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Methods and Applications in Fluorescence

Saman Hosseinkhani, Hossein Soltaninejad, Mohammad Ali Asadollahi, Morteza Hosseini, Mohammad Reza Ganjali
Among epigenetic modifications of DNA, methylation of cytosine at its carbon 5 is the most common mark that is usually associated with gene silencing in human. Determining whether a particular DNA molecule is methylated or not, is an indispensable task in many epigenetic investigations. Presenting detection methods with less labor-intensive and time-consuming procedures has substantial value. Here a facile method based on gold nanocluster (AuNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region in which hypermethylation is demonstrated as a reliable biomarker of colorectal cancer...
September 14, 2018: Methods and Applications in Fluorescence
Stefan Krause, Mads Koerstz, Riikka Arppe-Tabbara, Tero Soukka, Tom Vosch
Upon direct excitation with green light (522 nm), Er3+ ion doped nanoparticles feature a number of radiative and non-radiative decay pathways, leading to distinct and sharp emission lines in the visible and near-infrared (NIR) range. Here we apply, in addition to continuous 522 nm irradiation, a modulated NIR irradiation (1143 nm) to actively control and modulate the red emission intensity (around 650 nm). The modulation of red Er3+ ion emission at a chosen frequency allows us to reconstruct fluorescence images from the Fourier transform amplitude at this particular frequency...
September 5, 2018: Methods and Applications in Fluorescence
Shachar Schidorsky, Xiyu Yi, Yair Razvag, Julia Sajman, Kobi Hermon, Shimon Weiss, Eilon Sherman
Single-molecule-localization-microscopy (SMLM) and superresolution-optical-fluctuation-imaging (SOFI) enable imaging biological samples well beyond the diffraction-limit of light. SOFI imaging is typically faster, yet has lower resolution than SMLM. Since the same (or similar) data format is acquired for both methods, their algorithms could presumably be combined synergistically for reconstruction and improvement of overall imaging performance. For that, we first defined a measure of the acquired-SNR for each method...
September 18, 2018: Methods and Applications in Fluorescence
Dzmitry Melnikau, Saioa Elcoroaristizabal, Alan G Ryder
The accurate fluorescence analysis of complex, multi-fluorophore containing proteins requires the use of multi-dimensional measurement techniques. For the measurement of intrinsic fluorescence from tyrosine (Tyr) and tryptophan (Trp) one needs tuneable UV excitation and for steady-state measurements like Excitation Emission Matrix (EEM) simple pulsed Xe lamps are commonly used. Unfortunately, simultaneous multi-dimensional wavelength and time resolved measurement of intrinsic protein fluorescence in the 260 to 400 nm spectral range are challenging and typically required the use of very complex tuneable laser systems or multiple single excitation wavelength sources...
September 3, 2018: Methods and Applications in Fluorescence
N Razgoniaeva, S Rogers, P Moroz, J Cassidy, M Zamkov
The visualization of distinct molecular species represents an important challenge of bio-imaging research. In past decades, the development of multicolor fluorescent (FL) labels has greatly improved our ability to track biological analytes, paving the way for important advances in understanding the cell dynamics. It remains challenging, however, to visualize a large number of different fluorephores simultaneously. Owing to a spectrally broad absorption of fluorescent dyes, only up to five color categories can be resolved at once...
August 14, 2018: Methods and Applications in Fluorescence
Henriett Halász, Ali Reza Ghadaksaz, Tamás Madarász, Krisztina Huber, Gábor Harami, Eszter Angéla Tóth, Anikó Osteikoetxea-Molnár, Mihály Kovács, Zsolt Balogi, Miklós Nyitrai, János Matkó, Edina Szabó-Meleg
Membrane nanotubes are transient long-distance connections between cells that can facilitate intercellular communication. These tethers can form spontaneously between many cell types, including cells of the immune and nervous systems. Traffic of viral proteins, vesicles, calcium ions, mRNA, miRNA, mitochondria, lysosomes and membrane proteins/raft domains have all been reported so far via the open ended tunneling nanotubes (TNTs). Recently we reported on existence of plasma membrane derived GM1 /GM3 ganglioside enriched microvesicles and costimulatory proteins in nanotubes connecting B lymphocytes, the way they are formed and transported across TNTs, however, still remained unclear...
