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Dual Camera Alignment#

Why is this necessary?#

Images on the two cameras will not be perfectly registered and will require an affine transformation.

To determine the location-dependent deviation, we need to image something that can be seen in each channel.

Bead slide vs using spinning disk pinholes#

Previously, we used a multi-color TetraSpeck bead slide, but this has some downsides:

  • Weak fluorescence and poor photostability → Annoying to use when doing coarse alignment
  • Uneven bead distribution → can take time to find a good bead region
  • New slides need to be made monthly → Extra effort

However, our 60x and 100x objectives are excellent and exhibit less than 100nm axial chromatic shift within the green, red, and far-red channels at 37C--far below the axial resolution of our scope. With this in mind, stopping the spinning disk's rotation and using transmitted light is a good and easy alternative.

Aligning with spinning disk pinholes#

YouTube videos for aligning with pinholes

Aligning with TetraSpeck beads#

  • Focus onto TetraSpeck beads, enable PFS, and start a timelapse for about 30 minutes
  • 30 minutes should be sufficient for temperatures to stabilize (as evidenced by no more drifting)
  • Load the relevant two-color OC (e.g. align-488-561)
  • Go Live and inspect the overlap of the 488 and 561 channels

Note

  • Aim for perfect overlap in the center of the ROI
  • The chromatic aberration will get worse at the edges of the FOV
  • If adjustments need to be made, use the 1.5mm hex screw. Translate the 488 channel by tweaking "H" and "V"1.

Tip

  • Compensate for the slight movement as you remove the hex screw.
  • When satisfied, acquire a multi-channel z-stack of the beads in order to align channels later. Make sure the beads are well distributed and extend to the edges of your FOV.

  1. Rotating the DM here leads to lateral translatation ↩