排序方式: 共有81条查询结果,搜索用时 15 毫秒
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Charles‐Henri Hage Pierre Leclerc Marc Fabert Sylvia M. Bardet Julien Brevier Guillaume Ducourthial Tigran Mansuryan Aymeric Leray Alexandre Kudlinski Frdric Louradour 《Journal of biophotonics》2019,12(5)
A two‐photon fluorescence lifetime (2P‐FLIM) microendoscope, capable of energetic metabolism imaging through the intracellular nicotinamide adenine dinucleotide (NADH) autofluorescence, at sub‐cellular resolution, is demonstrated. It exhibits readily usable characteristics such as convenient endoscope probe diameter (≈2 mm), fiber length (>5 m) and data accumulation rate (16 frames per second (fps)), leading to a FLIM refreshing rate of ≈0.1 to 1 fps depending on the sample. The spiral scanning image formation does not influence the instrument response function (IRF) characteristics of the system. Near table‐top microscope performances are achieved through a comprehensive system including a home‐designed spectro‐temporal pulse shaper and a custom air‐silica double‐clad photonic crystal fiber, which enables to reach up to 40 mW of ≈100 fs pulses @ 760 nm with a 80 MHz repetition rate. A GRadient INdex (GRIN) lens provides a lateral resolution of 0.67 μm at the focus of the fiber probe. Intracellular NADH fluorescence lifetime data are finally acquired on cultured cells at 16 fps. 相似文献
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Influenza virus assembles in the budozone, a cholesterol-/sphingolipid-enriched (“raft”) domain at the apical plasma membrane, organized by hemagglutinin (HA). The viral protein M2 localizes to the budozone edge for virus particle scission. This was proposed to depend on acylation and cholesterol binding. We show that M2–GFP without these motifs is still transported apically in polarized cells. Employing FRET, we determined that clustering between HA and M2 is reduced upon disruption of HA’s raft-association features (acylation, transmembranous VIL motif), but remains unchanged with M2 lacking acylation and/or cholesterol-binding sites. The motifs are thus irrelevant for M2 targeting in cells. 相似文献
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Quantifying intracellular equilibrium dissociation constants using single‐channel time‐resolved FRET 下载免费PDF全文
Gloria de las Heras‐Martínez Josu Andrieu Banafshé Larijani Jose Requejo‐Isidro 《Journal of biophotonics》2018,11(1)
Quantification of the intracellular equilibrium dissociation constant of the interaction, Kd, is challenging due to the variability of the relative concentrations of the interacting proteins in the cell. Fluorescence lifetime imaging microscopy (FLIM) of the donor provides an accurate measurement of the molecular fraction of donor involved in FRET, but the fraction of bound acceptor is also needed to reliably estimate Kd. We present a method that exploits the spectroscopic properties of the widely used eGFP – mCherry FRET pair to rigorously determine the intracellular Kd based on imaging the fluorescence lifetime of only the donor (single‐channel FLIM). We have assessed the effect of incomplete labelling and determined its range of application for different Kd using Monte Carlo simulations. We have demonstrated this method estimating the intracellular Kd for the homodimerisaton of the oncogenic protein 3‐phosphoinositide‐dependent kinase 1 (PDK1) in different cell lines and conditions, revealing a competitive mechanism for its regulation. The measured intracellular Kd was validated against in‐vitro data. This method provides an accurate and generic tool to quantify protein interactions in situ.
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Daniel Doerr Martin Stark Friederike Ehrhart Heiko Zimmermann Frank Stracke Dr. 《Biotechnology journal》2009,4(8):1215-1220
In this study we demonstrate a new noninvasive imaging method to monitor freezing processes in biological samples and to investigate life in the frozen state. It combines a laser scanning microscope with a computer-controlled cryostage. Nearinfrared (NIR) femtosecond laser pulses evoke the fluorescence of endogenous fluorophores and fluorescent labels due to multiphoton absorption.The inherent optical nonlinearity of multiphoton absorption allows 3D fluorescence imaging for optical tomography of frozen biological material in-situ. As an example for functional imaging we use fluorescence lifetime imaging (FLIM) to create images with chemical and physical contrast. 相似文献
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Jörg Nikolaus Elisa Bayraktarov Stephanie Engel Martin Stöckl Michael Veit 《Biophysical journal》2010,99(2):489-498
The HA of influenza virus is a paradigm for a transmembrane protein thought to be associated with membrane-rafts, liquid-ordered like nanodomains of the plasma membrane enriched in cholesterol, glycosphingolipids, and saturated phospholipids. Due to their submicron size in cells, rafts can not be visualized directly and raft-association of HA was hitherto analyzed by indirect methods. In this study, we have used GUVs and GPMVs, showing liquid disordered and liquid ordered domains, to directly visualize partition of HA by fluorescence microscopy. We show that HA is exclusively (GUVs) or predominantly (GPMVs) present in the liquid disordered domain, regardless of whether authentic HA or domains containing its raft targeting signals were reconstituted into model membranes. The preferential partition of HA into ld domains and the difference between lo partition in GUV and GPMV are discussed with respect to differences in packaging of lipids in membranes of model systems and living cells suggesting that physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions. 相似文献
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Anne V. Thomas Oksana Berezovska Bradley T. Hyman Christine A.F. von Arnim 《Methods (San Diego, Calif.)》2008,44(4):299
To understand normal function of memory studying models of pathological memory decline is essential. The most common form of dementia leading to memory decline is Alzheimer’s disease (AD), which is characterized by the presence of neurofibrillary tangles and amyloid plaques in the affected brain regions. Altered production of amyloid β (Aβ) through sequential cleavage of amyloid precursor protein (APP) by β- and γ-secretases seems to be a central event in the molecular pathogenesis of the disease. Thus, the study of the complex interplay of proteins that are involved in or modify Aβ production is very important to gain insight into the pathogenesis of AD. Here, we describe the use of Fluorescence lifetime imaging microscopy (FLIM), a Fluorescence resonance energy transfer (FRET)-based method, to visualize protein–protein-interaction in intact cells, which has proven to be a valuable method in AD research. 相似文献
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Anja W. Fjorback Patrick Pla Heidi K. Müller Frédéric Saudou Jens R. Nyengaard 《Biochemical and biophysical research communications》2009,380(4):724-728
The serotonin transporter is a member of the monoamine transporter family that also includes transporters of dopamine and norepinephrine. We have used sensitized acceptor emission fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) to study the oligomerization of SERT in HEK-MSR-239 cells, RN46A cells and in cultured hippocampal neurons. We were able to show identical FRET efficiencies in cell lines as well as in primary cultured hippocampal neurons, demonstrating that the oligomerization is cell type independent. The results obtained with both FRET approaches are very similar and furthermore, in agreement with previous results obtained by donor bleaching FRET microscopy. 相似文献