Standalone interferometry-based calibration of convex lens-induced confinement microscopy with nanoscale accuracy

Morrin, Gregory T.; Kienle, Daniel F.; Schwartz, Daniel K.

doi:10.1039/c8an02300j

Cited by 8 publications

(13 citation statements)

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“…Following the interference pattern measurement, a 30 s movie was recorded, the lens was moved to a new location, and the process was repeated until several movies near the contact region and far from the contact region (to effectively characterize the interfacial behavior with a semi-infinite liquid layer) had been recorded. For a complete description of the imaging system used in these experiments, a detailed description can be found elsewhere …”

Section: Methodsmentioning

confidence: 99%

“…Objects identified in the first or last frames were removed from analysis to prevent the underestimation of waiting times. Height profiles for each movie were determined by converting the radially averaged intensity profiles (generated from their respective interference patterns) to height vs distance profiles, as described previously . Using the absolute stage position as a reference, the center point of the contact region between the lens and coverslip was determined and the distance from the center point to each object was recorded.…”

Section: Methodsmentioning

confidence: 99%

“…In this approach, a convex lens and glass coverslip are used to create a slit whose height varies radially from the contact region between the lens and coverslip, as shown in Figure . In a previous study, slit heights were accurately measured down to 5 nm with a precision of 1 nm using interferometry, permitting accurate measurements of molecular diffusion within the strongly confined regime. In the current experiments, surfaces were modified with various organosilanes, including an oligo(ethylene oxide) (OEG) silane, an aminosilane ( N -(6-aminohexyl)aminopropyltrimethoxysilane (AHAPS)), and a 1:1 AHAPS/OEG mixture, to measure the coupled effects of surface charge and short-range forces on polyelectrolyte surface diffusion.…”

Section: Introductionmentioning

confidence: 99%

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Polyelectrolyte Surface Diffusion in a Nanoslit Geometry

et al. 2020

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The surface diffusion of poly-L-lysine (PLL) in a planar nanoslit was studied using convex lens-induced confinement (CLiC) single-molecule tracking microscopy. Three surface chemistries were employed to understand the interplay of electrostatic and short-range interactions: an amine-functionalized silica surface, an oligo(ethylene oxide) (OEG)-modified surface, and a 1:1 mixture of the two ligands. Effective surface diffusion coefficients increased rapidly with slit height until saturating for slit heights <30 nm. While diffusion at a semi-infinite interface was significantly faster for OEG surfaces, the diffusion coefficient increased most rapidly with slit height for aminefunctionalized surfaces, resulting in surface diffusion within very thin slits being nearly independent of surface chemistry. Intermittent random walks were simulated within a planar slit geometry, using experimentally measured parameters obtained from diffusion at a single interface to account for the characteristic short-range interactions between PLL and each surface chemistry, and were in good agreement with experimental measurements.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Polyelectrolyte Surface Diffusion in a Nanoslit Geometry

et al. 2020

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“…It should be noted here that the generation of confined spaces using a plane–sphere geometry is a well-known procedure. The surface force apparatus, for example, combines this geometry with exquisite sensitivity in force and separation distance, to measure interaction forces between functionalized surfaces; more recently, this approach has also been combined with optical analysis for concurrent studies of the molecular organization and diffusion within the confined fluids by microscopy and spectroscopy techniques. − Plane-sphere geometries have also already been combined with optical microscopy for improved single-molecule imaging − or to visualize dynamic processes under confinement as diverse as blood clot formation, lubricant transfer during interfacial shear, and capillary condensation . Distinct aspects of the here-described method are the application to solvated polymer films and its ease of integration with existing microscopes and imaging modalities, where a substantially static and constant contact force is beneficial to control the compression of the polymer film.…”

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A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins

et al. 2020

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We present a method to probe molecular and nanoparticle diffusion within thin, solvated polymer coatings. The device exploits the confinement with well-defined geometry that forms at the interface between a planar and a hemispherical surface (of which at least one is coated with polymers) in close contact and uses this confinement to analyze diffusion processes without interference of exchange with and diffusion in the bulk solution. With this method, which we call plane–sphere confinement microscopy (PSCM), information regarding the partitioning of molecules between the polymer coating and the bulk liquid is also obtained. Thanks to the shape of the confined geometry, diffusion and partitioning can be mapped as a function of compression and concentration of the coating in a single experiment. The method is versatile and can be integrated with conventional optical microscopes; thus it should find widespread use in the many application areas exploiting functional polymer coatings. We demonstrate the use of PSCM using brushes of natively unfolded nucleoporin domains rich in phenylalanine–glycine repeats (FG domains). A meshwork of FG domains is known to be responsible for the selective transport of nuclear transport receptors (NTRs) and their macromolecular cargos across the nuclear envelope that separates the cytosol and the nucleus of living cells. We find that the selectivity of NTR uptake by FG domain films depends sensitively on FG domain concentration and that the interaction of NTRs with FG domains obstructs NTR movement only moderately. These observations contribute important information to better understand the mechanisms of selective NTR transport.

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“…In CLiC, a large radius convex lens is used to create an effective nanoslit geometry in which the radial height profile can be measured using interferometry. Slit heights as small as 5 nm have been measured with single nanometer precision, providing a useful and accessible environment in which to study biomolecules in nanoscale confinement. In one CLiC study, Leith et al directly measured the free energy of confinement of a linearized, 2.7 kbp pUC19 sample at slit heights ranging from the weak confinement regime (i.e., slit heights larger than the size of the unconfined polymer in solution) to the strong confinement regime by observing the concentration of the polymer at various slit heights for different bulk concentrations.…”

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confidence: 99%

Diffusion of Short Semiflexible DNA Polymer Chains in Strong and Moderate Confinement

2021

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In many technological applications, DNA is confined within nanoenvironments that are smaller than the size of the unconfined polymer in solution. However, the dependence of the diffusion coefficient on molecular weight and characteristic confinement dimension remains poorly understood in this regime. Here, convex lens-induced confinement (CLiC) was leveraged to examine how the diffusion of short DNA fragments varied as a function of slit height by using single-molecule fluorescence tracking microscopy. The diffusion coefficient followed approximate power law behavior versus confinement height, with exponents of 0.27 ± 0.01, 0.32 ± 0.02, and 0.42 ± 0.06 for 692, 1343, and 2686 base pair chains, respectively. The weak dependence on slit height suggests that shorter semiflexible chains may adopt increasingly rodlike conformations and therefore experience weaker excluded-volume interactions as the confinement dimension is reduced. The diffusion coefficient versus molecular weight also exhibited apparent power law behavior, with exponents that varied slightly (from −0.89 to −0.85) with slit height, consistent with hydrodynamic interactions intermediate between Rouse and Zimm model predictions.

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Standalone interferometry-based calibration of convex lens-induced confinement microscopy with nanoscale accuracy

Abstract: Interferometry-based measurement of gap height in convex lens-induced confinement significantly improves accuracy at sub-100 nm gap thickness.

Cited by 8 publications

References 42 publications

Polyelectrolyte Surface Diffusion in a Nanoslit Geometry

Polyelectrolyte Surface Diffusion in a Nanoslit Geometry

A Method to Quantify Molecular Diffusion within Thin Solvated Polymer Films: A Case Study on Films of Natively Unfolded Nucleoporins

Diffusion of Short Semiflexible DNA Polymer Chains in Strong and Moderate Confinement

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