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Alignment

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Kyle M. Douglass edited this page May 19, 2026 · 48 revisions

General Information

Beam height above table surface: 160 mm

Abbreviations

  • SL - Scan lens
  • Galvo - Galvanometer mirror
  • VAT - Vertical alignment tool
  • TL - Tube lens

Equipment

  • Alignment laser at 532 nm (1 is necessary, 2 are better)
  • Wavefront sensor (or shear plate)
  • Coverglass
  • Variable neutral density filter, such as Thorlabs NDC-50C-2M-A

Procedure

Set Up

  1. Ensure that the alignment laser is level with the table by checking its height at two separate, far apart locations on the bench.
  2. Ensure that the alignment laser is collimated. Below is a table of wavefront radii of curvature at different distances from the laser as measured with a Shack-Hartmann wavefront sensor.
  3. Set the alignment laser beam height to ~160 mm above the table surface.
  4. Ensure that the galvanometer mirror is at that correct height above the table by carefully putting the alignment laser next to it and visually verifying that it would intercept the entire cross section of the beam.
Distance, cm RoC, m
50 cm -30 +/- 1
75 cm -35 +/- 2
100 cm -40 +/- 3

Excitation Path

Align Galvo and Scan Lenses 1 and 2

  1. Set the galvanometer voltage to 0V and roughly position it on the table.
  2. Place two irises on a row of bolt holes on the table before the galvo.
  3. Put the alignment laser on the table and aim it the galvo.
  • Note for next time: Put the laser farther from SL1 than in the photo below so that it can be used to coalign with the excitation beams.
  1. Align the laser to the irises.
  2. Put an adjustable ND filter in front of the laser beam.
  3. Insert the galvanometer mirror and place it so that the beam is roughly in the center of the mirror.
alignment_0
  1. Use C-clamps to move the two irises after the mirror on a column of bolt holes. Position the galvanometer so that the beam passes through the irises at these new positions.
alignment_1
  1. Close down both irises. Insert a mirror after the second iris and align the back reflected beam back through both irises.
alignment_2
  1. Insert a coverslip after the ND filter to pick off the reflected beam.
alignment_3
  1. Insert two new irises to define the path of the pick off beam. Their absolute position doesn't matter.
alignment_4
  1. Insert scan lens one (SL1) and place it such that the distance between the mirror and the lens housing is approximately 170 mm. Use the speckle size from scattering from a white piece of paper to verify that the mirror is near or at focus.
  2. Adjust positioning of SL1 until the pick off beam is recentered through the irises and the forward the beam goes through both irises. Forward and backward beams may not agree completely; this is OK. Align them as close as possible.
  3. Insert SL2 and place it approximately 170 mm after the galvo. Align its transverse position so that the forward and backward beams pass through all irises.
  4. Insert the Shack-Hartmann wavefront sensor after SL2. Adjust SL2's axial position until the output beam is collimated (absolute value of the radius of curvature is greater than about 30 m, the value of the beam's RoC directly out of the alignment laser).
alignment_5
  1. Remove the wavefront sensor and iterate over the last two steps until transverse and axial alignments are good.

Align Objective and Tube Lens

  1. Insert two mirrors after SL2 in a figure 4 configuration. Use two irises to align the beam into the 90 degree upward folding mirror. (One iris is on the mirror mount in the photos.)
  2. Insert the vertical alignment tool (VAT) into the objective barrel. Use the tip/tilt adjustment screws on the folding mirror and adjust its position on the table until the beam is traveling approximately vertical and is centered along the axis where the objective will be placed. 2x tip/tilt adjusters and x/y positioning provide the full four degrees of freedom for beam positioning. You will not be able to exactly align the beam until the next step. Just make it approximate.
  3. Remove the alignment disk from the lower cage plate on the VAT. Use the adjustment screws on the folding mirror to align the beam to the tool's iris at the far end of the tool. Insert the alignment disk. Use the adjustment screws on the mirror in the figure 4 assembly that is closest to the assembly to align the beam into the hole in the alignment disk. Iterate until good alignment through both irises is achieved.
  4. Remove the VAT and insert the objective. Verify that the beam is traveling approximately vertically after the objective.
  • Almost certainly the beam will not being traveling perfectly vertical. This is OK for now. If it at least is close, use the tip/tilt adjusters on the folding mirror to redirect the beam vertically.
  • When debugging, you can also verify visually that the beam is entering the center of the objective's rear aperture.
  1. Set the laser to the lowest possible power such that you can just see the beam leaving the objective. This is the most dangerous part of the alignment. Insert the tube lens. Adjust its height by raising and lowering the post in the post holder until the correct height is found for maintaining a vertical beam path. Lock this position in place with a C-clamp. You may also need to roughly adjust the lens's transverse and axial positions to find the beam and place the focus of tube lens in the objective's back focal to start this step.
  • Setting the vertical position first will confine beam displacements to a vertical plane so that you can stand outside of this plane for eye safety during alignment.
  1. Next, find the axial position of the lens such that the focal planes of the tube lens and objective are coplanar. This can be done by eye by inserting an index card into the beam after the objective to observe its size while simultaneously moving the lens's axial position until its size is a minimum. There will be a large, maybe ~1 centimeter range of lens axial positions in which it will be difficult to identify when the spot size has been minimized. This means the precision of finding the coplanar point is relatively low.
  2. Alternatively, use the beam view on the wavefront sensor as shown in the image below. Note that the beam's radius of curvature will not change much with lens axial position. Instead, use the x and y diameter readouts from the wavefront sensor and mark the range of lens axial position that have been tried. Use a method of bisections to find the tube lens axial position that minimizes the beam size.
  3. Fix the tube lens to the table. Attach a C-clamp to the post so that the lens can be removed and replaced in the same position.
  4. Remove the objective and tube lens. Reinsert the VAT, align to it as before, and mark where the beam hits the top of the table's enclosure.
  5. Reinsert the tube lens and stop down the beam using one or more irises from previous steps. Verify that the beam hits the mark. If it doesn't, slightly adjust its transverse alignment, taking care to ensure that the axial alignment is minimally affected.
  6. Reposition the iris right before the tube lens, after the figure 4 folding mirrors. (It will be slightly off because the last mirror in the figure 4 was used to align the beam to the VAT.) Stop it down. Insert an alignment disk and change the lens's tip/tilt alignment until the back reflections are centered on the axis.
  7. Open all irises in the path. Reinsert the objective, paying attention to eye safety. Most likely the beam will not hit its mark on the top of the enclosure but instead travel at a slight angle out of the objective. If this happens, loosen the four screws on the 90 degree brackets that attach the MCL microscope body to the table and carefully move the whole body until the beam hits its mark. This has the effect of centering the objective on the laser beam.
  • Using a rubber mallet and soft taps on the microscope body help if you cannot displace the stage in fine-enough movements.
  • If the placement of the bolt holes do not allow you to align the beam to its mark, use Thorlabs CL5 table clamps to clamp the 90 degree brackets to the table.
alignment_6

