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fluorescence lifetime imaging microscopy

Eva Bártová, Soňa Legartová, Jana Krejčí, Petra Řezníčková, Alena Svobodová Kovaříková, Jana Suchánková, Radek Fedr, Evgeny Smirnov, Matúš Hornáček, Ivan Raška
We studied how deficiency in lamins A/C and lamina-associated polypeptide 2α (Lap2α) affects DNA repair after irradiation. A-type lamins and Lap2α were not recruited to local DNA lesions and did not accumulate to γ-irradiation-induced foci (IRIF), as it is generally observed for well-known marker of DNA lesions, 53BP1 protein. At micro-irradiated chromatin of lmna double knockout (dn) and Lap2α dn cells, 53BP1 protein levels were reduced, compared to locally irradiated wild-type counterpart. Decreased levels of 53BP1 we also observed in whole populations of lmna dn and Lap2α dn cells, irradiated by UV light...
June 19, 2018: Journal of Cellular Biochemistry
Alexandra Chanoca, Brian Burkel, Erich Grotewold, Kevin W Eliceiri, Marisa S Otegui
Anthocyanins are intrinsically fluorescent pigments that accumulate in plant vacuoles. We have developed a platform to analyze the fluorescence decay of anthocyanins by fluorescence lifetime imaging microscopy (FLIM), under in vitro and in vivo conditions. Fluorescence lifetime of a fluorophore can be influenced by temperature, pH, oxygen concentration, and other environmental conditions. Within plant cells, the anthocyanin fluorescence lifetime correlates with distinct subcellular compartments. Vacuolar anthocyanins exhibit shorter fluorescence lifetime than the cytoplasmic pool...
2018: Methods in Molecular Biology
H Saari, E Lisitsyna, K Rautaniemi, T Rojalin, L Niemi, O Nivaro, T Laaksonen, M Yliperttula, E Vuorimaa-Laukkanen
In response to physiological and artificial stimuli, cells generate nano-scale extracellular vesicles (EVs) by encapsulating biomolecules in plasma membrane-derived phospholipid envelopes. These vesicles are released to bodily fluids, hence acting as powerful endogenous mediators in intercellular signaling. EVs provide a compelling alternative for biomarker discovery and targeted drug delivery, but their kinetics and dynamics while interacting with living cells are poorly understood. Here we introduce a novel method, fluorescence lifetime imaging microscopy (FLIM) to investigate these interaction attributes...
June 12, 2018: Journal of Controlled Release: Official Journal of the Controlled Release Society
Ales Holoubek, Petr Heřman, Jan Sýkora, Barbora Brodská, Jana Humpolíčková, Markéta Kráčmarová, Dana Gášková, Martin Hof, Kateřina Kuželová
Oligomerization plays a crucial role in the function of nucleophosmin (NPM), an abundant nucleolar phosphoprotein. Two dual-color methods based on modern fluorescence confocal microscopy are applied for tracking NPM aggregates in live cells: cross-correlation Number and Brightness analysis (ccN&B) combined with pulsed interleaved excitation (PIE) and fluorescence-lifetime imaging microscopy (FLIM) utilizing resonance energy transfer (FRET). HEK-293T cells were transfected with mixture of plasmids designed for tagging with fluorescent proteins so that the cells express mixed population of NPM labeled either with eGFP or mRFP1...
June 14, 2018: Methods and Applications in Fluorescence
Michael Evers, Nunciada Salma, Sam Osseiran, Malte Casper, Reginald Birngruber, Conor L Evans, Dieter Manstein
Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders...
June 8, 2018: Scientific Reports
Naim Bel Haj Mohamed, Nassim Ben Brahim, Randa Mrad, Mohamed Haouari, Rafik Ben Chaâbane, Michel Negrerie
Water soluble CdS quantum dots (QDs) were synthesized by a simple aqueous chemical route using mercaptopropionic acid (MPA) as a stabilizer. These QDs had a fluorescence emission band maximum at 540 nm with a FWHM ∼130 nm and a quantum yield of ∼12%. Transmission electronic microscopy images were used to determine the QD diameter of 8.9 ± 0.4 nm. From this value we calculated the molecular mass M(QD)  = 1.17 × 106  g mol-1 and the extinction coefficient at the band edge (450 nm) ε450  = 4...
October 22, 2018: Analytica Chimica Acta
Matthew Huisman, Jacob P Kodanko, Karan Arora, Mackenzie Herroon, Marim Alnaed, John Endicott, Izabela Podgorski, Jeremy J Kodanko
Two new Re(I)- and Ru(II)-based inhibitors were synthesized with the formulas [Re(phen)(CO)3 (1)](OTf) (7; phen = 1,10-phenanthroline, OTf = trifluoromethanesulfonate) and [Ru(bpy)2 (2)](Cl)2 (8; bpy = 2,2'-bipyridine), where 1 and 2 are the analogues of CLIK-148, an epoxysuccinyl-based cysteine cathepsin L inhibitor (CTSL). Compounds 7 and 8 were characterized using various spectroscopic techniques and elemental analysis; 7 and 8 both show exceptionally long excited state lifetimes. Re(I)-based complex 7 inhibits CTSL in the low nanomolar range, affording a greater than 16-fold enhancement of potency relative to the free inhibitor 1 with a second-order rate constant of 211000 ± 42000 M-1 s-1 ...
