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

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)

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

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)

Nanowire assembly, e.g. for optical probes, comprises optically trapping high aspect ratio semiconductor nanowire with infrared single-beam optical trap and attaching nanowire to organic or inorganic structure

NOVELTY - A nanowire assembly method comprises optically trapping a semiconductor nanowire with an infrared single-beam optical trap and attaching the nanowire to an organic or inorganic structure by laser fusing. The nanowire is further trapped in a fluid environment. The optical trap has a beam wavelength of 1064 nm. The nanowire has an aspect ratio greater than 100 and a diameter less than 100 (preferably less than 80) nm. The nanowire and the organic or inorganic structure form a heterostructure. USE - For fabricating a nanowire assembly for use as e.g. active photonic devices, passive photonic devices, optical probes, subwavelength microscopy. ADVANTAGE - Nanowires with diameters as small as 20 nm and aspect ratios of above one hundred can be trapped and transported in three dimensions, enabling the construction of nanowire architectures which may function in various capacities. Nanowire structures can now be assembled in physiological environments, offering new forms of chemical, mechanical and optical stimulation of living cells. DETAILED DESCRIPTION - An INDEPENDENT CLAIM is included for a nanowire assembly comprising a nanowire and an organic or inorganic structure or an arbitrary structure. DESCRIPTION OF DRAWING(S) - The figure shows a schematic diagram of an optical tweezers instrument for nanowire trapping

Fabrication of 10 nm diameter hydrocarbon nanopores

The addition of carbon to samples, during transmission electron microscope imaging, presents a barrier to accurate analysis; the controlled deposition of hydrocarbons by a focused electron beam can be a useful technique for local nanometer-scale sculpting of material. Here we use hydrocarbon deposition to form nanopores from larger focused ion beam holes in silicon nitride membranes. Using this method, we close 100–200 nm diameter holes to diameters of 10 nm and below, with deposition rates of 0.6 nm/min. I-V characteristics of electrolytic flow through these nanopores agree quantitatively with a one dimensional model at all examined salt concentrations