixel basis for each species. The second, superior, approach uses a global analysis technique that simultaneously fits the fractional contributions in all pixels and the spatially invariant lifetimes. In frequency domain FLIM the maximum number of lifetimes that can be fit with the global analysis method is twice the number of lifetimes that can be fit with conventional approaches. As a result, it is possible to discern two lifetimes with a single-frequency FLIM setup. The algorithms were tested on simulated data and then applied to separate the cellular distributions of coexpressed green fluorescent proteins in living cells.
Brian J Bacskai - One of the best experts on this subject based on the ideXlab platform.
-
selective plane illumination Microscopy spim with time domain fluorescence lifetime Imaging Microscopy flim for volumetric measurement of cleared mouse brain samples
Review of Scientific Instruments, 2018Co-Authors: Anand Kumar, Oksana Berezovska, Tsukasa Funane, Katarzyna Marta Zoltowska, Susanne J Van Veluw, Brian J BacskaiAbstract:We have developed an Imaging technique which combines selective plane illumination Microscopy with time-domain fluorescence lifetime Imaging Microscopy (SPIM-FLIM) for three-dimensional volumetric Imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Forster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.We have developed an Imaging technique which combines selective plane illumination Microscopy with time-domain fluorescence lifetime Imaging Microscopy (SPIM-FLIM) for three-dimensional volumetric Imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Forster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used ...
-
selective plane illumination Microscopy spim with time domain fluorescence lifetime Imaging Microscopy flim for volumetric measurement of cleared mouse brain samples
Review of Scientific Instruments, 2018Co-Authors: Tsukasa Funane, Anand Kumar, Oksana Berezovska, Katarzyna Marta Zoltowska, Steven S Hou, Susanne J Van Veluw, Brian J BacskaiAbstract:We have developed an Imaging technique which combines selective plane illumination Microscopy with time-domain fluorescence lifetime Imaging Microscopy (SPIM-FLIM) for three-dimensional volumetric Imaging of cleared mouse brains with micro- to mesoscopic resolution. The main features of the microscope include a wavelength-adjustable pulsed laser source (Ti:sapphire) (near-infrared) laser, a BiBO frequency-doubling photonic crystal, a liquid chamber, an electrically focus-tunable lens, a cuvette based sample holder, and an air (dry) objective lens. The performance of the system was evaluated with a lifetime reference dye and micro-bead phantom measurements. Intensity and lifetime maps of three-dimensional human embryonic kidney (HEK) cell culture samples and cleared mouse brain samples expressing green fluorescent protein (GFP) (donor only) and green and red fluorescent protein [positive Forster (fluorescence) resonance energy transfer] were acquired. The results show that the SPIM-FLIM system can be used for sample sizes ranging from single cells to whole mouse organs and can serve as a powerful tool for medical and biological research.
-
fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime Imaging Microscopy to characterize amyloid beta plaques
Journal of Biomedical Optics, 2003Co-Authors: Brian J Bacskai, Jesse Skoch, Gregory A Hickey, Racquel Allen, Bradley T HymanAbstract:We describe the implementation of a commercial fluorescence lifetime Imaging Microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein–protein interactions on a spatial scale less than 10 nm using commercially available components.
Bradley T Hyman - One of the best experts on this subject based on the ideXlab platform.
-
DETECTED BY FLUORESCENCE LIFETIME Imaging Microscopy AND A HIGH THROUGHPUT FLUORESCENT
2015Co-Authors: Plate Reader, Bradley T Hyman, Rudolph E. Tanzi, Oksana BerezovskaAbstract:heterodimeric form is the catalytic center of the γ-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Aβ). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the Ubiquilin 1 gene are associated with an increased risk of developing Alzheimer’s Disease (AD). However, it is still unclear whether PS1 and Ubiquilin 1 interact when expressed at endogenous levels unde
-
two postprocessing techniques for the elimination of background autofluorescence for fluorescence lifetime Imaging Microscopy
Journal of Biomedical Optics, 2008Co-Authors: Phillip B Jones, Aneta Rozkalne, Melanie Meyerluehmann, Tara L Spiresjones, Alexandra Makarova, Anand Kumar, Oksana Berezovska, Brian Bacskai, Bradley T HymanAbstract:The analysis of fluorescence lifetime Imaging Microscopy (FLIM) data under complex biological conditions can be challenging. Particularly, the presence of short-lived autofluorescent aggregates can confound lifetime measurements in fluorescence energy transfer (FRET) experiments, where it can become confused with the signal from exogenous fluorophores. Here we report two techniques that can be used to discriminate the contribution of autofluorescence from exogenous fluorphores in FLIM. We apply the techniques to transgenic mice that natively express yellow fluorescence protein (YFP) in a subset of cortical neurons and to histological slices of aged human brain tissue, where we study the misfolding of intracellular tau protein in the form of neurofibrillary tangles.
