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Methods: a Companion to Methods in Enzymology

Elliot L Elson
No abstract text is available yet for this article.
November 16, 2017: Methods: a Companion to Methods in Enzymology
Henry Hui, Kathryn A Fuller, Hun Chuah, James Liang, Hasib Siddiqi, Dejan Radeski, Wendy N Erber
Chronic Lymphocytic Leukaemia (CLL), the most common leukaemia in the Western world, has a characteristic phenotype and prognosis largely defined by the presence of cytogenetic aberrations. The gold standard for detecting these cytogenetic abnormalities is interphase fluorescence in situ hybridisation (FISH) performed on cell smears or tissue sections on glass slides. Fluorescently labelled DNA probes bind to specific chromosomal regions and the signal detected by fluorescent microscopy. Generally only 200 cells are assessed and the limit of sensitivity is 3% positive cells...
November 15, 2017: Methods: a Companion to Methods in Enzymology
Sara De Biasi, Lara Gibellini, Alberto Feletti, Giacomo Pavesi, Elena Bianchini, Domenico Lo Tartaro, Simone Pecorini, Anna De Gaetano, Rosalberta Pullano, Milena Nasi, Marcello Pinti, Andrea Cossarizza
Circulating endothelial cells (CECs) detach from the intima monolayer after endothelial damages. Their circulating endothelial progenitors (CEPs) represent less than 0.01% of nucleated blood cells. Increased levels of CECs and CEPs have been detected in patients with several types of cancer, suggesting that they could be a useful blood-based marker for detecting a tumor, or for monitoring its clinical course. However, their routine monitoring is time consuming and technically challenging. Here, we present a flow cytometry method for quantifying such cells in a cohort of patients with hemangioblastoma (HB)...
November 10, 2017: Methods: a Companion to Methods in Enzymology
Snow H Tseng
Optical techniques are assuming greater importance in biomedical applications, however, due to extreme complexity involved in light propagation through scattering medium, it is very challenging to analyze experimentally. Here we report a two-stage simulation technique to simulate phase-conjugated light propagation through scattering medium with macroscopic dimensions. The reported simulation yields accurate information with flexibility to access research parameters. The proposed simulation method is suitable for finite-difference time-domain (FDTD) technique, pseudospectral time-domain (PSTD) technique, and other simulation techniques based upon numerical solutions of Maxwell's equations...
November 7, 2017: Methods: a Companion to Methods in Enzymology
Shikhar Uttam, Yang Liu
Quantitative phase imaging (QPI) modality has been widely adopted in a variety of applications ranging from identifying photomask defects in lithography to characterizing cell structure and tissue morphology in cancer. Traditional QPI utilizes the electromagnetic phase of transmitted light to measure, with nanometer scale sensitivity, alterations in the optical thickness of a sample of interest. In our work, the QPI paradigm is generalized to study depth-resolved properties of phase objects with slowly varying refractive index without a strong interface by utilizing the Fourier phase associated with Fourier-domain optical coherence tomography (FD-OCT)...
November 7, 2017: Methods: a Companion to Methods in Enzymology
Dominic Jenner, Damien Chong, Nicola Walker, A Christopher Green
The endocytosis and trafficking of ricin in mammalian cells is an important area of research for those producing ricin anti-toxins and other ricin therapeutics. Ricin trafficking is usually observed by fluorescence microscopy techniques. This gives good resolution and leads to a detailed understanding of the internal movement of ricin within cells. However, microscopy techniques are often hampered by complex analysis and quantification techniques, and the inability to look at ricin trafficking in large populations of cells...
November 7, 2017: Methods: a Companion to Methods in Enzymology
Sanders Oh, John A Kessler
Expression of exogenous genes using synthetic modified mRNAs has a superior safety profile and higher efficiency than DNA-based techniques. However, mRNA stability and immunogenicity have been significant barriers for wide spread use. The modified mRNAs in this protocol address these shortcomings and can be used to direct cell fate specification or cellular reprogramming.
November 7, 2017: Methods: a Companion to Methods in Enzymology
Patrick A Stockton, Jeffrey J Field, Randy A Bartels
We introduce a new single pixel imaging technique that automatically co-registers quantitative phase and incoherent image modalities through the simultaneous acquisition of identical object spatial frequency information. The technique consists of using a time varying groove density diffraction grating to produce a reference and scan beam. The interference between the beams produce time varying spatial frequencies in the sample. The collected light on a single pixel detector produces a time trace that allows easy recovery of coherent and incoherent contrast mechanisms...
October 26, 2017: Methods: a Companion to Methods in Enzymology
Daniele Ancora, Diego Di Battista, Georgia Giasafaki, Stylianos E Psycharakis, Evangelos Liapis, Jorge Ripoll, Giannis Zacharakis
We describe a computational method for accurate, quantitative tomographic reconstructions in Optical Projection Tomography, based on phase retrieval algorithms. Our method overcomes limitations imposed by light scattering in opaque tissue samples under the memory effect regime, as well as reduces artifacts due to mechanical movements, misalignments or vibrations. We make use of Gerchberg-Saxton algorithms, calculating first the autocorrelation of the object and then retrieving the associated phase under four numerically simulated measurement conditions...
October 25, 2017: Methods: a Companion to Methods in Enzymology
D Gillies, W Gamal, A Downes, Y Reinwald, Y Yang, A El Haj, P O Bagnaninchi
Cell mechanical behaviour is increasingly recognised as a central biophysical parameter in cancer and stem cell research, and methods of investigating their mechanical behaviour are therefore needed. We have developed a novel qualitative method based on quantitative phase imaging which is capable of investigating cell mechanical behaviour in real-time at cellular resolution using Optical Coherence Phase Microscopy (OCPM), and stimulating the cells non-invasively using hydrostatic pressure. The method was exemplified to distinguish between cells with distinct mechanical properties, and transient change induced by Cytochalasin D...