August 9, 2018: Methods and Applications in Fluorescence
Fanyong Yan, Xiaodong Sun, Fanlin Zu, Zhangjun Bai, Yingxia Jiang, Keqing Fan, Jie Wang
Cysteine plays a crucial role in physiological processes. Therefore, it is necessary to develop a method for detecting cysteine. Fluorimetry has the advantages of convenient detection, short response time, high sensitivity and good selectivity. In this review, fluorescent probes that detect cysteine over the past three years are summarized based on structural features of fluorophores such as coumarin, BODIPY, rhodamine, fluorescein, CDs, QDs, etc and reaction groups including acrylate, aldehyde, halogen, 7-nitrobenzofurazan, etc...
August 6, 2018: Methods and Applications in Fluorescence
Bryan Donaphon, Linda B Bloom, Marcia Levitus
Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of fluorescence approaches that require the interpretation of fluorescence intensities, lifetimes and fluctuations. Here, we report on a systematic study of the photophysical properties of three rhodamine dyes (tetramethylrhodamine, Alexa 488 and Alexa 546) covalently bound to the E...
July 26, 2018: Methods and Applications in Fluorescence
Zhiqian Zhang, Changli Zhong, Taixian Yuan, Xiaoyan Zhou, Min Zhao, Husun Qian, Wei Cheng, Tingmei Chen
A hybridized chain reaction (HCR)-based biosensing method has been developed for the imaging detection of intracellular telomerase activity. The telomerase-targeting responder-transmitter DNA complex (HPT) consisting of telomerase primer sequence (HP) and a HCR initiator (trigger) is transfected into cell plasma. In the presence of telomerase, HPT can be recognized and extended, producing plenty of triggers which initiate HCR amplification reaction. Finally, a long nicked dsDNA with a lot of outstretched single chains was formed by hybridizing with Q of the reporter complex, generating an enhanced fluorescence signal...
July 13, 2018: Methods and Applications in Fluorescence
Liisa M Hirvonen, Susan Cox
Superresolution microscopy based on localisation is usually performed in a buffer containing enzymatic oxygen scavenger, which facilitates reversible photoswitching of the dye molecules. This makes correlative fluorescence localisation and atomic force microscopy (AFM) challenging, because enzymatic oxygen scavenging interferes with the AFM cantilevers. Here we report on the blinking kinetics of a new red cyanine dye, iFluor-647, which is similar to the Alexa-647 dye commonly used for superresolution microscopy, but with brightness and blinking properties which are superior to Alexa-647 in a buffer without enzymatic oxygen scavenger...
July 9, 2018: Methods and Applications in Fluorescence
Iryna Lysova, Coralie Spiegelhalter, Eleonore Réal, Sarwat Zgheib, Halina Anton, Yves Mély
Visualization of viruses in the host cell during the course of infection by correlative light-electron microscopy (CLEM) requires a specific labelling of the viral structures in order to recognize the nanometric viral cores in the intracellular environment. For Human immunodeficiency virus type 1 (HIV-1), the labelling approaches developed for fluorescence microscopy are generally not suited for transmission electron microscopy (TEM), so that imaging of HIV-1 particles in infected cells by CLEM is not straightforward...
July 9, 2018: Methods and Applications in Fluorescence
Ben A Russell, Ashley Garton, Abeer Saeed Alshammari, David J S Birch, Yu Chen
Fluorescent protein-encapsulated gold nanoclusters (AuNCs) offer a non-toxic means of sensing and imaging biological phenomena on the nanoscale. However, the biofunctionality of proteins encapsulating AuNCs has not been fully elucidated to date. Here we studied the biofunctionality of the second major drug binding site (Sudlow II) of Human Serum Albumin (HSA) encapsulated AuNCs after AuNC synthesis. L-Dopa, a fluorescent drug molecule associated with the clinical treatment of Parkinson's disease, which commonly binds to the Sudlow II site, was used to study the availability of the site before and after AuNC synthesis through changes to its fluorescence characteristics...
