Visualization Of Individual Molecules Research Articles

The Benefits of Unnatural Amino Acid Incorporation as Protein Labels for Single Molecule Localization Microscopy.

Single Molecule Localization Microscopy (SMLM) is an imaging method that allows for the visualization of structures smaller than the diffraction limit of light (~200 nm). This is achieved through techniques such as stochastic optical reconstruction microscopy (STORM) and photoactivated localization microscopy (PALM). A large part of obtaining ideal imaging of single molecules is the choice of the right fluorescent label. An upcoming field of protein labeling is incorporating unnatural amino acids (UAAs) with an attached fluorescent dye for precise localization and visualization of individual molecules. For this technique, fluorescent probes are conjugated to UAAs and are introduced into the protein of interest (POI) as a label. Here we contrast this labeling method with other commonly used protein-based labeling methods such as fluorescent proteins (FPs) or self-labeling tags such as Halotag, SNAP-tags, and CLIP-tags, and highlight the benefits and shortcomings of the site-specific incorporation of UAAs coupled with fluorescent dyes in SMLM.

Direct visualization of individual molecules in molecular crystals by electron cloud densitometry

Built on electron beam shifting effect, the picoscope with resolution of 10 picometers demonstrates fascinating ability to directly visualize the density of electron clouds molecules together with chemical bonds. It results in the appearance of long-awaited visual molecular technologies. Densitometry of electron density is used to show the structure of crystalline graphite with visualization of σ-bonds formed by overlaping the sp2 chemical bonds of carbon atoms, and a system of weak π-bonds that connects the layers of graphite. The article also presents new superdense two-layered diamond-like carbon allotrope, which is proposed to designate rudenite. In the rudenite, each carbon atom forms four strong sp3 chemical bonds as in the structure of diamond, but since the layers are pressed at a distance of 100 picometers, it should have a density of 1.3 times greater than that of diamond.

Long-Range RNA-RNA Interactions Circularize the Dengue Virus Genome

Secondary and tertiary RNA structures present in viral RNA genomes play essential regulatory roles during translation, RNA replication, and assembly of new viral particles. In the case of flaviviruses, RNA-RNA interactions between the 5' and 3' ends of the genome have been proposed to be required for RNA replication. We found that two RNA elements present at the ends of the dengue virus genome interact in vitro with high affinity. Visualization of individual molecules by atomic force microscopy revealed that physical interaction between these RNA elements results in cyclization of the viral RNA. Using RNA binding assays, we found that the putative cyclization sequences, known as 5' and 3' CS, present in all mosquito-borne flaviviruses, were necessary but not sufficient for RNA-RNA interaction. Additional sequences present at the 5' and 3' untranslated regions of the viral RNA were also required for RNA-RNA complex formation. We named these sequences 5' and 3' UAR (upstream AUG region). In order to investigate the functional role of 5'-3' UAR complementarity, these sequences were mutated either separately, to destroy base pairing, or simultaneously, to restore complementarity in the context of full-length dengue virus RNA. Nonviable viruses were recovered after transfection of dengue virus RNA carrying mutations either at the 5' or 3' UAR, while the RNA containing the compensatory mutations was able to replicate. Since sequence complementarity between the ends of the genome is required for dengue virus viability, we propose that cyclization of the RNA is a required conformation for viral replication.

How dense are cylindrical brushes grafted from a multifunctional macroinitiator?

A series of cylindrical brushes with poly(methyl methacrylate) (PMMA) and poly( n-butyl acrylate) (PBA) side chains of different lengths was studied to understand the grafting density of brush molecules prepared by the ‘grafting from’ approach. Molecules with PMMA side chains were prepared by grafting MMA from a multifunctional macroinitiator, poly(2-(2-bromoisobutyryloxy)ethyl methacrylate). Molecules with PBA side chains were prepared with poly(2-(2-bromopropionyloxy)ethyl methacrylate) as a macroinitiator. Analysis of the detached side chains showed an incomplete initiation process resulting in longer side chains than expected for complete initiation. Limited initiation (40–80%) was also observed for short PMMA chains using a low molar mass initiator ethyl 2-bromoisobutyrate with CuCl catalyst systems. The lower initiation efficiency was confirmed via visualization of individual molecules by atomic force microscopy. The larger distance between adsorbed brush molecules was consistent with <50% initiation efficiency.

Localization and imaging of nucleic acids on nanoporous aluminum oxide membranes.

