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fluorescence lifetime imaging microscopy

Nirmal Mazumder, Gitanjal Deka, Wei-Wen Wu, Ankur Gogoi, Guan-Yu Zhuo, Fu-Jen Kao
Second harmonic (SH) microscopy has proven to be a powerful imaging modality over the past years due to its intrinsic advantages as a multiphoton process with endogenous contrast specificity, which allows pinhole-less optical sectioning, non-invasive observation, deep tissue penetration, and the possibility of easier signal detection at visible wavelengths. Depending on the relative orientation between the polarization of the incoming light and the second-order susceptibility of non-centrosymmetric structures, SH microscopy provides the unique capacity to probe the absolute molecular structure of a broad variety of biological tissues without the necessity for additional labeling...
June 14, 2017: Methods: a Companion to Methods in Enzymology
Arunima Bhattacharjee, Rupsa Datta, Enrico Gratton, Allon I Hochbaum
Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells...
June 16, 2017: Scientific Reports
Sivan Gershanov, Shalom Michowiz, Helen Toledano, Gilad Yahav, Orit Barinfeld, Avraham Hirshberg, Haim Ben-Zvi, Gabriel Mircus, Mali Salmon-Divon, Dror Fixler, Nitza Goldenberg-Cohen
In pediatric brain tumours, dissemination of malignant cells within the central nervous system confers poor prognosis and determines treatment intensity, but is often undetectable by imaging or cytology. This study describes the use of fluorescence lifetime (FLT) imaging microscopy (FLIM), a novel diagnostic tool, for detection of metastatic spread. The study group included 15 children with medulloblastoma and 2 with atypical teratoid/rhabdoid tumour. Cells extracted from the tumour and the cerebrospinal fluid (CSF) 2 weeks postoperatively and repeatedly during chemo/radiotherapy were subjected to nuclear staining followed by FLT measurement and cytological study...
June 16, 2017: Scientific Reports
D Zaharie-Butucel, M Potara, A M Craciun, R Boukherroub, S Szunerits, S Astilean
A high-end correlated spectral and imaging multianalysis, adapted for bidimensional systems, is presented here to analyze graphene oxide (GO) and reduced GO (rGO) modified with pyrene carboxylic acid (PCA). Confocal Raman mapping was used next to two-photon excited Fluorescence Lifetime Imaging Microscopy (FLIM) to characterize the distribution of PCA on GO and rGO and compared to UV-vis and X-ray Photoelectron Spectroscopy (XPS) analysis of the materials. Raman imaging clearly highlights the difference in the spatial distribution of PCA molecules on GO and rGO...
June 21, 2017: Physical Chemistry Chemical Physics: PCCP
Jan Huebinger, Martin E Masip, Jens Christmann, Frank Wehner, Philippe I H Bastiaens
Fluorescence live-cell imaging by single molecule localization microscopy (SMLM) or fluorescence lifetime imaging microscopy (FLIM) in principle allows for the spatio-temporal observation of molecular patterns in individual, living cells. However, the dynamics of molecules within cells hamper their precise observation. We present here a detailed protocol for consecutive cycles of reversible cryo-arrest of living cells on a microscope that allows for a precise determination of the evolution of molecular patterns within individual living cells...
April 20, 2017: Bio-protocol
Timea Nagy-Simon, Andra-Sorina Tatar, Ana-Maria Craciun, Adriana Vulpoi, Maria-Ancuta Jurj, Adrian Florea, Ciprian Tomuleasa, Ioana Berindan-Neagoe, Simion Astilean, Sanda Boca
In this Research Article, we propose a new class of contrast agents for the detection and multimodal imaging of CD19(+) cancer lymphoblasts. The agents are based on NIR responsive hollow gold-silver nanospheres conjugated with antiCD19 monoclonal antibodies and marked with Nile Blue (NB) SERS active molecules (HNS-NB-PEG-antiCD19). Proof of concept experiments on specificity of the complex for the investigated cells was achieved by transmission electron microscopy (TEM). The microspectroscopic investigations via dark field (DF), surface-enhanced Raman spectroscopy (SERS), and two-photon excited fluorescence lifetime imaging microscopy (TPE-FLIM) corroborate with TEM and demonstrate successful and preferential internalization of the antibody-nanocomplex...
