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3D Orbital Tracking in a Modified Two-photon Microscope: An Application to the Tracking of Intracellular Vesicles

The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a ...

University of California, Irvine

We investigate the role of self-assembly monolayers in modulating the response of organic field-effect transistors. Alkanethiol monolayers of chain length n are self-assembled on the source and drain electrodes of pentacene field-effect transistors. The charge carrier mobility mu exhibits large fluctuations correlated with odd-even n. For n < 8, mu increases by 1 order of magnitude owing to the decrease of the hole injection barrier and the improved molecular order at the organic-metallic interface. For n > or = 8, mu decays exponentially with an inverse decay length beta = 0.6 A(-1). Our results show that (i) charge injection across the interface occurs by through-bond tunneling of holes mediated by the alkanethiol layer; (ii) in the long-chain regime, the charge injection across the alkanethiol monolayer completely governs the transistor response; (iii) the transistor is a sensitive gauge for probing charge transport across single monolayers. The odd-even effect is ascribed to the anisotropic coupling between the alkanethiol terminal sigma bond and the HOMO level of ordered pentacene molecules.

Charge injection across self-assembly monolayers in organic field-effect transistors: odd-even effects.

We investigate by a scanning probe technique termed phase-electrostatic force microscopy the local electrostatic potential and its correlation to the morphology of the organic semiconductor layer in operating ultra-thin film pentacene field effect transistors. This technique yields a lateral resolution of about 60 nm, allowing us to visualize that the voltage drop across the transistor channel is step-wise. Spatially localized voltage drops, adding up to about 75% of the potential difference between source and drain, are clearly correlated to the morphological domain boundaries in the pentacene film. This strongly supports and gives a direct evidence that in pentacene ultra-thin film transistors charge transport inside the channel is ultimately governed by domain boundaries.

High-resolution mapping of the electrostatic potential in organic thin-film transistors by phase electrostatic force microscopy.

We used AFM to investigate the interaction of polyelectrolytes such as ssDNA and dsDNA molecules with graphene as a substrate. Graphene is an appropriate substrate due to its planarity, relatively large surfaces that are detectable via an optical microscope, and straightforward identification of the number of layers. We observe that in the absence of the screening ions deposited ssDNA will bind only to the graphene and not to the SiO(2) substrate, confirming that the binding energy is mainly due to the π-π stacking interaction. Furthermore, deposited ssDNA will map the graphene underlying structure. We also quantify the π-π stacking interaction by correlating the amount of deposited DNA with the graphene layer thickness. Our findings agree with reported electrostatic force microscopy (EFM) measurements. Finally, we inspected the suitability of using a graphene as a substrate for DNA origami-based nanostructures.

ssDNA binding reveals the atomic structure of graphene.

Identification of clustering artifacts in photoactivated localization microscopy.

In this work we discuss how to use photophysical information for improved quantitative measurements using Photo Activated Localization Microscopy (PALM) imaging. We introduce a method that reliably estimates the number of photoblinking molecules present in a biological sample and gives a robust way to quantify proteins at the single-cell level from PALM images. We apply this method to determine the amount of β2 adrenergic receptor, a prototypical G Protein Coupled Receptor, expressed on the plasma membrane of HeLa cells.

Quantitative photo activated localization microscopy: unraveling the effects of photoblinking.

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 β2-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 β2 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 β2 receptor clustering is associated with the raft.

Cell type-specific β2-adrenergic receptor clusters identified using photoactivated localization microscopy are not lipid raft related, but depend on actin cytoskeleton integrity.

The possibility to visualize and image the arrangement of proteins within the cell at the molecular level has always been an attraction for scientists in biological research. In particular, for signalling molecules such as GPCRs (G-protein-coupled receptors), the existence of protein aggregates such as oligomers or clusters has been the topic of extensive debate. One of the reasons for this lively argument is that the molecular size is below the diffraction-limited resolution of the conventional microscopy, precluding the direct visualization of protein super-structures. On the other hand, new super-resolution microscopy techniques, such as the PALM (photoactivated localization microscopy), allow the limit of the resolution power of conventional optics to be broken and the localization of single molecules to be determined with a precision of 10-20 nm, close to their molecular size. The application of super-resolution microscopy to study the spatial and temporal organization of GPCRs has brought new insights into receptor arrangement on the plasma membrane. Furthermore, the use of this powerful microscopy technique as a quantitative tool opens up the possibility for investigating and quantifying the number of molecules in biological assemblies and determining the protein stoichiometry in signalling complexes.

Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy.

In this contribution we provide an overview of the recent advances allowed by the use of fluorescence microscopy methods in the study of transcriptional processes and their interplay with the chromatin architecture in living cells. Although the use of fluorophores to label nucleic acids dates back at least to about half a century ago, (1) two recent breakthroughs have effectively opened the way to use fluorescence routinely for specific and quantitative probing of chromatin organization and transcriptional activity in living cells: namely, the possibility of labeling first the chromatin loci and then the mRNA synthesized from a gene using fluorescent proteins. In this contribution we focus on methods that can probe rapid dynamic processes by analyzing fast fluorescence fluctuations.

Article	Year
Charge injection across self-assembly monolayers in organic field-effect transistors: odd-even effects. Journal of the American Chemical Society\| PubMed ID: 17472377	2007
High-resolution mapping of the electrostatic potential in organic thin-film transistors by phase electrostatic force microscopy. The journal of physical chemistry. A\| PubMed ID: 18052312	2007
ssDNA binding reveals the atomic structure of graphene. Langmuir : the ACS journal of surfaces and colloids\| PubMed ID: 20977263	2010
Identification of clustering artifacts in photoactivated localization microscopy. Nature methods\| PubMed ID: 21666669	2011
Quantitative photo activated localization microscopy: unraveling the effects of photoblinking. PloS one\| PubMed ID: 21818365	2011
Cell type-specific β2-adrenergic receptor clusters identified using photoactivated localization microscopy are not lipid raft related, but depend on actin cytoskeleton integrity. The Journal of biological chemistry\| PubMed ID: 22442147	2012
Enlightening G-protein-coupled receptors on the plasma membrane using super-resolution photoactivated localization microscopy. Biochemical Society transactions\| PubMed ID: 23356282	2013
Advanced fluorescence microscopy methods for the real-time study of transcription and chromatin dynamics. Transcription\| PubMed ID: 24802848	2014

Paolo Annibale

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