The technologies currently available for nucleic acid analysis in the clinical laboratory rely on target amplification methods, such as PCR or transcription-mediated amplification, or signal amplification methods, such as branched-chain DNA, hybrid capture, Invader technology, and fluorescent in situ hybridization. In current nucleic acid analyses, a multistep process consisting of sample preparation and purification followed by amplification and detection is required. A comprehensive method for direct localization and visualization of individual molecules would provide an additional method for qualitative or quantitative nucleic acid analysis. We evaluated a nanoporous aluminum oxide membrane (AOM) as a support for direct localization and visualization of individual nucleic acid molecules. The process described here allows nucleic acids to be localized by filtration onto AOM with direct imaging using nucleic acid-binding stains. Additionally, we present a method for modifying the membranes to improve the optical properties. The membranes used in this study are commercially available under the trade name Anopore™ (Whatman International) and are composed of aluminum oxide with a mean pore size of 200 nm and a nominal thickness of 60 μm (1). The mean width between the pores is 50 nm, and the membranes have a pore density of ∼108 pores/cm2. The pores are evenly distributed in a honeycomb pattern across the surface, with a porosity of ∼50%. Because the length of one DNA base pair is 0.34 nm (2)(3), a linear double-stranded DNA molecule will extend ∼600 bp across a single 200 nm pore, and ∼150 bp of the molecule will cover the area between the pores. The uniform pore structure, the relatively rigid and flat surface, and the high porosity and flow rate make AOMs an attractive material for sample filtration and nucleic acid visualization. The surface properties of aluminum oxide allow for chemical modifications to the …

Atomic force microscopy revelation of molecular complexes in the multiprotein cytochrome P450 2B4-containing system.

The application of atomic force microscopy (AFM) to the identification and visualization of individual molecules and their complexes in a reconstituted monooxygenase P450 2B4 system without the phospholipid was demonstrated. The method employed in this study distinguishes the monomeric proteins from their binary complexes and, also, the binary from the ternary complexes. The AFM images of the full-length P450 2B4 system's constituent components - cytochrome P450 2B4 (2B4), NADPH-cytochrome P450 reductase and cytochrome b5 (b5), were obtained on highly-oriented pyrolitic graphite. The typical heights of the d-2B4, d-flavoprotein (Fp) and d-b5 molecules were measured and found to be 2.2 +/- 0.2, 2.3 +/- 0.2 and 1.8 +/- 0.1 nm, respectively. The measured heights of the binary d-Fp/d-2B4 and d-2B4/d-b5 complexes were estimated to be 3.4 +/- 0.2 and 2.8 +/- 0.2 nm, respectively. No formation of d-Fp/d-b5 complexes was registered. The ternary d-Fp/d-2B4/d-b5 complexes were visualized and their heights were found to be roughly equal to 4.3 +/- 0.3 nm and 6.2 +/- 0.3 nm.

Measuring molecular weight by atomic force microscopy.

Absolute-molecular-weight distribution of cylindrical brush molecules were determined using a combination of the Langmuir Blodget (LB) technique and Atomic Force Microscopy (AFM). The LB technique gives mass density of a monolayer, i.e., mass per unit area, whereas visualization of individual molecules by AFM enables accurate measurements of the molecular density, i.e., number of molecules per unit area. From the ratio of the mass density to the molecular density, one can determine the absolute value for the number average molecular weight. Assuming that the structure of brush molecules is uniform along the backbone, the length distribution should be virtually identical to the molecular weight distribution. Although we used only brush molecules for demonstration purpose, this approach can be applied for a large variety of molecular and colloidal species that can be visualized by a microscopic technique.

Assay for Movements of RNA Polymerase along DNA

Publisher Summary This chapter discusses the assay for movement of RNA polymerase (RNAP) along DNA. The most direct assay of a movement of a protein is carried out by visualization of individual molecules under an optical microscope, which is called “single-molecule dynamics.” This assay detects the inconsistency in the precursor-product relationship of the moribund complex and the elongation complex. If the former remains longer than the latter, the former is not the precursor of the latter. Therefore, this assay examines a sufficient condition for the existence of moribund complex, and thus a negative result does not deny the existence. The best labeling nucleotide for this assay is γ- 32 P–labeled initiation nucleotide, as it eliminates the danger of misunderstanding cleaved products as abortive ones. This assay is performed on the template DNA attached to a bead. The template must have a restriction site at more than 30 bases downstream of the promoter. For isolation of moribund and dead-end complexes, the template DNA has to be fixed at the downstream end. For isolation of productive complex, the upstream end of DNA is fixed and the template should have the early transcribed sequence lacking one nucleotide, a nucleotideless cassette.

Atomic force microscopy detection of molecular complexes in multiprotein P450cam containing monooxygenase system.