July 2, 2018: Methods and Applications in Fluorescence
Aleš Holoubek, Petr Herman, 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 29, 2018: Methods and Applications in Fluorescence
Bingyan Han, Tingting Peng, Ying Li, Mingbo Yu, Xixi Hu, Gaohong He
Water-soluble and ultra-stable L-proline-protected copper nanoclusters (Cu NCs) are reported, which overcome the bottleneck that Cu NCs are easily oxidized. The average size of Cu NCs is found to be 1.88 ± 0.17 nm. The as-prepared Cu NCs exhibit an excellent stability over the physiologically relevant pH range, long storage time, high ionic strength, high concentration of hydrogen peroxide and various common metal ions. The fluorescence intensity of Cu NCs maintains no change after 5 months stored in the refrigerator...
June 28, 2018: Methods and Applications in Fluorescence
A K Choudhary, S K Singh, A Dwivedi, A Bahadur, S B Rai
There are two key factors to design an efficient green upconversion (UC) emission based optical sensor for temperature. The primary need is to develop a thermally stable and economical material, for a stable sensor, and the second essence is to get an efficient green UC emission, for high sensitivity of the sensor. The proof of this concept is demonstrated on a model system CaAl2 O4 : Er3+ , co-doped with Yb3+ and Zn2+ . UC emission of Er3+ ion is enhanced, primarily, through co-operative energy transfer from Yb3+ to Er3+ ions...
June 21, 2018: Methods and Applications in Fluorescence
Bahareh Babamiri, Rahman Hallaj, Abdollah Salimi
In the present study, we constructed an ultrasensitive solid surface fluorescence-immunosensor based on highly luminescent CdTe@CdS-PAMAM structures as nanoprobe for determination of HBsAg by monitoring fluorescence intensity. This strategy was achieved by using PAMAM as a signal amplifier; the PAMAM dendrimer with the many functional amine groups can amplify the fluorescence signal of QDs by covalent attachment of CdTe@CdS on PAMAM and hence, improve the sensitivity of the proposed method significantly. A sandwich type immunosensor was formed after the addition of HBsAg and the PAMAM-QD-Ab2 , respectively...
June 20, 2018: Methods and Applications in Fluorescence
Alizé Proust, Julie-Christine Lévesque, Corinne Barat, Sachiko Sato, Michel J Tremblay
Extracellular traps ejected by various immune cells (neutrophils, macrophages, eosinophils and mast cells) have several immune functions, either protective against pathogens or deleterious in some autoimmune or inflammatory disorders. Since their first description in 2004, the mechanisms of extracellular traps formation have been extensively investigated though still not fully understood. We describe here a new tool for the detection of extracellular traps by fluorescence microscopy in a single-step staining protocol, which does not require any wash...
May 29, 2018: Methods and Applications in Fluorescence
Rukiye Ayranci, Fatma Ozturk Kirbay, Dilek Odaci Demirkol, Metin Ak, Suna Timur
A simple, rapid and effective fluorescence sensing platform has been fabricated using a fluorescent conducting polymer surface. For this purpose, a rhodamine based electroactive monomer (RDC) and a functional group containing monomer (SNS) have been copolymerized to develop a conducting polymer based sensor platform having a fluorescence and enzyme-binding surface on ITO electrode. The proposed fluorescence sensing mechanism for detection of glucose is related to the consumption of dissolved oxygen at the double layer of the electrode which is fluorescence quenching agent by glucose-GOx reaction...
May 29, 2018: Methods and Applications in Fluorescence
Linlin Zhong, Kyusik Yun
A novel fluorescence 'Switch on' for the detection of heparin based on the RhB-COL/GO system was achieved. A strong fluorescence dye, Rhodamine B, was modified by chitosan oligosaccharide lactate (COL), which plays a major role in the formation of a positively charged RhB-COL complex. RhB-COL was soluble and stable in solution, which was characterized by using Fourier transform infrared spectroscopy and x-ray photoelectron spectroscopy. GO sheets quenched the fluorescence intensity of RhB-COL due to electron transfer from RhB to the GO surface...
May 29, 2018: Methods and Applications in Fluorescence
James D Manton, Yao Xiao, Robert D Turner, Graham Christie, Eric J Rees
It is often necessary to precisely quantify the size of specimens in biological studies. When measuring feature size in fluorescence microscopy, significant biases can arise due to blurring of its edges if the feature is smaller than the diffraction limit of resolution. This problem is avoided if an equation describing the feature's entire image is fitted to its image data. In this paper we present open-source software, ELM, which uses this approach to measure the size of spheroidal or cylindrical fluorescent shells with a precision of around 10 nm...
May 16, 2018: Methods and Applications in Fluorescence
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