A programmable light engine for quantitative single molecule TIRF and HILO imaging

Single molecule resolution in electron beam sensitive, uncoated, noncrystalline materials has been impossible except in thin Pt-C replicas ≤ 150Å) which are resistant to the electron beam destruction. Previously the granularity of metal film replicas limited their resolution to ≥ 20Å. This paper demonstrates that Pt-C film granularity and resolution are a function of the method of replication and other controllable factors. Low angle 20° rotary , 45° unidirectional and vertical 9.7±1 Å Pt-C films deposited on mica under the same conditions were compared in Fig. 1. Vertical replication had a 5A granularity (Fig. 1c), the highest resolution (table), and coated the whole surface. 45° replication had a 9Å granulartiy (Fig. 1b), a slightly poorer resolution (table) and did not coat the whole surface. 20° rotary replication was unsuitable for high resolution imaging with 20-25Å granularity (Fig. 1a) and resolution 2-3 times poorer (table). Resolution is defined here as the greatest distance for which the metal coat on two opposing faces just grow together, that is, two times the apparent film thickness on a single vertical surface.

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Single molecule optical diffraction: A tool for understanding the structure of triple stranded left-hand helical cellulose

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100157103 ◽

1989 ◽

Vol 47 ◽

pp. 1024-1025

Author(s):

George C. Ruben ◽

William Krakow

Keyword(s):

Single Molecule ◽

Helical Structure ◽

Optical Diffraction ◽

Maximum Diameter ◽

Primary Cell Wall ◽

Cellulose Synthesis ◽

Left Hand ◽

Left Handed ◽

Freeze Dried ◽

Diffraction Patterns

Tobacco primary cell wall and normal bacterial Acetobacter xylinum cellulose formation produced a 36.8±3Å triple-stranded left-hand helical microfibril in freeze-dried Pt-C replicas and in negatively stained preparations for TEM. As three submicrofibril strands exit the wall of Axylinum , they twist together to form a left-hand helical microfibril. This process is driven by the left-hand helical structure of the submicrofibril and by cellulose synthesis. That is, as the submicrofibril is elongating at the wall, it is also being left-hand twisted and twisted together with two other submicrofibrils. The submicrofibril appears to have the dimensions of a nine (l-4)-ß-D-glucan parallel chain crystalline unit whose long, 23Å, and short, 19Å, diagonals form major and minor left-handed axial surface ridges every 36Å.The computer generated optical diffraction of this model and its corresponding image have been compared. The submicrofibril model was used to construct a microfibril model. This model and corresponding microfibril images have also been optically diffracted and comparedIn this paper we compare two less complex microfibril models. The first model (Fig. 1a) is constructed with cylindrical submicrofibrils. The second model (Fig. 2a) is also constructed with three submicrofibrils but with a single 23 Å diagonal, projecting from a rounded cross section and left-hand helically twisted, with a 36Å repeat, similar to the original model (45°±10° crossover angle). The submicrofibrils cross the microfibril axis at roughly a 45°±10° angle, the same crossover angle observed in microflbril TEM images. These models were constructed so that the maximum diameter of the submicrofibrils was 23Å and the overall microfibril diameters were similar to Pt-C coated image diameters of ∼50Å and not the actual diameter of 36.5Å. The methods for computing optical diffraction patterns have been published before.

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Structural dynamics of P-type ATPase ion pumps

Biochemical Society Transactions ◽

10.1042/bst20190124 ◽

2019 ◽

Vol 47 (5) ◽

pp. 1247-1257 ◽

Cited By ~ 17

Author(s):

Mateusz Dyla ◽

Sara Basse Hansen ◽

Poul Nissen ◽

Magnus Kjaergaard

Keyword(s):

Structural Dynamics ◽

Single Molecule ◽

New Technologies ◽

Atp Hydrolysis ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Resolved ◽

Dynamics Simulations ◽

Types Of Information ◽

P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

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Label-free, mass-sensitive single-molecule imaging using interferometric scattering microscopy

Essays in Biochemistry ◽

10.1042/ebc20200023 ◽

2020 ◽

Author(s):

Nikolas Hundt

Keyword(s):

Single Molecule ◽

Cellular Systems ◽

Single Molecule Imaging ◽

Label Free ◽

Measurement Sensitivity ◽

Mass Distributions ◽

Quantitative Measurements ◽

Contrast Mechanism ◽

Cellular Components ◽

Scattering Signal

Abstract Single-molecule imaging has mostly been restricted to the use of fluorescence labelling as a contrast mechanism due to its superior ability to visualise molecules of interest on top of an overwhelming background of other molecules. Recently, interferometric scattering (iSCAT) microscopy has demonstrated the detection and imaging of single biomolecules based on light scattering without the need for fluorescent labels. Significant improvements in measurement sensitivity combined with a dependence of scattering signal on object size have led to the development of mass photometry, a technique that measures the mass of individual molecules and thereby determines mass distributions of biomolecule samples in solution. The experimental simplicity of mass photometry makes it a powerful tool to analyse biomolecular equilibria quantitatively with low sample consumption within minutes. When used for label-free imaging of reconstituted or cellular systems, the strict size-dependence of the iSCAT signal enables quantitative measurements of processes at size scales reaching from single-molecule observations during complex assembly up to mesoscopic dynamics of cellular components and extracellular protrusions. In this review, I would like to introduce the principles of this emerging imaging technology and discuss examples that show how mass-sensitive iSCAT can be used as a strong complement to other routine techniques in biochemistry.

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