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fluorescence lifetime imaging microscopy

Ana Carolina B Sant'Anna-Silva, Gilson C Santos, Samir P Costa Campos, André Marco Oliveira Gomes, Juan Alberto Pérez-Valencia, Franklin David Rumjanek
Tumor cells are subjected to a broad range of selective pressures. As a result of the imposed stress, subpopulations of surviving cells exhibit individual biochemical phenotypes that reflect metabolic reprograming. The present work aimed at investigating metabolic parameters of cells displaying increasing degrees of metastatic potential. The metabolites present in cell extracts fraction of tongue fibroblasts and of cell lines derived from human tongue squamous cell carcinoma lineages displaying increasing metastatic potential (SCC9 ZsG, LN1 and LN2) were analyzed by1 H NMR (nuclear magnetic resonance) spectroscopy...
2018: Frontiers in Oncology
Anna M Chizhik, Carina Wollnik, Daja Ruhlandt, Narain Karedla, Alexey I Chizhik, Lara Hauke, Dirk Hähnel, Ingo Gregor, Jörg Enderlein, Florian Rehfeldt
We report a novel method, dual color axial nanometric localization by Metal Induced Energy Transfer (dcMIET), and combine it with Förster Resonant Energy Transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells (hMSCs). Our approach is based on Fluorescence-Lifetime-Imaging Microscopy (FLIM), and allows for precise determination of the 3D architecture of stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics...
February 14, 2018: Molecular Biology of the Cell
Alexander Edlich, Pierre Volz, Robert Brodwolf, Michael Unbehauen, Lars Mundhenk, Achim D Gruber, Sarah Hedtrich, Rainer Haag, Ulrike Alexiev, Burkhard Kleuser
Owing their unique chemical and physical properties core-multishell (CMS) nanocarriers are thought to underlie their exploitable biomedical use for a topical treatment of skin diseases. This highlights the need to consider not only the efficacy of CMS nanocarriers but also the potentially unpredictable and adverse consequences of their exposure thereto. As CMS nanocarriers are able to penetrate into viable layers of normal and stripped human skin ex vivo as well as in in vitro skin disease models the understanding of nanoparticle crosstalk with components of the immune system requires thorough investigation...
February 3, 2018: Biomaterials
Gabriel Bidaux, Corentin Le Nézet, Mariano Gonzalez Pisfil, Mélanie Henry, Alessandro Furlan, Oliver Bensaude, Bernard Vandenbunder, Laurent Héliot
Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Förster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, EFRET, because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through...
February 6, 2018: Biophysical Journal
Fumihiro Dake, Yusuke Taki
We propose a time-domain approach for fluorescence lifetime measurements using nonlinear fluorescence microscopy constructed of a pump-probe setup with two-wavelength laser pulses. Nonlinear fluorescence signals generated by fluorescence reduction due to stimulated emission were detectable through a lock-in technique. Changing the time delay between the two-wavelength pulses enables acquisition of a time-resolved nonlinear fluorescence signal, which directly reflects the fluorescence lifetime of the sample and is thus applicable to fluorescence lifetime imaging...
February 1, 2018: Applied Optics
Pawan Kumar, Navneet Chandra Verma, Natasha Goyal, Jayeeta Biswas, Saurabh Lodha, Chayan K Nandi, Viswanath Balakrishnan
Self-organized semiconductor-semiconductor heterostructures (3R-2H) that coexist in atomically thin 2D monolayers forming homojunctions are of great importance for next-generation nanoelectronics and optoelectronics applications. Herein, we investigated the defect controlled growth of heterogeneous electronic structure within a single domain of monolayer WS2 to enable in-plane homojunctions consisting of alternate 2H semiconducting and 3R semiconducting phases of WS2. X-ray photoelectron, Raman, and photoluminescence spectroscopy along with fluorescence and Kelvin probe force microscopy imaging confirm the formation of homojunctions, enabling a direct correlation between chemical heterogeneity and electronic heterostructure in the atomically thin WS2 monolayer...
January 31, 2018: Nanoscale
Ruma Das, Gone Rajender, P K Giri
We explore the mechanism of the fluorescence enhancement and fluorescence quenching effect of single walled carbon nanotubes (SWCNTs) on highly fluorescent graphene quantum dots (GQDs) over a wide range of concentrations of SWCNTs. At very low concentrations of SWCNTs, the fluorescence intensity of the GQDs is enhanced, while at higher concentrations, systematic quenching of fluorescence is observed. The nature of the Stern-Volmer plot for the latter case was found to be non-linear indicating a combined effect of dynamic and static quenching...