October 25, 2017: Methods: a Companion to Methods in Enzymology
Poorya Hosseini, Di Jin, Zahid Yaqoob, Peter T C So
Interferometric microscopy (IM) can provide complex field information of the biological samples with high spatial and temporal resolution with virtually no photodamage. Measuring wavelength-dependent information in particular has a wide range of applications from cell and tissue refractometry to the cellular biophysical measurements. IM measurements at multiple wavelengths are typically associated with a loss in temporal resolution, field of view, stability, sensitivity, and may involve using expensive equipment such as tunable filters or spatial light modulators...
October 24, 2017: Methods: a Companion to Methods in Enzymology
Katja M Piltti, Brian J Cummings, Krystal Carta, Ayla Manughian-Peter, Colleen Worne, Kulbir Singh, Danier Ong, Yuriy Maksymyuk, Michelle Khine, Aileen J Anderson
Neural stem cell (NSC) cultures have been considered technically challenging for time-lapse analysis due to high motility, photosensitivity, and growth at confluent densities. We have tested feasibility of long-term live-cell time-lapse analysis for NSC migration and differentiation studies. Here, we describe a method to study the dynamics of cell cycle, migration, and lineage selection in cultured multipotent mouse or human NSCs using single-cell tracking during a long-term, 7-14 day live-cell time-lapse analysis...
October 16, 2017: Methods: a Companion to Methods in Enzymology
Wojciech Krauze, Arkadiusz Kuś, Dariusz Śladowski, Ewa Skrzypek, Małgorzata Kujawińska
In the paper we present a novel method of extended depth-of-field limited-angle optical diffraction tomography in which the change of a focal plane position is performed with a liquid focus-tunable lens. One sinogram is acquired for each state of a focus-tunable lens. After acquisition process is complete, all sinograms are independently reconstructed and stitched to form the final tomographic reconstruction. The presented solution effectively extends the applicability of the Rytov approximation to relatively thick samples and provides uniform resolution of 3D tomographic reconstructions...
October 13, 2017: Methods: a Companion to Methods in Enzymology
Sabine Münst, Philipp Koch, Jaideep Kesavan, Michael Alexander-Mays, Bernhard Münst, Sandra Blaess, Oliver Brüstle
The neural crest (NC) is a transient embryonic cell population with remarkable characteristics. After delaminating from the neural tube, NC cells (NCCs) migrate extensively, populate nearly every tissue of the body and differentiate into highly diverse cell types such as peripheral neurons and glia, but also mesenchymal cells including chondrocytes, osteocytes, and adipocytes. While the NC has been extensively studied in several animal models, little is known about human NC development. A number of methods have been established to derive NCCs in vitro from human pluripotent stem cells (hPSC)...
October 13, 2017: Methods: a Companion to Methods in Enzymology
Baoshan Guo, Cheng Lei, Yi Wu, Hirofumi Kobayashi, Takuro Ito, Yaxiaer Yalikun, Sangwook Lee, Akihiro Isozaki, Ming Li, Yiyue Jiang, Atsushi Yasumoto, Dino Di Carlo, Yo Tanaka, Yutaka Yatomi, Yasuyuki Ozeki, Keisuke Goda
Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform...
October 12, 2017: Methods: a Companion to Methods in Enzymology
Sriganesh Srihari
No abstract text is available yet for this article.
October 9, 2017: Methods: a Companion to Methods in Enzymology
Pinkie J Eravuchira, Simcha K Mirsky, Itay Barnea, Mattan Levi, Michal Balberg, Natan T Shaked
The selection of sperm cells possessing normal morphology and motility is crucial for many assisted reproductive technologies (ART), especially for intracytoplasmic sperm injection (ICSI), as sperm quality directly affects the probability of inducing healthy pregnancy. We present a novel platform for real-time quantitative analysis and selection of individual sperm cells without staining. Towards this end, we developed an integrated approach, combining interferometric phase microscopy (IPM), for stain-free sperm imaging and real-time automatic analysis based on the sperm cell 3D morphology and contents, with a disposable microfluidic device, for sperm selection and enrichment...
September 27, 2017: Methods: a Companion to Methods in Enzymology
Ankit Roy, Sanjana Nair, Neeladri Sen, Neelesh Soni, M S Madhusudhan
It has been twenty years since the first rationally designed small molecule drug was introduced into the market. Since then, we have progressed from designing small molecules to designing biotherapeutics. This class of therapeutics includes designed proteins, peptides and nucleic acids that could more effectively combat drug resistance and even act in cases where the disease is caused because of a molecular deficiency. Computational methods are crucial in this design exercise and this review discusses the various elements of designing biotherapeutic proteins and peptides...
September 27, 2017: Methods: a Companion to Methods in Enzymology
Dominic Waithe, Falk Schneider, Jakub Chojnacki, Mathias Clausen, Dilip Shrestha, Jorge Bernardino de la Serna, Christian Eggeling
Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems...
September 26, 2017: Methods: a Companion to Methods in Enzymology
Iryna Nikolayeva, Oriol Guitart-Pla, Benno Schwikowski
Biological processes often manifest themselves as coordinated changes across modules, i.e., sets of interacting genes. Commonly, the high dimensionality of genome-scale data prevents the visual identification of such modules, and straightforward computational search through a set of known pathways is a limited approach. Therefore, tools for the data-driven, computational, identification of modules in gene interaction networks have become popular components of visualization and visual analytics workflows. However, many such tools are known to result in modules that are large, and therefore hard to interpret biologically...
September 20, 2017: Methods: a Companion to Methods in Enzymology
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