Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

The attachment of ports to mate to syringes with tubing is critical because, when it's done right, there are no leaks, there are no bubbles in the flow cell, and experiments can be done repeatedly using the same flow cell over a period of one year with careful use. Here, two simple methods will be presented to attach the connectors to flow cells. These are easy, they're reproducible, and allow the investigator to easily adapt the flow cell to their needs without too much trouble.

The use of these flow cells was done with single molecule experiments in mind. In particular, what we call visual biochemistry, but they can be used in any study where microfluidics are required and in situations where lamina fluid flow is to be used. So, to avoid mistakes, instead of using valuable flow cells to establish the techniques, I would suggest practicing on a glass slide or a cover slip first.

This will enable one to get a feel for the glass or the PDMS materials to know how to handle them and how much force to apply when inserting tubing or bonding the connectors. This will require sacrificing some connectors but they're much cheaper than the flow cells. Begin by placing the flow cell on a clean flat surface.

Hold the PTFE tubing three millimeters from the free end with forceps and push the tubing into the port. Repeat this procedure for each of the remaining ports. Connect the inlet ports to the syringe pump and the outlet to a waste bottle.

Fill each glass syringe with one milliliter of spectro photometric, grade methanol and attach them to a switching valve. Ensure the valve has the outlet directed to waste and purge each line with 50 microliters of methanol. Switch the outlet position to the flow cell and pump 800 microliters of methanol through the flow cell at a flow rate of 100 microliters per hour to wet the surfaces and eliminate bubbles.

The next day, repeat this process using 800 microliters of ultrapure water. The flow cell is now ready for use. If press-fit tubing connectors are to be used, carefully remove the adhesive tape from one side of the connector and place it over the hole in the microscope slide.

Press down for a few seconds. Repeat the process for the remaining connectors. Then place the flow cell on a clean flat surface.

Hold the PTFE tubing three millimeters from the free end and push the tubing into the hole in the port. Repeat this procedure for each of the remaining ports. Attach tubing from the inlets to the switching valves.

Place the attachment either on a clean, flat surface or on a custom-built manifold to hold the flow cell and connectors in place while the bonding occurs. Place the flow cell on the recessed section of the manifold then place a small amount of glass glue on the bottom of the assembly and insert the seal. Position the nano port over one of the entry holes on the microscope slide.

Gently push down and hold in place with no lateral movement. Repeat the process for the remaining ports. Allow to dry or clamp in place in the manifold.

Gently remove the flow cell from the manifold and ensure that the assemblies align well with the entry ports in the flow cell. Place a flow cell on the microscope stage and attach the tubing using the finger tight connectors. Fill each glass syringe with one milliliter of spectro photometric grade methanol.

Attach each syringe to a switching valve. Ensure the valve has the outlet directed to waste and purge each line with 50 microliters of methanol. Switch the outlet position to the flow cell and pump 800 microliters of methanol through the flow cell at a flow rate of 100 microliters per hour.

The next day, repeat this process using 800 microliters of ultrapure water. Measurement of the power output is shown here. The power output from the laser is measured before installing the laser head into the optical layout.

Once trap alignment is done, the beam power is measured after the 100 x objective for each trap. The images demonstrate the stable optical trapping of one micrometer fluorescent beads. Here F is a fixed trap, and M is a mobile trap.

With the mobile trap in the scanning mode moving through a small or large circle at 30 hertz. Fluid flow within the flow cell is laminar. The schematic shows the flow cell from the top to demonstrate that the inter channel diffusion is the main source of mixing between the adjacent fluid streams.

Blue arrows indicate the flow direction and the individual streams are colored, white, light gray, and white. The widening regions of transverse diffusion between channels are indicated in red. The inset shows a fluorescence image of the adjacent fluid streams at 10 times magnification with fluorescent labeled DNA bead complexes in the lower stream and buffers only in the upper stream.

The white spots in the upper stream are shot noise from the CCD camera. The flow profile and laminar flow cells is parabolic. The flow cell is viewed from the side and the direction of the flow is indicated above each cell.

Linear bead velocity is affected by pump speed and sucrose concentration. The opposing force on a bead is affected by solution viscosity. Bead diameter influences the force on beads under flow.

When attempting this procedure, make sure that the area you are working in is clean and that you have cleaned the glass surfaces with spectrometric grade methanol or acetone. At this stage, you should have a flow cell that is made it to your syringe pump and you are now ready to introduce your solutions and begin optical tweezer experiments. The use of flow cells, which can be used for extended periods of time, has enabled researchers to investigate a variety of single molecule reactions where either forces are measured, reactions are visualized by fluorescence or a combination of both.