June 15, 2017: ACS Applied Materials & Interfaces
Stefanie Weidtkamp-Peters, Yvonne Stahl
The investigation of protein interactions in living plant tissue has become of increasing importance in recent years. A high spatial and temporal resolution for the observation of in vivo protein interaction is needed, e.g., in order to follow changes of plant receptor kinase interactions and complex formation over time. In vivo fluorescence or Förster resonance energy transfer (FRET) measurements allow for detailed analyses of interacting proteins in their natural environment at a subcellular level. Especially FRET-FLIM (fluorescence lifetime imaging microscopy) measurements provide an extremely powerful and reliable tool meeting the demands for investigating in vivo protein interaction quantitatively and with high precision...
2017: Methods in Molecular Biology
Mengyan Wang, Feng Tang, Xiaobo Pan, Longfang Yao, Xinyi Wang, Yueyue Jing, Jiong Ma, Guifang Wang, Lan Mi
A method of rapidly differentiating lung tumor from healthy tissue is extraordinarily needed for both the diagnosis and the intraoperative margin assessment. We assessed the ability of fluorescence lifetime imaging microscopy (FLIM) for differentiating human lung cancer and normal tissues with the autofluorescence, and also elucidated the mechanism in tissue studies and cell studies. A 15-patient testing group was used to compare FLIM results with traditional histopathology diagnosis. Based on the endogenous fluorescence lifetimes of the testing group, a criterion line was proposed to distinguish normal and cancerous tissues...
December 2017: BBA Clinical
Ujwal Kumar Thakur, Abdelrahman M Askar, Ryan Kisslinger, Benjamin D Wiltshire, Piyush Kar, Karthik Shankar
This is the first report of a 17.6% champion efficiency solar cell architecture comprising monocrystalline TiO2 nanorods (TNRs) coupled with perovskite, and formed using facile solution processing without non-routine surface conditioning. Vertically oriented TNR ensembles are desirable as electron transporting layers (ETLs) in halide perovskite solar cells (HPSCs) because of potential advantages such as vectorial electron percolation pathways to balance the longer hole diffusion lengths in certain halide perovskite semiconductors, ease of incorporating nanophotonic enhancements, and optimization between a high contact surface area for charge transfer (good) versus high interfacial recombination (bad)...
July 7, 2017: Nanotechnology
Anh Thy Bui, Alexei Grichine, Alain Duperray, Pierre Lidon, François Riobé, Chantal Andraud, Olivier Maury
Fluorescent probes that are able to directly measure viscosity are attractive candidates for the study of intracellular environments. We report a new class of luminescent rotors, based on the sensitized emission of a terbium(III) complex. A 4-fold increase in both quantum yield and luminescence lifetime was observed in viscous media for the studied complexes, with a lifetime ranging from 0.23 to 0.89 ms over a broad range of viscosities (0.6-1200 cP). The presented approach, relying on the millisecond-scale luminescence lifetime of the lanthanide ions, was applied to fixed T24 cancer cells using temporal sampling lifetime imaging microscopy...
May 31, 2017: Journal of the American Chemical Society
Carlos Zaldo, María Dolores Serrano, Xiumei Han, Concepción Cascales, Marta Cantero, Lluís Montoliu, Elvira Arza, Valeria R Caiolfa, Moreno Zamai
Yb and Er codoped NaT(XO4)2 (T = Y, La, Gd, Lu and X = Mo, W) disordered oxides show a green (Er3+ related) up-conversion (UC) efficiency comparable to that of Yb:Er:β-NaYF4 compound and unless 3 times larger UC ratiometric thermal sensitivity. The similar UC efficiency of Yb:Er doped NaT(XO4)2 and β-NaYF4 compounds allowed testing equal subcutaneous depths of ex-vivo chicken tissue in both cases. This extraordinary behavior for NaT(XO4)2 oxides with large cutoff phonon energy (ħω≈ 920 cm-1) is ascribed to 4F9/2 electron population recycling to higher energy 4G11/2 level by a phonon assisted transition...
2017: PloS One
Yongkui Xu, Qi Liu, Ruoyu He, Xianchong Miao, Minbiao Ji
Nanoparticles have shown promise in loading and delivering drugs for targeted therapy. Many progresses have been made in the design, synthesis, and modification of nanoparticles to fulfill such goals. However, realizing targeted intracellular delivery and controlled release of drugs remains challenging, partly because of the lack of reliable tools to detect the drug-releasing process. In this paper, we applied femtosecond laser pulses to trigger the explosion of gold nanocages (AuNCs) and control the intracellular release of loaded aluminum phthalocyanine (AlPcS) molecules for photodynamic therapy (PDT)...
