TIRF objective quick start guide

This guide covers the workflow when using the LUMICKS EDGE system (widefield and/or TIRF and/or IRM system) with the TIRF (i.e. "oil") objective installed. In the case where the water objective is installed, please refer to steps 0, 1, 2, 3, 4, 5 in the confocal guide (LINK). Then, follow step 6a in this current TIRF objective quick start guide (LINK TO STEP 6a).

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Step 0: Set up optics

• Clear the flow cell space (lower the objective, raise the condenser, slide the flow cell holder out)
• When opening Bluelake, select the correct configuration that corresponds to the TIRF objective!
• Position the nanostage to x=100, y=100, z=150 μm
• Add 1 drop of oil on the TIRF objective (also referred to as the "oil" objective) 
• Add 1-2 drops of condenser oil on the flow cell on the working area of interest
• Slide the flow cell holder (with the standard LUMICKS flow cell or a custom flow cell) 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. Move the microstage if needed
• Close the C-Trap lid

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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). For certain assays, one may want to use a smaller Overall Power to perform this step 1 (and the following step 2) to avoid unnecessary laser exposure to the sample. This is always possible, but will require adjusting the contrast settings of the Z-finder camera
• 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 very clear reflection is visible on the Z-finder. This first clear reflection shows the interface between the glass and the buffer inside the chamber, i.e. the surface of the sample. Sometimes, a very faint reflection can be noticed first, at the interface between the objective oil and the glass due to the very slight difference in refraction index between the oil and glass. If that is the case, continue rotating clockwise until the very clear reflection is visible

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Step 2: Adjusting the condenser position

• Now that the objective position is set and fixed, move the nanostage back down to z = 100 μm (to offset the focus by ~ 50 μm up from the sample’s surface)
• 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 TIRF”
• 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 flow cell 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)

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Step 3: Adjusting the Bright-field imaging and navigating the flow cell 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
• If a custom flow cell is used, go to Step 4 directly
• If a standard LUMICKS flow cell is used:
  • Move the microstage up (in Y- direction) until the top horizontal edge of the flow cell 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

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Step 4: Catch some beads to localize Trap 1 and Trap 2

• Step 4a. if a standard LUMICKS flow cell is used (in which case, typically both traps are used):
  • Adjust the Overall Power to the desired level. 20-30% typically works best for DNA-tether assays and ~4 μm beads
  • Move the nanostage up until the surface is reached (it should move up by ~50 μm)
  • Move the nanostage down by ~5-6x the diameter of the beads (see Note: troubleshooting bead catching)
  • Turn on the flow (valves on, pressure on) and wait to catch some beads at this height of ~5-6x the bead’s diameter
  • 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 4b. if a custom flow cell is used (in which case, typically only a single trap is used):
  • If a single trap is needed, Set Trap 1 split to 100%. You may want to adjust the Overall Power to less than 5%, depending on your specific experiment
  • Move the nanostage up until the surface is reached (it should move up by ~50 um)
  • Move the nanostage down by ~5-6x the diameter of the bead (see Note: troubleshooting bead catching)
  • Move the microstage around to catch a bead at this height of ~5-6x the bead’s diameter
  • Select the digital Trap 1 position. A red rectangle will appear. Hold Shift key, then left mouse click on where Trap 1 is
• Note: Troubleshooting bead catching
  • Depending on how close the traps are to the surface, catching beads may be difficult. If there are no visible beads flowing by (Step 4a) or floating around (Step 4b), move the nanostage further down by another 5-6x diameter of the beads. This will effectively bring the traps further up and away from the surface. If you see the beads but are unable to trap them, you may be too far from the surface, and you may need to move the nanostage slightly back up. If it is still difficult to trap the beads despite troubleshooting the nanostage position, you may try to set the T1+2z position to 0 and repeat Step 4a or Step 4b

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 Step 5a: 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 5b: Surface assay workflow

• Find a location of interest using the microstage
• Find the approximate surface plane using the Bright-field image and while moving the nanostage back up from step 4b (save this surface nanostage position)
• Move the nanostage down to catch a bead 5x diameter away from the surface in Trap 1 (save this nanostage Z position)
• Move the nanostage back up to the surface and offset T1+2z such that when imaging at the surface (as indicated by the Bright-field image) the bead is still freely movable with Trap 1 (i.e., the trapping focus is sufficiently above the surface
• Once T1+2z offset is found (at this point, this is an approximation that you will fine tune later on with TIRF and IRM), catch a new bead again at 5x diameter away from the surface, and Re-calibrate (F9 key) following the calibration guidelines instructed during training. You may be able to perform an alternative calibration that is done close to the surface (instead of 5x diameter away) - please follow the specific instructions given during the training. Zero the forces after the calibration is applied
• Move the nanostage up to the surface and proceed with your experiments and imaging
• You may need to iterate many times between moving nanostage in Z, finding the correct T1+2z offset, and fine-tuning the offset such that you are able to have the surface in focus in TIRF and IRM, while also being able to move the bead freely but close enough to the surface to allow interactions

