Alignment

Overview

Due to removing duplicated parts in it's optical design, this microscope utilizes many optical elements for 4 purposes: imaging through left and right objective, and light sheet generation through those. Because of this, it requires careful alignment in the remaining degrees of freedom (DOF), so that aligning each modality does not misalign the other three. We approached the problem in the following framework:

• First mechanical (crude positioning with laser pointer guides), then optical (fine adjustment based on camera image).
  • Laser-pointer jig that can be screwed into SM1 mount is handy for crude mechanical alignment.
• Start optical alignment from the camera, and make apertures concentric in the order they appear before the camera:
  • First, the detection path: camera, L2, DM, L1, M-prism, TubeL1, TubeL2, M-fold1, M-fold2, L-objective, R-objective.
    • Use screw-in frosted glass targets, cage drop-in targets, or other visual cues (e.g. DM edge) to visualize the centers of apertures.
    • Use cages where possible (single DOF).
    • Create some image overlays, such as center lines/points, as visual guides.
    • Place temporary imaging lenses instead of L2 to allow imaging of path apertures at various positions.
  • Second, the excitation path: L4, ETL, M-scan, L3.
    • Try to not change the elements already aligned for the detection path. If this is not possible, do several iterations between detection and excitation.
    • Shoot the laser beam through frosted glass targets (center holes).
    • When laser hits M-prism, apply voltage bias at galvo (M-scan) to send the laser coaxially into left or right arm (e.g. -0.4, +0.4V).
• Finalize alignment by imaging beam profile of a 488nm laser using fluorescein bath. Make final adjustments to bring the beam image in FOV center of each view.

Pupil alignment

In order to correct aberrations in both views simultaneously and equally, pupils of both objectives must be aligned to be concentric to the DM aperture. This is achieved in the following steps:

1. Replace lens L2 (f400mm achromat) with a lens half its focal length, L2' (f200mm), see Fig.1a. The latter images DM pupil plane to the camera sensor without magnification (1:1).
2. Illuminate DM with diffuse light, e.g. using flashlight with a diffuser. Ensure that DM is concentric to the camera sensor, Fig. 1b. Adjust DM position if necessary.
3. Block left arm with beam block (e.g. black paper), and illuminate the right arm with diffused light, placing the diffused into the sample chamber. Find the right objective pupil (dark round edge) in the camera image.
4. Adjust tip/tilt of the folding mirror M-fold2 until right objective pupil image is aligned with the DM aperture in camera image, Fig. 1c.
5. Repeat steps 3-4 for the other arm.

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