Alternative steps due to typical differences between systems are highlighted in italic. Other differences may exist between systems and may not be captured here.
Step 0: Set up optics
- Clear the flow cell space (lower the objective, raise the condenser, slide the flow cell holder out). If you have a nanostage, position it to the middle (x=100, y=100, z=100 μm)
- Add ~60 μL water on the objective
- Add 1-2 drops of condenser oil on the flow cell on the working area of interest
- Slide the flow cell holder back in
- Raise the objective until the water touches the bottom of the flow cell
- Visually inspect that the center of the objective is aligned with the area of interest (inside a channel). Move the microstage if needed.
- Close the C-Trap lid
Step 1: Adjusting the objective position
- Turn on the optical trap source box if not already on (as indicated by the lit up green button). Follow the instructions given to you, which are either 1) Key to ‘on’, push-twist-pull red button, press green button, or 2) Key to ‘REM’
- Select the Z-finder camera
- Set Trapping Laser to 100% and Overall Power to 20% (optical trap source box should have orange light turned on)
- Verify that Trap 1 split is at 50% (this is needed for step 2)
- A signal should be visible on the Z-finder. Adjust the LUT (contrast settings) if needed
- Slowly raise the objective by manually rotating the knob clockwise until a first clear reflection is visible on the Z-finder
- Continue to raise the objective until a second clear reflection is visible. This second reflection should be very close to the first one (should require less than ½ turn of the knob). When in doubt, restart from step 0 and inspect visually again to make sure the objective is aligned (within a channel containing buffer, not just through glass)
- Once the second reflection is confirmed, continue to raise the objective until the reflection patterns are largest (which should correspond to about 50 μm, i.e. the middle of the flowcell). Alternatively, move the nanostage down by 50 μm, preferably if the nanostage was set to 150 μm in step 0, such that after this procedure, the nanostage remains at 100 μm
Step 2: Adjusting the condenser position
- Open the condenser control (available after toggling the Moon camera, or drop down from Tools menu)
- Lower the condenser to the Approach position
- Reduce the Trapping Laser to 30%
- Select the Moon camera. Adjust the LUT (contrast settings) if needed
- Step down with 0.100 mm (i.e. 100 μm) coarse steps while watching the Moon camera image until the condenser touches the oil (visible event on the camera) or until the diameter of the lit area is similar to the “good moon” on the desktop
- Step down with 0.010 mm (i.e. 10 μm) fine steps until the number of bands and their overall patterns (curvature and thickness) matches the “good moon”
- Stepping down should decrease the number of bands while stepping up should increase them. If the reverse occurs, this likely indicates that you are too close to the flowcell and you should immediately Raise the condenser
- Adjust the Trapping Laser back to 100% (always 100% - except during step 2 - and use Overall Power to control the effective power delivered to the sample)
Step 3: Adjusting the Bright-field imaging and navigating the flowcell minimap
- Select the Bright-field camera
- Turn on the Bright-field LED to 15-30% (or to the desired power to have a good signal). Adjust the LUT (contrast settings) or the bright-field camera settings if desired
- Move the microstage up (in Y- direction) until the top horizontal edge of the flowcell is visible on the Bright-field camera
- Move the microstage right or left (in X+ or X- direction respectively) until the corner between the buffer channel and Ch4 is visible on the Bright-field camera
- Inspect the Bluelake microstage minimap on the right. Adjust the location of the blue small rectangle (right click, “set as current…”) to the corresponding location displayed by the bright-field image
- Optional: move the microstage to the corner of Ch5 to confirm the alignment
Step 4: Catch some beads to localize Trap 1 and Trap 2
- Adjust the Overall Power to the desired level. 20-30% typically works best for DNA-tether assays and ~4 μm beads
- Turn on the flow (valves on, pressure on) and wait to catch some beads
- Move the microstage to the buffer channel while keeping the beads in your traps, turn off the flow
- Adjust the location of Trap 1 and Trap 2 to the desired configuration using the caught beads as visual indicators