January 29, 2018: Physical Chemistry Chemical Physics: PCCP
Galina Nifontova, Maria Zvaigzne, Maria Baryshnikova, Evgeny Korostylev, Fernanda Ramos-Gomes, Frauke Alves, Igor Nabiev, Alyona Sukhanova
Fabrication of polyelectrolyte microcapsules and their use as carriers of drugs, fluorescent labels, and metal nanoparticles is a promising approach to designing theranostic agents. Semiconductor quantum dots (QDs) are characterized by extremely high brightness and photostability that make them attractive fluorescent labels for visualization of intracellular penetration and delivery of such microcapsules. Here, we describe an approach to design, fabricate, and characterize physico-chemical and functional properties of polyelectrolyte microcapsules encoded with water-solubilized and stabilized with three-functional polyethylene glycol derivatives core/shell QDs...
January 25, 2018: Nanoscale Research Letters
Daniëlle R J Verboogen, Maksim V Baranov, Martin Ter Beest, Geert van den Bogaart
Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions...
December 29, 2017: Journal of Visualized Experiments: JoVE
C H Hage, P Leclerc, J Brevier, M Fabert, C Le Nézet, A Kudlinski, L Héliot, F Louradour
In situ fluorescence lifetime imaging microscopy (FLIM) in an endoscopic configuration of the endogenous biomarker nicotinamide adenine dinucleotide (NADH) has a great potential for malignant tissue diagnosis. Moreover, two-photon nonlinear excitation provides intrinsic optical sectioning along with enhanced imaging depth. We demonstrate, for the first time to our knowledge, nonlinear endogenous FLIM in a fibered microscope with proximal detection, applied to NADH in cultured cells, as a first step to a nonlinear endomicroscope, using a double-clad microstructured fiber with convenient fiber length (> 3 m) and excitation pulse duration (≈50 fs)...
January 1, 2018: Biomedical Optics Express
David M Small, Washington Y Sanchez, Sandrine F Roy, Christudas Morais, Heddwen L Brooks, Jeff S Coombes, David W Johnson, Glenda C Gobe
Oxidative stress and mitochondrial dysfunction exacerbate acute kidney injury (AKI) but their role in any associated progress to chronic kidney disease (CKD) remains unclear. Antioxidant therapies often benefit AKI but their benefits in CKD are controversial since clinical and pre-clinical investigations often conflict. Here we examined the influence of the antioxidant, N-acetyl cysteine (NAC) on oxidative stress and mitochondrial function during AKI (20-minute bilateral renal ischemia plus reperfusion/IR) and progression to chronic kidney pathologies in mice...
January 10, 2018: American Journal of Physiology. Renal Physiology
Carolina García, Alejandro Losada, Miguel A Sacristán, Juan Fernando Martínez-Leal, Carlos M Galmarini, M Pilar Lillo
The design of living cell studies aimed at deciphering the mechanism of action of drugs targeting proteins with multiple functions, expressed in a wide range of concentrations and cellular locations, is a real challenge. We recently showed that the antitumor drug plitidepsin (APL) localizes sufficiently close to the elongation factor eEF1A2 so as to suggest the formation of drug-protein complexes in living cells. Here we present an extension of our previous micro-spectroscopy study, that combines Generalized Polarization (GP) images, with the phasor approach and fluorescence lifetime imaging microscopy (FLIM), using a 7-aminocoumarin drug analog (APL*) as fluorescence tracer...
January 18, 2018: Scientific Reports
Jakob Neef, Nicolai T Urban, Tzu-Lun Ohn, Thomas Frank, Philippe Jean, Stefan W Hell, Katrin I Willig, Tobias Moser
Ca2+ influx triggers the release of synaptic vesicles at the presynaptic active zone (AZ). A quantitative characterization of presynaptic Ca2+ signaling is critical for understanding synaptic transmission. However, this has remained challenging to establish at the required resolution. Here, we employ confocal and stimulated emission depletion (STED) microscopy to quantify the number (20-330) and arrangement (mostly linear 70 nm × 100-600 nm clusters) of Ca2+ channels at AZs of mouse cochlear inner hair cells (IHCs)...