June 8, 2018: Inorganic Chemistry
Sviatlana Kalinina, Jasmin Breymayer, Kirsten Reeß, Lothar Lilge, Arkady Mandel, Angelika Rück
During PDT disruption of cell respiration and metabolic changes could be one of the first events. Photophysical characteristics of the photosensitizer (PS) and its specific redox potential define consumption of molecular oxygen followed by generation of reactive oxygen species (ROS). The potential photosensitizer TLD1433 is based on transition metal Ru(II) and possess an oxygen dependent luminescence. This enables study of oxygen consumption by PS-PLIM (phosphorescence lifetime imaging) and simultaneously changes of the cellular metabolic state by NAD(P)H-FLIM (fluorescence lifetime imaging)...
June 7, 2018: Journal of Biophotonics
Yuchen Long, Yvonne Stahl, Stefanie Weidtkamp-Peters, Wouter Smet, Yujuan Du, Theodorus W J Gadella, Joachim Goedhart, Ben Scheres, Ikram Blilou
Protein complex formation has been extensively studied using Förster resonance energy transfer (FRET) measured by Fluorescence Lifetime Imaging Microscopy (FLIM). However, implementing this technology to detect protein interactions in living multicellular organism at single-cell resolution and under native condition is still difficult to achieve. Here we describe the optimization of the labeling conditions to detect FRET-FLIM in living plants. This study exemplifies optimization procedure involving the identification of the optimal position for the labels either at the N or C terminal region and the selection of the bright and suitable, fluorescent proteins as donor and acceptor labels for the FRET study...
2018: Frontiers in Plant Science
Tsukasa Funane, Steven S Hou, Katarzyna Marta Zoltowska, Susanne J van Veluw, Oksana Berezovska, Anand T N Kumar, Brian J Bacskai
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...
May 2018: Review of Scientific Instruments
William Ghann, Hyeonggon Kang, Jamal Uddin, Sunalee J Gonawala, Sheikh Mahatabuddin, Meser M Ali
Dye sensitized solar cells were fabricated with DyLight680 (DL680) dye and its corresponding europium conjugated dendrimer, DL680-Eu-G5PAMAM, to study the effect of europium on the current and voltage characteristics of the DL680 dye sensitized solar cell. The dye samples were characterized by using Absorption Spectroscopy, Emission Spectroscopy, Fluorescence lifetime and Fourier Transform Infrared measurements. Transmission electron microscopy imaging was carried out on the DL680-Eu-G5PAMAM dye and DL680-Eu-G5PAMAM dye sensitized titanium dioxide nanoparticles to analyze the size of the dye molecules and examine the interaction of the dye with titanium dioxide nanoparticles...
2018: Journal of Nanomedicine & Nanotechnology
Yongliang Li, Shipeng Shao, Xiao Xu, Xiaodong Su, Yujie Sun, Shicheng Wei
Bacterial binary cell division requires accurate placement of division machinery. FtsZ, a vital component of the division machinery, can assemble into filaments and self-organize into a ring structure (Z ring) at the appropriate site for cell division. MapZ, a recently identified FtsZ regulator in Streptococcaceae, has been found to localize at the mid-cell where it helps to properly position the FtsZ ring. However, its mechanism is still unclear. Here, by using total internal reflection fluorescence microscopy, super-resolution imaging, and single molecule tracking, we investigated the mechanism by which MapZ controls the position of the FtsZ ring...
May 29, 2018: ACS Nano
B Pradère, F Poulon, E Compérat, I Lucas, D Bazin, S Doizi, O Cussenot, O Traxer, D Abi Haidar
In the framework of urologic oncology, mini-invasive procedures have increased in the last few decades particularly for urothelial carcinoma. One of the essential elements in the management of this disease is still the diagnosis, which strongly influences the choice of treatment. The histopathologic evaluation of the tumor grade is a keystone of diagnosis, and tumor characterization is not possible with just a macroscopic evaluation. Even today intraoperative evaluation remains difficult despite the emergence of new technologies which use exogenous fluorophore...
May 28, 2018: Journal of Biophotonics
Yin Yang, Qian Li, Qi-Hua He, Ji-Sheng Han, Li Su, You Wan
Activation of the cholecystokinin type B receptor (CCKBR) by cholecystokinin octapeptide (CCK-8) inhibits opioid analgesia. Chronic opiate treatment leads to an increase in the CCK-8 concentration and thus enhances the antagonism of CCK-8 against opioid analgesia; the underlying molecular mechanisms remain of great interest. In the present study, we validated the colocalization of the μ-opioid receptor (MOR) and CCKBR in pain signal transmission-related spinal cord dorsal horn and dorsal root ganglion neurons of rats...
