Aligning 2P Laser to Sutter 2P Rig

Sutter 2P Rig

  • Best person to contact: Rick Ayer of Sutter Instruments: rick@sutter.com
  • Herringbone artifact is normal for End-On Hamamatsu PMT H10770PA-40

References

Safety

  • Safety glasses (Thorlabs)
  • For 2P laser alignment at 690 nm: LG8 (green lens):
    • LG8 series does NOT cover 920 nm
      • Wavelength coverage: Only 630-695 nm (stops at 695 nm)
      • Does not provide protection at 920 nm ❌
    • Higher OD (5+ vs 3+) provides better protection during alignment
    • Still has reasonable VLT (24%) for seeing your alignment
    • The slightly reduced visibility compared to LG9 is worth the extra 2 OD levels of protection
  • For 2P laser alignment at 920 nm: LG9 (orange lens):
    • Wavelength coverage: 610-695 nm AND >740-1070 nm (covers 920 nm)
    • Optical Density (OD): 7+ at 920 nm
    • Visible Light Transmission (VLT): 44%
    • EN 207 Rating: D LB6 + IRM LB7 in the >740-1070 nm range
    • LG8 series does NOT cover 920 nm

Key Points

  • Last stage at which beam is a focal dot: The horizontal enclosure (not the one labeled MOM) before the galvos (see tutorial video) — beam expands after that point
  • Adjusting 90° mirrors: ignore the screw on the corner (middle screw at 90° angle)
    • Top screw: up/down
    • Bottom screw: left/right
  • After laser reaches objective:
    1. For angular alignment: Adjust the mirror closer to the laser (further from objective) at 920 nm and align pollen across the Z-axis.
    2. For translational alignment in center of objective: Use crosshair objective adapter (in alignment case). Either a) adjust mirror(s) closest to objective (see Option A) or b) walk the beam (see Beam Walking)
    3. Re-check angular alignment after translational alignment and make any subtle changes if needed.

Fine Alignment Using Pollen Grains

Initial Setup

  • Use LG9 orange lens safety glasses
  • Select a spiky pollen grain at 40X (via pollen grain slide) as your alignment target with epifluorescence (LED + QCam)
  • Navigate to the top surface of the pollen grain under 2P with both green and red channels enabled
  • Enable the crosshair in ScanImage (right-click on image)
  • Display the histogram (right-click on image)
    • Histogram Settings
      • Left edge of histogram bar: Position at peak (should be near zero)
      • Right edge: Balance for good background noise contrast

Alignment Check

  1. Focus through the pollen grain from top to bottom along the Z-axis
  2. Monitor whether the pollen grain remains centered on the crosshair
    • If centered through Z-axis: Laser angle is properly aligned and reaches sample at a reasonably orthogonal orientation ✓
    • If drifts off center through Z-axis: Angular adjustment needed → Use the mirror closest to the laser (see angular alignment)
  3. Monitor whether the brightness of the pollen grain changes when it is moved around X-Y with the objective (on Sutter) or stage (on Scientifica Rig)
    • If brightness consistent across X-Y axes: Laser is centered in the objective ✓
    • If brightness changes across X-Y axes: Translational adjustment needed → see Translational alignment

Angular Alignment (Z)

  • Done at 920 nm (Use LG9 orange lens safety glasses)
  • Goal: The beam should remain stationary in X-Y position while scanning through the Z-axis (focus). This will ensure pollen grain remains focused through the Z-axis.
  • If pollen grain drifts off center through Z-axis: Make slight adjustments using the mirror closest to the laser

Translational Alignment (X-Y)

  • CRITICAL: First use PRM1Z8 (controlled via jog wheel near Sutter Rig) to adjust power to the lowest setting at 920 nm to avoid burning anything in the beam path
  • Done at 690 nm (Use LG8 green lens safety glasses)
  • Requires crosshair objective adapter in alignment case (want beam to fill the center of the crosshair projected from adapter onto white paper)

OPTION A: Adjust Mirror Closest to Objective

  • Assumes laser angle is aligned, thus the mirror closest to the objective should allow translational changes along X-Y axes without drastic changes to laser angle
  • Adjust mirror closest to objective so that laser fills the center of the crosshair through crosshair objective
  • May need to make subtle re-adjustments to the laser angle (see above)

OPTION B: Beam Walking

If you experience intensity loss, "walk the beam":

  • Adjust one direction on the first mirror
  • Compensate in the opposite direction on the second mirror
  • Example: Right on first mirror → Left on second mirror
  • This keeps the beam parallel while translating it laterally

Why walk the beam?

  • Adjusting only one mirror changes the beam angle
  • Adjusting both mirrors together translates the beam position without changing angle
  • Proper alignment ensures the beam only moves in Z (focus), not X-Y, when focusing

Coarse Alignment Process (ONLY IF POLLEN GRAIN NOT VISIBLE)

Prerequisites

  • Wear LG9 safety glasses during alignment
  • Ensure bolts to table are secure

Steps

  1. Set laser wavelength to 920 nm (use LG9 orange lens safety glasses)
  2. Adjust Optic Rotation Mount (Newport RSP-1T) so that power meters for both Scientifica and Sutter read approximately 50% (even split between 2 rigs)
  3. Slightly loosen post bolt of barrel at first split and twist until both meters are at maximum for 50% split
  4. Tighten down barrel post bolt
  5. Ensure beam passes uninterrupted through shutter to 90° mirrors

Log

  • 1/20/2025