You’ve already heard about out plan to add an SLM path to the NLW two-photon microscope.
As we described earlier the first step is to provide a tool to align the SLM and 2P paths. This is now complete and it consists of four phases carried out automatically by a Matlab function:
- A port camera connected to the microscope images the two-photon scan area to be used in the experiment. Matlab detects the area and draws bounding rectangle around it on the screen. Given that we know the size of the area in pixels, this allows us to map a location of a cell within the scan area to the coordinate frame of the camera.
- A nine point calibration takes place next, where the SLM is used to generate points at different locations in the SLM target image plane. Those points are then imaged by the port camera. Matlab estimates their location in the plane of the camera and displays them as yellow ‘x’ markers. These data allow us to map points in the SLM image plane to the camera plane.
- Next, optimal affine transformation linking points in the SLM imaging plane to the camera plane is computed. We also compute the optimal transformation between the Scanbox image plane to the camera plane using the data in step #1.
- Finally, a validation procedure allows one to click anywhere within the scan area and check the SLM projects a point in the desired location (yellow circles).
For whatever reason video screen capture interferes with the Matlab preview of the camera, so here is an old-fashion iPhone video of how the procedure runs.