- Select the digital Trap 1 position. This icon is on the same icons bar as all the moving devices' control (Trap 1, Trap 2, Trap 1+2, etc.), to the right, and looks like a trapped bead. A red rectangle will appear. Hold Shift key, then left mouse click on where Trap 1 is. If available (depends on the specific system), perform the same procedure for Trap 2
- Tip: if both traps are on top of each other or very close by, you will find it difficult to catch beads. Try moving Trap 1 at random to see if you are able to better catch two beads
Step 5: DNA-tether assay workflow
- Set up Bright-field template tracking
- Draw a new Template on the desired bead (supposedly a single bead, of the expected size), confirm and input the correct diameter
- Select the Tracking ROI and extend it to generously cover the two beads with room for any bead movement, then, unselect to hide the ROI
- The initially drawn template should be recognized on the corresponding bead with the highest correlation score (around 99%) while the other should be recognized with a high score (around 95%)
- The vectorial distance between the center of the two templates minus 2x the bead radius is now being tracked under “Distance 1”. Different Tracking modes giving an additional “Distance 2” may be used for certain assays (e.g. Q-Trap)
- Set up force-distance visualization
- On the data viewer screen (left), select the F,d tab
- Add a new eWLC model of the corresponding contour length
- Choose the Force: and Distance: of interest (typically, Force 2x and Distance/Distance1)
- Workflow
- Catch new beads, move to buffer channel and Re-calibrate (F9 key) without DNA, without flow, and following the calibration guidelines instructed during training. Zero the forces after the calibration is applied
- Turn on flow, then fish DNA (use Trap 1 to repeatedly decrease and increase the distance to ~0.6xLc to ~1.0 Lc to form a tether
- Stop the flow (vent, and close channels), verify it is a single tether, move to imaging channel, etc.
- See Step 6 for confocal imaging
Step 6: Confocal imaging
- While the C-Trap lid is shut, turn on the single-photon counter module (button in rack). If the single-photon counter module does not have a button in the rack, it is enabled directly through, near the confocal laser controls
- If the fluorescent laser module is visible in the rack, turn it on manually (key turn + button). Otherwise, it is directly turned on through Bluelake
- On Bluelake, on the confocal control area, enable the desired lasers and choose the desired percent powers (keep in mind the differences between true powers and displayed powers)
- Verify that the z-plane of the trapped bead is co-aligned with the z-plane of the confocal
- Perform 2D scans covering both beads while changing Trap 1+2 z plane in incremental steps
- Use the bead’s autofluorescence to approximate the right Trap 1+2 z plane, which is where the beads appear the biggest and their edges sharpest
- And/or, if available, use the fluorescence signal from the DNA (from bound proteins, markers or intercalators). The best Trap 1+2 z plane is where the fluorescence spots on the DNA appear the sharpest (smallest, with the best signal-to-noise)
- Keep in mind that this Trap 1+2 z plane will vary significantly based on a few factors, the most common ones being the bead size and the optical trap laser power used. However, for the same bead size and same optical trap powe, the correct Trap 1+2 z plane should remain very similar between experiments
- In general, use kymographs to image the DNA
- The confocal 2D scans obtained (left screen) will show the true scanning area (as compared to the approximate blue-highlighted area on the Bright-field). Select the kymograph mode and use the image on the 2D scan on the left screen to align the scanning line accurately before proceeding with kymographs
- Optimize the imaging settings by striking the balance between signal-to-noise, time resolution, and photobleaching/photodamage
- Signal-to-noise can be improved with higher laser powers, longer Pixel Time and or removal of the background (by imaging out of the protein channel, or by optimizing the protein concentration and labelling)
- Time resolution can be improved with shorter Pixel Time and smallest scanning dimensions
- Photobleaching/photodamage can be reduced by using lower laser powers and/or introducing dwell times between scans by artificially setting a slower Line Time (or a slower Image time for 2D scans)
- Turn off the photon counters when the C-Trap lid is open, or when you are finished with confocal imaging