-
fluorescence lifetime Imaging Microscopy flim detects stimulus dependent phosphorylation of the low density lipoprotein receptor related protein lrp in primary neurons
Biochemical and Biophysical Research Communications, 2006Co-Authors: Ithan D Peltan, Bradley T Hyman, Anne V Thomas, Irina Mikhailenko, Dudley K Strickland, Christine A F Von ArnimAbstract:The low-density lipoprotein receptor-related protein (LRP) is a large, endocytic receptor involved in intracellular signalling. LRP acts as a co-receptor with the PDGF-receptor (PDGF-r) for platelet-derived growth factor (PDGF). PDGF-r and Src-kinases induce tyrosine-phosphorylation of LRP. We used fluorescence lifetime Imaging Microscopy (FLIM) to specifically detect LRP phosphorylation, measure its extent and localization in intact cells, and assess its effects upon LRP-APP interaction. Robust phosphorylation of LRP throughout the cell was observed after overexpression of Src-kinase. This depended on LRP's distal NPXY domain. By contrast, activation of the PDGF-r resulted in phosphorylation of the subpopulation of LRP at or near the cell surface. PDGF activation triggered phosphorylation of endogenous LRP in primary neurons. LRP is also a trafficking receptor for the Alzheimer-related molecule amyloid-precursor-protein (APP). PDGF stimulation did not affect LRP-APP interactions. This approach allows exquisite subcellular resolution of specific LRP post-translational changes and protein-protein interactions of endogenous proteins in intact cells.
-
fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime Imaging Microscopy to characterize amyloid beta plaques
Journal of Biomedical Optics, 2003Co-Authors: Brian J Bacskai, Jesse Skoch, Gregory A Hickey, Racquel Allen, Bradley T HymanAbstract:We describe the implementation of a commercial fluorescence lifetime Imaging Microscopy (FLIM) instrument used in conjunction with a commercial laser scanning multiphoton microscope. The femtosecond-pulsed near-infrared laser is an ideal excitation source for time-domain fluorescence lifetime measurements. With synchronization from the x-y scanners, fluorescence lifetimes can be acquired on a pixel-by-pixel basis, with high spatial resolution. Multiexponential curve fits for each pixel result in two-dimensional fluorescence resonance energy transfer (FRET) measurements that allow the determination of both proximity of fluorescent FRET pairs, as well as the fraction of FRET pairs close enough for FRET to occur. Experiments are described that characterize this system, as well as commonly used reagents valuable for FRET determinations in biological systems. Constructs of CFP and YFP were generated to demonstrate FRET between this pair of green fluorescent protein (GFP) color variants. The lifetime characteristics of the FRET pair fluorescein and rhodamine, commonly used for immunohistochemistry, were also examined. Finally, these fluorophores were used to demonstrate spatially resolved FRET with senile plaques obtained from transgenic mouse brain. Together these results demonstrate that FLIM allows sensitive measurements of protein–protein interactions on a spatial scale less than 10 nm using commercially available components.
Junle Qu - One of the best experts on this subject based on the ideXlab platform.
-
phasor fluorescence lifetime Imaging Microscopy analysis to monitor intercellular drug release from a ph sensitive polymeric nanocarrier
Analytical Chemistry, 2018Co-Authors: Ting Zhou, Jun Song, Junle QuAbstract:The design of highly efficient drug carriers, and the development of appropriate techniques to monitor their mechanism of action and therapeutic effect, are both critical for improving chemotherapy. Herein, a polymeric nanoparticle, PAH-Cit/DOX (poly(allylamine)–citraconic anhydride/doxorubicin), was synthesized and used as a nanodrug system for the efficient delivery and pH-responsive release of doxorubicin (DOX) into cancer cells. The PAH-Cit/DOX nanoparticles were stable at physiological pH but effectively released DOX under weakly acidic conditions. The release efficiency was 90.6% after 60 h of dialysis in phosphate-buffered saline at pH 5.5. Confocal images showed the rapid movement of the drug from the cytoplasm to the nucleus, indicating the effective drug release MCF-7 cells. Notably, the combination of fluorescence lifetime Imaging Microscopy (FLIM) and phasor analysis (phasor–FLIM) provides an approach to monitor the dynamic change of DOX fluorescence lifetime in intercellular environments. Pha...