June 5, 2017: ACS Applied Materials & Interfaces
Mary Rose Hilaire, Ismail A Ahmed, Chun-Wei Lin, Hyunil Jo, William F DeGrado, Feng Gai
Many fluorescent proteins are currently available for biological spectroscopy and imaging measurements, allowing a wide range of biochemical and biophysical processes and interactions to be studied at various length scales. However, in applications where a small fluorescence reporter is required or desirable, the choice of fluorophores is rather limited. As such, continued effort has been devoted to the development of amino acid-based fluorophores that do not require a specific environment and additional time to mature and have a large fluorescence quantum yield, long fluorescence lifetime, good photostability, and an emission spectrum in the visible region...
June 6, 2017: Proceedings of the National Academy of Sciences of the United States of America
Daniëlle Rianne José Verboogen, Natalia González Mancha, Martin Ter Beest, Geert van den Bogaart
SNARE proteins play a crucial role in intracellular trafficking by catalyzing membrane fusion, but assigning SNAREs to specific intracellular transport routes is challenging with current techniques. We developed a novel Förster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET-FLIM)-based technique allowing visualization of real-time local interactions of fluorescently tagged SNARE proteins in live cells. We used FRET-FLIM to delineate the trafficking steps underlying the release of the inflammatory cytokine interleukin-6 (IL-6) from human blood-derived dendritic cells...
May 19, 2017: ELife
Arghajit Pyne, Jagannath Kuchlyan, Chiranjit Maiti, Dibakar Dhara, Nilmoni Sarkar
In this article, we have reported the synthesis and physicochemical characterization of a novel l-glycine amino acid derived cholesterol based surface active ionic liquid (SAIL). This SAIL has been explored for the preparation of ionic liquid (IL)-in-oil microemulsions and vesicles. The formation of IL-in-oil microemulsion is characterized by construction of a ternary phase diagram, dynamic light scattering (DLS) measurement, proton nuclear magnetic resonance ((1)H NMR) study, fluorescence measurement using coumarin 480 (C-480) as a molecular probe, and also by recording the diffusion behavior of the molecular probe rhodamine 6G (R6G) in microemulsion droplets through the fluorescence correlation spectroscopy (FCS) technique...
May 30, 2017: Langmuir: the ACS Journal of Surfaces and Colloids
Malou Zuidscherwoude, Vera-Marie E Dunlock, Geert van den Bogaart, Sjoerd J van Deventer, Alie van der Schaaf, Jenny van Oostrum, Joachim Goedhart, Joanna In 't Hout, Günter J Hämmerling, Satoshi Tanaka, André Nadler, Carsten Schultz, Mark D Wright, Merel J W Adjobo-Hermans, Annemiek B van Spriel
Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCβ. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCβ from the cytosol to plasma membrane regions containing the tetraspanin protein CD53...
May 9, 2017: Science Signaling
Tiffany M Heaster, Alex J Walsh, Yue Zhao, Scott W Hiebert, Melissa C Skala
The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co-enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell-cycle status of tumor cells. Heterogeneity in tumor cell-cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell-cycle status is closely linked to cellular metabolism. Thus, this study applies cell-level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations...
May 9, 2017: Journal of Biophotonics
Gloria de Las Heras-Martínez, Josu Andrieu, Banafshé Larijani, Jose Requejo-Isidro
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)...
May 9, 2017: Journal of Biophotonics
Alexander Johnson, Grégory Vert
Endocytosis is a key process in the internalization of extracellular materials and plasma membrane proteins, such as receptors and transporters, thereby controlling many aspects of cell signaling and cellular homeostasis. Endocytosis in plants has an essential role not only for basic cellular functions but also for growth and development, nutrient delivery, toxin avoidance, and pathogen defense. The precise mechanisms of endocytosis in plants remain quite elusive. The lack of direct visualization and examination of single events of endocytosis has greatly hampered our ability to precisely monitor the cell surface lifetime and the recruitment profile of proteins driving endocytosis or endocytosed cargos in plants...
2017: Frontiers in Plant Science
Yuanqing Ma, Elvis Pandzic, Philip R Nicovich, Yui Yamamoto, Joanna Kwiatek, Sophie V Pageon, Aleš Benda, Jérémie Rossy, Katharina Gaus
Clustering of the T-cell receptor (TCR) is thought to initiate downstream signalling. However, the detection of protein clustering with high spatial and temporal resolution remains challenging. Here we establish a Förster resonance energy transfer (FRET) sensor, named CliF, which reports intermolecular associations of neighbouring proteins in live cells. A key advantage of the single-chain FRET sensor is that it can be combined with image correlation spectroscopy (ICS), single-particle tracking (SPT) and fluorescence lifetime imaging microscopy (FLIM)...
April 28, 2017: Nature Communications
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