I’ve continued to test our high speed stage stitching (I, II, IIIa, IIIb). This time I’ve imaged the same sample as before, now using a 20x/0.75 NA objective. This requires shortening the strobe duration to 20 μs and requires 594 images to cover the entire tissue. This objective also has a substantially smaller depth of field, and it appears that the Nikon Perfect Focus System and the mechanical Z-drive on the Ti cannot move fast enough to keep the sample in perfect focus while scanning at full speed. Leveling the stage insert so that the focus difference from one edge of the specimen to the other was only a few microns seems to have fixed this.
While testing this, I also compared the timing for this fast scanning acquisition to two other acquisition modes. In both of these modes, the stage stops at each coordinate where the images are acquired. The first mode uses hardware triggering and the ARRAY functions of the ASI controller as described by Austin, the second just generates a position list in Micro-Manager and acquires it normally through software. The acquisition time differences are pretty pronounced:
- Fast scanning: 90.7 s
- ASI Triggered: 288 s
- Micro-Manager: 565 s