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Precision platform for convex lens-induced confinement microscopy
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10.1063/1.4822276
/content/aip/journal/rsi/84/10/10.1063/1.4822276
http://aip.metastore.ingenta.com/content/aip/journal/rsi/84/10/10.1063/1.4822276

Figures

Image of FIG. 1.
FIG. 1.

Schematic of the CLIC device. (a) Cross-section showing the device components and assembly. (b) Top view of the flow cell used in the CLIC device with double-sided adhesive tape shown in red. (c) Cross-section of the deformed flow cell with exaggerated thickness. (d) Cross-section of lens-rotation mechanism.

Image of FIG. 2.
FIG. 2.

Interactive 3D drawing showing a cross-section of the Z-axis mechanics and sample holder. (To enable interactive 3D viewing, click the figure and use ctrl to pan and shift to zoom.)

Image of FIG. 3.
FIG. 3.

Interactive 3D drawing of the CLIC device. (To enable interactive 3D viewing, click the figure and use ctrl to pan and shift to zoom.)

Image of FIG. 4.
FIG. 4.

Simulation of the CLIC imaging geometry. (a) Cross-section of the deformed flow cell: the top coverslip is pushed into the bottom coverslip by the push-lens. Note the difference in x and z scales. (b) Detailed view of the coverslip-coverslip contact region for two cases: in-contact (left) and over-pushed by 200 nm (right). (c) Height of the chamber (red dots) with quadratic fit (blue line). (d) Flow cell chamber, with the in-focus region shown in blue (∼1.4 mm diameter). (e) Triangular mesh used to model the coverslips, whose precision progressively increases towards the contact point.

Image of FIG. 5.
FIG. 5.

CLIC imaging chamber measurements (left: flow cell CLIC, = 13.8 mm. Right: lens-coverslip CLIC, = 45 mm). Images are shown with the same distance scale. [(a) and (b)] Fluorescence images of Alexa647 dye, shown with the same intensity scale. [(c) and (d)] Interferometry images, with overlaid contours (in green) corresponding to chamber heights which are integer multiples of where λ = 488 nm and = 1.33. [(e) and (f)] Cross sections of the mean dye fluorescence intensity (red dots) and fit-function (blue lines). The radius of curvature, , is determined using a quadratic fit to the central 300 μm diameter region.

Image of FIG. 6.
FIG. 6.

Diffusing linearized pBR322 plasmids. (a) Kymograph of relatively unconfined molecules (∼0.5 μm average chamber height), acquired using a 30 ms exposure time, 150 ms delay between frames, and 0.1 nM DNA concentration. Blue circles and red squares enclose two selected molecules. (b) Respective trajectories of the selected molecules. (c) Histogram of diffusion coefficients, , of molecules with trajectories tracked for longer than 1 s, in a chamber region whose height is between 0.40 and 0.50 μm. The lifetime-weighted mean for these molecules is 1.15 ± 0.08 μm2 s−1. (d) Kymograph of confined molecules (∼0.10 μm chamber height), acquired using a 30 ms exposure time, 150 ms delay between frames, and 10 nM DNA concentration. Blue circles and red squares enclose two selected particles. (e) Respective trajectories of selected particles. (f) Histogram of diffusion coefficients of molecules in a chamber with height ranging between 0.10 and 0.15 μm, using trajectories longer than 1 s. The weighted-mean is reduced to 0.76 ± 0.08 μm2 s−1 by the applied confinement.

Image of FIG. 7.
FIG. 7.

λ-phage DNA ( ∼ 1 μm) confined to chambers of variable average height: (a) = 3.2 μm ∼ 3 (37.5 pM concentration). (b) = 0.5 μm ∼ 0.5 (2400 pM concentration). (c) = 2.0 μm > (2400 pM concentration). Since > focal depth (∼1.5 μm), the imaged molecules are out of focus.

Tables

Generic image for table
Table I.

Radius (μm) of region of focus for different distances of pushing past contact and tape thicknesses.

Generic image for table
Table II.

Radius (μm) of region of focus for different over-pushing distances and radii of curvature of the push-lens.

Generic image for table
Table III.

Radius of curvature of the imaging chamber height profile ( , mm), as well as the radius of the coverslip-coverslip contact region ( , μm), as a function of radius of curvature of the push-lens and over-push distance. Fits were performed within a central 300 μm radius disk and the region of direct coverslip-coverslip contact was excluded.

Generic image for table
Table IV.

Radius of curvature of the imaging chamber height profile ( , mm), as well as the radius of the coverslip-coverslip contact region ( , μm), as a function of tape thickness and radius of curvature of the push-lens. Fits were performed within a central 300 μm radius disk.

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/content/aip/journal/rsi/84/10/10.1063/1.4822276
2013-10-10
2014-04-24
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752b84549af89a08dbdd7fdb8b9568b5 journal.articlezxybnytfddd
Scitation: Precision platform for convex lens-induced confinement microscopy
http://aip.metastore.ingenta.com/content/aip/journal/rsi/84/10/10.1063/1.4822276
10.1063/1.4822276
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