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mesoSPIM_coalignment

Rob Campbell edited this page Mar 12, 2019 · 8 revisions

mesoSPIM co-alignment of both lightsheets

Principle:

The optical path of the mesoSPIM is set up in a way to streamline co-alignment of both light-sheets. This is done by having an additional linear stage controlling the light-sheet tilt. The "weird" folding of the beam path of the mesoSPIM excitation path is caused by the need to put this linear stage in.

Excitation arm in 3D By putting the last mirror on a linear stage and translating it along the direction of the input beam, we can adjust the tilt of the light-sheet. In the ideal case, the tilt is clean (i.e. the pivot point of the light-sheet inside the sample does not move depending on the pivot angle.) In reality, the pivot point moves - one reason being that the high-index medium inside the sample chamber induces refraction of the excitation light-sheet which shifts the pivot point.

Co-alignment of both light-sheets

  • use a test sample that is very transparent and very homogenous in index
  • pick one side as master and align the other side relative to it
    • in the mesoSPIM-control software, go to the alignment tab and click "Run lightsheet switching mode"
    • the microscope will now switch the shutter between images
    • using the alignment screw on M5 that is closest to L4, adjust both the tilt and offset of the lightsheet till both light-sheets overlap in the center of the image
    • using the linear stage, adjust the tilt of the light-sheet till both light-sheets overlap at the edges of the image
    • usually, several iterations of the last two steps are necessary
  • The lightsheet switching mode may be hard to do if you are fairly far out of alignment. In this case:
    • choose one side as the reference and search in Z until you find a bead pattern that is easy to recognise
    • ensure you are in focus so beads are as small as possible
    • switch to the other sheet with the joystick and search of that pattern
    • guide yourself by making adjustments that bring beads into focus. i.e. so that they become smaller
  • If you are very far out of alignment:
    • remove the immersion cuvette
    • put rods through the 60 mm cage plates from one excitation path to the other
    • if you noticed the cage plate with the objective is sagging downwards then likely that means you need to use slightly longer ER rods to support it
    • use 60 mm alignment targets to check the beam, move the target back and forth along the light-sheet propagation direction for alignment
    • use M5 tip/tilt and linear translation to align the beam along the axis of the 60 mm cage system

Excitation path Using a 60 mm alignment target to check the light-sheet propagation

Tips and tricks

  • even the smallest index variations and slight cuvette rotation (<1 degree) can lead to misalignment of two very thin light-sheets
  • when changing immersion media or clearing methods, realignment of both light-sheets relative to each other is likely to be necessary
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