The application of atomic force microscopy (AFM) technique in proteomic research, identification and visualization of individual molecules and molecular complexes within the P450cam containing monooxygenase system was demonstrated. The method distinguishes between the binary protein complexes and appropriate monomeric proteins and, also, between the binary and ternary complexes. The AFM images of the components of a cytochrome P450cam containing monooxygenase system - cytochrome P450cam (P450cam), putidaredoxin (Pd) and putidaredoxin reductase (PdR) - were obtained on a mica support. The molecules of P450cam, Pd and PdR were found to have typical heights of 2.6 +/- 0.3 nm, 2.0 +/- 0.3 and 2.8 +/- 0.3 nm, respectively. The measured heights of the binary Pd/PdR and P450cam/PdR complexes were 4.9 +/- 0.3 nm and 5.1 +/- 0.3 nm, respectively. The binary P450cam/Pd complexes were found to have a typical height of about (3.9 / 5.7 nm) and the ternary PdR/Pd/P450cam complexes, a typical height of about 9.1 +/- 0.3 nm.

Synthesis of Molecular Brushes with Block Copolymer Side Chains Using Atom Transfer Radical Polymerization

Brush macromolecules having poly(n-butyl acrylate-block-styrene) and poly(styrene-block-n-butyl acrylate) side chains have been synthesized by the “grafting from” approach using atom transfer radical polymerization (ATRP). The molecular weights of the resulting polymers were characterized by gel permeation chromatography (GPC) using refractive index and multiangle light scattering detection. The block copolymer side chains were cleaved from the backbone and analyzed by GPC, confirming the synthesis of well-defined copolymer brushes. Visualization of individual molecules by atomic force microscopy (AFM) enabled analysis of the conformation and microstructure of the brush macromolecules on mica surface. The brushes with the pnBuA core were almost fully stretched, while the inverted structure with the pS core exhibited longitudinal contraction compared to the contour length of the main chain. In addition, the poly(n-butyl acrylate-block-styrene) brushes demonstrated a characteristic necklace morphology which...

Optimization of adhesion mode atomic force microscopy resolves individual molecules in topography and adhesion

The force sensor of an atomic force microscope (AFM) is sensitive enough to measure single molecular binding strengths by means of a force–distance curve. In order to combine high-force sensitivity with the spatial resolution of an AFM in topography mode, adhesion mode has been developed. Since this mode generates a force–distance curve for every pixel of an image, the measurement speed in liquid is limited by the viscous drag of the cantilever. We have equipped our adhesion mode AFM with a cantilever that has a low viscous drag in order to reach pixel frequencies of 65 Hz. Optimized filtering techniques combined with an auto-zero circuitry that reduces the drift in the deflection signal, limited high- and low-frequency fluctuations in the height signal to 0.3 nm. This reduction of the height noise, in combination with a thermally stabilized AFM, allowed the visualization of individual molecules on mica with an image quality comparable to tapping mode. The lateral resolution in both the topography and the simultaneously recorded adhesion image are only limited by the size of the tip. Hardware and software position feedback systems allows individual molecules to be followed in time during more than 30 min with scan sizes down to 60×60 nm 2

Fascinated by a chain molecule review of 35 years' research on cellulose

AbstractFor over 35 years the author has been involved in cellulose research and his retirement next year is the reason why he has undertaken to present a review on some aspects of his investigations on the fascinating substance cellulose. One topic of his research activities is electron microscopy. At the very start, at the Institute for Cellulose Chemistry (T.H. Darmstadt), observations were made on bordered pits and structural details on the walls of wood and pulp fibres. He was able to continue his teacher's, Georg Jayme's, traditional basic research on cellulose at the Institute for Wood Research (University of Munich) where “dreams” of the fifties and sixties such as the visualization of individual molecules and the growing of macrocrystals of cellulose could be realized. Here, the electron microscope became a vital instrument for the study of cellulose chemistry.

Type V Collagens of the Human Placenta: Trimer α-Chain Composition, Ultrastructural Morphology and Peptide Analysis

The α-chain trimer composition of type V collagen preparations from human placental membrane and villi was determined by two-dimensional electrophoresis on a non-denaturing polyacrylamide gel system followed by electrophoresis in the presence of sodium dodecylsulfate. In preparations isolated from placental membranes pure type V collagen was found with the α-chain composition [α1(V)]2 α2(V). In preparations from placental villi two different collagen trimers could be identified with α-chain compositions [α1(V)]2 α2(V) and [α3(V)]3. Two-dimensional peptide maps after chymotryptic digestion of the various α-chain revealed distinct patterns for α1(V), α2(V) and α3(V) suggesting unique structures for all three α-chains. Shadowing of the two collagen preparations with carbon-platinum by the rotary shadowing technique allowed the visualization of individual molecules. In the placental membrane preparations, a uniform species of molecules was present while in placental villi preparations the same elongated form of collagen was found together with larger aggregates presumably containing molecules with the α3-chain component. These data are interpreted to indicate that so-called type V collagen, at least in preparations from placental villi, contain two distinct collagen molecules.