May 21, 2018: Experimental & Molecular Medicine
Yaoyao Xiong, Andreas Vargas Jentzsch, Johannes W M Osterrieth, Erdinc Sezgin, Igor V Sazanovich, Katharina Reglinski, Silvia Galiani, Anthony W Parker, Christian Eggeling, Harry L Anderson
Recent developments in super-resolution microscopy have significantly expanded the requirements for switchable dyes, leading to demand for specially designed molecular switches. We report the synthesis and characterization of a spironaphthoxazine photochromic switch (a derivative of palatinate purple) displaying high photoconversion (85-95%) under readily accessible 405 nm light, broad absorption in the visible, and excellent fatigue resistance. The indole substituent on this spironaphthoxazine is twisted out of conjugation with the naphthalene unit, yet it is crucial for activation with visible light...
March 21, 2018: Chemical Science
Jens Balke, Pierre Volz, Falko Neumann, Robert Brodwolf, Alexander Wolf, Hannah Pischon, Moritz Radbruch, Lars Mundhenk, Achim D Gruber, Nan Ma, Ulrike Alexiev
Nanoparticles hold a great promise in biomedical science. However, due to their unique physical and chemical properties they can lead to overproduction of intracellular reactive oxygen species (ROS). As an important mechanism of nanotoxicity, there is a great need for sensitive and high-throughput adaptable single-cell ROS detection methods. Here, fluorescence lifetime imaging microscopy (FLIM) is employed for single-cell ROS detection (FLIM-ROX) providing increased sensitivity and enabling high-throughput analysis in fixed and live cells...
May 4, 2018: Small
Sourav Bhattacharjee, Maria Manuela Gaspar, Dimitri Scholz, António J Almeida, David J Brayden
AIM:  To investigate how surface charge and hydrophilicity affect the mucopermeation of liposomes across intestinal mucus. METHODOLOGY: Rhodamine-labeled liposomes (∼120-130 nm) with different surface charges were investigated for their capacity to flux across fresh porcine jejunal mucus in a microchannel device. Fluorescent microscopy and tracking analysis were used to measure liposome movement, while fluorescence lifetime imaging microscopy was utilized to determine mucus pH...
May 1, 2018: Therapeutic Delivery
Jagadish Sankaran, Andreas Karampatzakis, Scott A Rice, Thorsten Wohland
Light microscopy has enabled the observation of the structure and organisation of biofilms. Typically, the contrast in an image obtained from light microscopy is given by the time-averaged intensity that is effective in visualising the overall structure. Technological advancements in light microscopy have led to the creation of techniques that not only provide a static intensity image of the biofilm, but also enable one to quantify various dynamic physicochemical properties of biomolecules in microbial biofilms...
May 1, 2018: FEMS Microbiology Letters
Yiqun Xue, Jingjing Xing, Yinglang Wan, Xueqin Lv, Lusheng Fan, Yongdeng Zhang, Kai Song, Li Wang, Xiaohua Wang, Xin Deng, František Baluška, John M Christie, Jinxing Lin
Phototropin (phot)-mediated signaling initiated by blue light (BL) plays a critical role in optimizing photosynthetic light capture at the plasma membrane (PM) in plants. However, the mechanisms underlying the regulation of phot activity at the PM in response to BL remain largely unclear. In this study, by single-particle tracking and stepwise photobleaching analysis of phot1-GFP proteins we demonstrated that in the dark phot1 proteins remain in an inactive state and mostly exist as monomers. Dimerization and the diffusion rate of phot1-GFP increased in a dose-dependent manner in response to BL...
June 4, 2018: Molecular Plant
Kasipandi Vellaisamy, Guodong Li, Chung-Nga Ko, Hai-Jing Zhong, Sarwat Fatima, Hiu-Yee Kwan, Chun-Yuen Wong, Wai-Jing Kwong, Weihong Tan, Chung-Hang Leung, Dik-Lung Ma
Dopamine receptor expression is correlated with certain types of cancers, including lung, breast and colon cancers. In this study, we report luminescent iridium(iii) complexes ( 11-14 ) as intracellular dopamine receptor (D1R/D2R) cell imaging agents. Complexes 11 and 13 , which are conjugated with a dopamine receptor agonist, showed superior cell imaging characteristics, high stability and low cytotoxicity (>100 μM) in A549 lung cancer cells. siRNA knockdown and dopamine competitive assays indicated that complexes 11 and 13 could selectively bind to dopamine receptors (D1R/D2R) in A549 cells...
February 7, 2018: Chemical Science
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