January 18, 2018: Nature Communications
Ting Zhou, Teng Luo, Jun Song, Junle Qu
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, was synthesized and used as a nano-drug 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...
January 16, 2018: Analytical Chemistry
Vera Schützhold, Joachim Fandrey, Katrin Prost-Fingerle
Fluorescence resonance energy transfer (FRET) is widely used as a method to investigate protein-protein interactions in living cells. A FRET pair donor fluorophore in close proximity to an appropriate acceptor fluorophore transfers emission energy to the acceptor, resulting in a shorter lifetime of the donor fluorescence. When the respective FRET donor and acceptor are fused with two proteins of interest, a reduction in donor lifetime, as detected by fluorescence lifetime imaging microscopy (FLIM), can be taken as proof of close proximity between the fluorophores and therefore interaction between the proteins of interest...
2018: Methods in Molecular Biology
Krishna M Padmanabha Das, Lisa Wechselberger, Márton Liziczai, Montserrat De la Rosa Rodriguez, Gernot F Grabner, Christoph Heier, Roland Viertlmayr, Claudia Radler, Joerg Lichtenegger, Robert Zimmermann, Jan Willem Borst, Rudolf Zechner, Sander Kersten, Monika Oberer
Elaborate control mechanisms of intracellular triacylglycerol (TAG) breakdown are critically involved in the maintenance of energy homeostasis. Hypoxia inducible lipid droplet associated protein (HILPDA)/Hypoxia inducible gene-2 (Hig-2) has been shown to affect intracellular TAG levels, yet, the underlying molecular mechanisms are unclear. Here, we show that HILPDA inhibits adipose triglyceride lipase (ATGL), the enzyme catalyzing the first step of intracellular TAG hydrolysis. HILPDA shares structural similarity with G0/G1 switch gene 2 (G0S2), an established inhibitor of ATGL...
January 11, 2018: Journal of Lipid Research
Franziska Doll, Jessica Hassenrück, Valentin Wittmann, Andreas Zumbusch
Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells...
2018: Methods in Enzymology
Kenichi G N Suzuki, Hiromune Ando, Naoko Komura, Miku Konishi, Akihiro Imamura, Hideharu Ishida, Makoto Kiso, Takahiro K Fujiwara, Akihiro Kusumi
Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking...
2018: Methods in Enzymology
Meng-Na Yu, Hamid Soleimaninejad, Jin-Yi Lin, Zong-Yan Zuo, Bin Liu, Yi-Fan Bo, Lu-Bing Bai, Ya-Min Han, Trevor A Smith, Man Xu, Xiang-Ping Wu, Dave E Dunstan, Ruidong Xia, Linghai Xie, Donal D C Bradley, Wei Huang
We demonstrate a systematic visualization of the unique photophysical and fluorescence anisotropic properties of polyfluorene coplanar conformation (-conformation) using time-resolved scanning confocal fluorescence imaging (FLIM) and fluorescence anisotropy imaging microscopy (FAIM) measurements. We observe inhomogeneous morphologies and fluorescence decay profiles at various micrometer-sized regions within all type polyfluorene -conformational spin-coated films. Poly(9,9-dioctylfluorene-2,7-diyl) (PFO) and poly[4-(octyloxy)-9,9-diphenylfluoren-2,7-diyl]-co-[5-(octyloxy)-9,9-diphenylfluoren-2,7-diyl] (PODPF) -domains both have shorter lifetime than those of the glassy conformation for the longer effective conjugated length and rigid chain structures...
January 3, 2018: Journal of Physical Chemistry Letters
Qi Pian, Ruoyang Yao, Nattawut Sinsuebphon, Xavier Intes
Spectrally resolved fluorescence lifetime imaging1-3 and spatial multiplexing1,4,5 have offered information content and collection-efficiency boosts in microscopy, but efficient implementations for macroscopic applications are still lacking. An imaging platform based on time-resolved structured light and hyperspectral single-pixel detection has been developed to perform quantitative macroscopic fluorescence lifetime imaging (MFLI) over a large field of view (FOV) and multiple spectral bands simultaneously. The system makes use of three digital micromirror device (DMD)-based spatial light modulators (SLMs) to generate spatial optical bases and reconstruct N by N images over 16 spectral channels with a time-resolved capability (~40 ps temporal resolution) using fewer than N2 optical measurements...
2017: Nature Photonics
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