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Optically trapped aqueous droplets for single molecule studies
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21.See EPAPS Document No. for material on fluorescence from single RFP molecules, the control experiment to determine RFP survival probability, and a video of controlled droplet fusion. This document can be reached via a direct link in the online article’s HTML reference section or via the EPAPS homepage (http://www.aip.org/pubservs/epaps.html).[Supplementary Material]
View: Figures


Image of FIG. 1.

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FIG. 1.

(Color online) A schematic diagram of the optical setup. F1 and F2 are bandpass filters for and light, respectively. NDF is a series of neutral density filters. L2 is the second lens of the IR trap beam telescope that is mounted on a three-axis translation stage for precisely overlapping the trap and fluorescence beams. M1 is the movable mirror for the moving optical trap in the fusion demonstration. All elements in the shaded gray region are internal to an inverted microscope (Axiovert 100, Zeiss) and M2 is a manual flipper mirror that directs light to the charge coupled device camera or is removed to allow passage to the APD. P-100 is a pinhole that is part of the confocal microscope setup. FRET measurements were made by replacing the M3 mirror with a dichroic mirror, and a second APD was inserted to capture light from the acceptor fluorophore. Also, the P-100 pinhole was removed for the FRET measurement to avoid issues due to chromatic aberrations.

Image of FIG. 2.

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FIG. 2.

(a)–(c) Three examples of single molecule detection in trapped droplets illustrating the trapping and detection of 1, 2, and 3 (SRB) molecules, respectively. The measurements are taken with different excitation strengths. For (a) and (c), the laser power sent into the back aperture of the microscope objective was , and for (b) the power used was . The different laser powers resulted in different step sizes for photobleaching events. For these measurements, a solution of SRB was used, such that a droplet is calculated to contain an average of 1.6 dye molecules.

Image of FIG. 3.

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FIG. 3.

(Color) Single molecule FRET data obtained from a single-stranded 16mer DNA with a Cy3 molecule attached to the end and a Cy5 molecule attached to the end in an optically trapped droplet. The Cy3 donor molecule is quenched by the Cy5 acceptor molecule until the photobleaching of the Cy5 molecule occurs at . The donor molecule fluoresces until when the donor molecule photobleaches.

Image of FIG. 4.

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FIG. 4.

(Color) Sequence of video images showing the fusion of two aqueous droplets, initially held in independent optical tweezers. The upper droplet is translated by the mobile trap to the location of the droplet held by the fixed trap, at which point the two droplets fuse into one. The fixed trap is then turned off and the single droplet is translated upward by the mobile trap. The mobile trap (upper) is slightly defocused from the fixed trap (lower). The solid bar in the first picture is in length.


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We demonstrate a technique for creating, manipulating, and combining femtoliter volume chemical containers. The containers are surfactant-stabilized aqueous droplets in a low index-of-refraction fluorocarbon medium. The index-of-refraction mismatch between the container and fluorocarbon is such that individual droplets can be optically trapped by single focus laser beams, i.e., optical tweezers. Here, we trap and manipulate individual droplets, detect the fluorescence from single dye and red fluorescent protein molecules encapsulated in droplets, and observe fluorescence resonance energy transfer from a single dye pair on a deoxyribonucleic acid molecule encapsulated in a droplet.


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Scitation: Optically trapped aqueous droplets for single molecule studies