Align the Galvo/Scan Lens Assembly to the Objective/Tube Lens Assembly

This is a critical section because we will have to transfer our datum from the laser to irises and back. Be sure to follow the sequence.

  1. Insert a second iris between the figure 4 folding mirrors and the tube lens. Ensure it and the second iris in the same space are centered on the beam.
  2. Insert the wavefront sensor after the tube lens.
  3. Activate the wavefront sensor and mark the beam center.
  4. Move the alignment laser (or insert a new one if available) to the space between the galvo and SL2. Align it back to the irises and mark on the wavefront sensor.
  5. Use the wavefront sensor to find the approximate mirror locations that produce a collimated beam after the TL. Take care to put the focus of SL2 in free space and not near one of the mirror surfaces. (It and any dust on it will be imaged onto the sample plane.)
  6. Once the approximate axial positions are found, put the beam near the mirror centers and align the beam back onto the path.
  7. Iterate until the mirror positions produce a collimated beam after the TL and the beam is recentered on the path.
  8. Move the alignment laser back to its original position (or remove the second one before SL2 if using two lasers) and check good alignment along the entire path, including collimation after the objective and that the beam after the objective hits its mark. Use the mirrors in the figure 4 for fine adjustment into the objective if necessary.

Align the Excitation Lasers

This section assumes that the 488 and 561 nm lasers are already coaligned. It also assumes that they go through the AOTF and periscope.

  1. Set the height of a new iris to the table's beam height using the alignment laser.
  2. Level the 488 nm laser beam using this single iris with a C-clamp at two different positions on the table along a line of bolt holes.
  3. Insert the second iris into the 488 nm beam path and set its height. Ensure the 488 nm beam goes through both irises.
  4. Set up a figure 4 configuration of folding mirrors to direct the beam 90 degrees towards the dichroic's future position in front of SL1.
  5. Use the alignment laser to set the height of a new iris. Don't worry too much about its height; it will be used only as a rough guide to the dichroic.
  6. Put the new iris in the path going towards the dichroic's position to roughly (and incompletely) define the 488 nm beam path to the dichroic.
  7. Check one last time that the alignment laser is hitting its mark on the top of the enclosure and is aligned to its irises.
  8. Insert the dichroic into the beam path. The alignment laser should transmit through it. The beam hitting the mark on the ceiling will have been displaced slightly.
  9. Position the new set of figure 4 folding mirrors and the dichroic so that 1) the beam is centered on the dichroic and 2) the 488 nm beam is roughly aligned to the alignment beam path.
  10. Use the figure 4 folding mirrors, the two irises before the tube lens, and the mark on the top of the enclosure to align the 488 nm beam to the excitation path.
  11. Check the 561 nm laser as well. If it's not overlapping the 488 nm at the mark on the top of the enclosure, then tune the mirrors immediately after the 561 nm laser device itself.
  12. Insert the second, 400 mm lens of the beam expander into the path and align it to the irises and mark. Ensure it is oriented correctly (infinity towards sample).
  13. Insert the first, 40 mm lens of the beam expander into the path and align it to the irises and mark. Ensure it is oriented correctly (infinity towards lasers).
  14. Use the wavefront sensor to ensure that the beam after the 400 mm lens is collimated. Adjust the axial position of either lens until this is the case.
  15. Iterate the positioning from the last two steps until satisfied.

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