The emergence of nanopores in next-generation sequencing

Next-generation sequencing methods based on nanopore technology have recently gained considerable attention, mainly because they promise affordable and fast genome sequencing by providing long read lengths (5 kbp) and do not require additional DNA amplification or enzymatic incorporation of modified nucleotides. This permits health care providers and research facilities to decode a genome within hours for less than $1000. This review summarizes past, present, and future DNA sequencing techniques, which are realized by nanopore approaches such as those pursued by Oxford Nanopore Technologies. © 2015 IOP Publishing Ltd

Cell-type-specific 2 adrenergic receptor clusters identified using photo-activated localization microscopy are not lipid raft related, but depend on actin cytoskeleton integrity

Recent developments in the field of optical super-resolution techniques allow both a 10-fold increase in resolution as well as an increased ability to quantify the number of labeled molecules visualized in the fluorescence measurement. By using photoactivated localization microscopy (PALM) and an experimental approach based on the systematic comparison with a nonclustering peptide as a negative control, we found that the prototypical G protein-coupled receptor beta 2-adrenergic receptor is partially preassociated in nanoscale-sized clusters only in the cardiomyocytes, such as H9C2 cells, but not in other cell lines, such as HeLa and Chinese hamster ovary (CHO). The addition of the agonist for very short times or the addition of the inverse agonist did not significantly affect the organization of receptor assembly. To investigate the mechanism governing cluster formation, we altered plasma membrane properties with cholesterol removal and actin microfilament disruption. Although cholesterol is an essential component of cell membranes and it is supposed to be enriched in the lipid rafts, its sequestration and removal did not affect receptor clustering, whereas the inhibition of actin polymerization did decrease the number of clusters. Our findings are therefore consistent with a model in which beta 2 receptor clustering is influenced by the actin cytoskeleton, but it does not rely on lipid raft integrity, thus ruling out the possibility that cell type-specific beta 2 receptor clustering is associated with the raft

Photoactivatable Fluorescent Protein mEos2 Displays Repeated Photoactivation after a Long-Lived Dark State in the Red Photoconverted Form

Illumination with 405 nm light can recover the emission for single green fluorescent protein (GFP) mutants that have gone into a long-lived dark state. The reported behavior is the standard for the reverse photoswitchable protein Dronpa and its mutants. However, conventional knowledge regarding the mEos2 photoactivatable fluorescent protein (PA-FP) is that, once bleached, this fluorophore is hardly reactivated, aside from a minority population that might display this behavior. Here we show that, in a typical experiment, approximately 50% of the investigated single molecule time traces display multiple reactivations, making this a seemingly inherent feature of the mEos2 PA-FP. These results hint to some similarities between mEos2 and other reversibly photoactivatable probes such as Dronpa. We investigate the consequences of this phenomenon in the framework of photoactivated localization microscopy (PALM) experiments

Challenges in quantitative single molecule localization microscopy

Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo-activable fluorescent proteins (PA-FPs) are used as labels, we focus specifically on Photo-Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co-localization analysis. (C) 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Progress in quantitative single-molecule localization microscopy

With the advent of single-molecule localization microscopy (SMLM) techniques, intracellular proteins can be imaged at unprecedented resolution with high specificity and contrast. These techniques can lead to a better understanding of cell functioning, as they allow, among other applications, counting the number of molecules of a protein specie in a single cell, studying the heterogeneity in protein spatial organization, and probing the spatial interactions between different protein species. However, the use of these techniques for accurate quantitative measurements requires corrections for multiple inherent sources of error, including: overcounting due to multiple localizations of a single fluorophore (i.e., photoblinking), undercounting caused by incomplete photoconversion, uncertainty in the localization of single molecules, sample drift during the long imaging time, and inaccurate image registration in the case of dual-color imaging. In this paper, we review recent efforts that address some of these sources of error in quantitative SMLM and give examples in the context of photoactivated localization microscopy (PALM)

Nonlinear Optical Response in Single Alkaline Niobate Nanowires

We have synthesized and characterized three types of perovskite alkaline niobate nanowires: NaNbO3, KNbO3, and LiNbO3 (XNbO3). All three types of nanowires exhibit strong nonlinear response. Confocal imaging has been employed to quantitatively compare the efficiency of synthesized nanowires to generate second harmonic signal and to show that LiNbO3 nanowires exhibit the strongest nonlinear response. We also investigated the polarization response of the second harmonic generation (SHG) signal in all three types of alkaline nanowires for the two geometries tractable by our optical trapping setup. The SHG signal is highly influenced by the nanowire crystallinity and experimental geometry. We also demonstrate for the first time wave-guiding of SHG signal in all three types of alkaline niobate nanowires. By carefully examining nonlinear properties of (XNbO3) nanowires we suggest which type of wires are best suited for the given application

Controllable Shrinking and Shaping of Glass Nanocapillaries under Electron Irradiation

The ability to reshape nanopores and observe their shrinkage under an electron microscope is a powerful and novel technique. It increases the sensitivity of the resistive pulse sensing and enables to detect very short and small molecules. However, this has not yet been shown for glass nanocapillaries. In contrast to their solid-state nanopore counterparts, nanocapillaries are cheap, easily fabricated and in the production do not necessitate clean room facilities. We show for the first time that quartz nanocapillaries can be shrunken under a scanning electron microscope beam. Since the shrinking is caused by the thermal heating of the electrons, increasing the beam current increases the shrink rate. Higher acceleration voltage on the contrary increases the electron penetration depth and reduces the electron density causing slower shrinkage. This allows us to fine control the shrink rate and to stop the shrinking process at any desired diameter. We show that a shrunken nanocapillary detects DNA translocation with six times higher signal amplitudes than an unmodified nanocapillary. This will open a new path to detect small and short molecules such as proteins or RNA with nanocapillaries

Probing the size of proteins with glass nanopores

Single molecule studies using nanopores have gained attention due to the ability to sense single molecules in aqueous solution without the need to label them. In this study, short DNA molecules and proteins were detected with glass nanopores, whose sensitivity was enhanced by electron reshaping which decreased the nanopore diameter and created geometries with a reduced sensing length. Further, proteins having molecular weights (MW) ranging from 12 kDa to 480 kDa were detected, which showed that their corresponding current peak amplitude changes according to their MW. In the case of the 12 kDa ComEA protein, its DNA-binding properties to an 800 bp long DNA molecule was investigated. Moreover, the influence of the pH on the charge of the protein was demonstrated by showing a change in the translocation direction. This work emphasizes the wide spectrum of detectable molecules using nanopores from glass nanocapillaries, which stand out because of their inexpensive, lithography-free, and rapid manufacturing proces

Photodissociation of the OD radical at 226 and 243 nm

The photodissociation dynamics of state selected OD radicals has been examined at 243 and 226 nm using velocity map imaging to probe the angle–speed distributions of theD(2S) and O(3P2) products. Both experiment and complementary first principle calculations demonstrate that photodissociation occurs by promotion of OD from high vibrational levels of the ground X 2Π state to the repulsive 1 2Σ− state