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Step 6a: Widefield imaging

• If the fluorescent laser module is visible in the rack, turn it on (key turn + button). Otherwise, it is directly turned on through Bluelake
• On Bluelake, enable the desired lasers and choose the desired percent powers (keep in mind differences between true and displayed powers)
• Initializing widefield imaging:
  • Select the widefield (WT) camera, and display all three colors (R, G, B)
  • In the Camera settings tab, set the Exposure to 500 ms, and Framerate to 1 Hz. Use 10% blue and 10% green laser powers, then enable Exposure Synchronization to start imaging. Using the Exposure Synchronization feature will automatically start imaging and will ensure that the lasers are enabled only for the Exposure Time set, and not continuously
  • In the Image adjustment tab toggle Auto-adjust for all 3 colors, and toggle the Align colors‍
• Verify that the z-plane of the trapped bead is co-aligned with the z-plane of the widefield
  • Change Trap 1+2 z plane in incremental steps and use the bead’s autofluorescence to approximate the right Trap 1+2 z plane, which is where the beads appear the biggest, brightest and their edges the sharpest. Increase the laser power and/or exposure time if needed. 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). This optimal Trap 1+2 z plane will vary significantly based on the bead size, the trap laser power used, and the objective used (oil vs. water objective)
• 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 Exposure 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 Exposure Time and smaller widefield imaging area
  • Photobleaching/photodamage can be reduced by using lower laser powers and/or introducing dwell times between images, using a longer time between image (i.e. lower Framerate). E.g. 200 ms exposure with 1 Hz framerate, allowing ~800 ms of dwell time between images

Step 6b: IRM / TIRF / Near-TIRF imaging

• IRM imaging:
  • Select the IRM camera and enable the IRM LED. In the Camera settings tab, set the Exposure to 2 or 3 ms, and Framerate to 150-200 Hz. In general, more exposure will give better signal-to-noise but will restrict the framerate
  • While exactly at the sample’s surface without any trapped object, toggle the Background subtraction tab and Acquire a background subtraction with 100 frames, 10.00 Radius (μm) and 10 Frames to average, then enable the Background Subtract. This process effectively reduces the framerate by 10-fold but drastically improves the signal-to-noise to allow, for example, single microtubules to be seen without any labelling.
  • You may need to fine-tune the sample plane as IRM works best when within a few μm of the surface
  • You may need to fine-tune the exposure, framerate, and averaging settings to get the best signal with your specific assay
  • Turn off the IRM led when you are finished with your experiment
• TIRF imaging:
  • Initialize widefield imaging as outlined in Step 6a, under “Initializing widefield imaging”
  • While exactly at the sample’s surface, toggle the TIRF excitation mode
  • Optimize the imaging settings as outlined in Step 6a, under “Optimize the imaging settings...”. Usually, shorter exposure time and lower laser powers are sufficient in TIRF mode due to the drastically lower background signal, and the increase in laser power reaching the sample (from the removal of the diffuser), as compared to widefield mode. For example, 100 ms exposure with 5% laser power may already be sufficient.
  • You may need to fine-tune the sample plane as TIRF works best when within a few 100 of nms of the surface
• Near-TIRF imaging:
  • Typically, this can be used for samples imaged a few μm's from the surface, and can provide a slight improvement in signal-to-noise by reducing the background slightly (e.g. DNA tether held close to the surface)
  • Initialize widefield imaging as outlined in Step 6a, under “Initializing widefield imaging”
  • Make sure the DNA tether, or any object of interest, is close enough to the surface (between 1-3x the diameter of the beads in the case of the DNA tether) which is where near-TIRF works best. Then toggle the TIRF excitation mode. Note: being this close to the surface may impact your force readings. Feel free to ask your application scientist for more information
  • Step the Y position of the TIRF mirror slightly away from the TIRF position (step towards the widefield position)
  • Optimize the imaging settings as before
  • You may need to fine-tune the sample plane and TIRF mirror position for the best signal-to-noise
• Opening the 2nd camera view is useful in case two imaging modalities need to be visible at the same time
• When working with the tweezers near the surface in combination with TIRF and IRM imaging, Trap 1+2z plane typically must be adjusted with a slight offset such that the trapped object is freely trapped slightly above the surface, while the two imaging planes (TIRF and IRM) are in focus at the surface (see step 5b)