The words of this tutorial, step by step. The text lives in src/smappy/tutorial/topics/; the directions it follows are in src/smappy/tutorial/STYLE.md.
1. card: Two colours in 3D: dual-colour calibration and Spline 3D 2C
Two dyes on a split camera, as in 2D, and z from a measured PSF, as in 3D fitting.
What is new: the beads give both the map between the halves and a PSF for each half, and the fit shares x, y and z between them.
spoken: Two colours in 3D combines the two: a split camera, and a PSF measured on beads, now one for each half.
2. Tools, Dual-colour calibration opens the bead calibration in its two-colour mode.
3. Bead stacks from the split camera: every bead appears in both halves, and the pairs are what the map is measured from.
4. Say how the chip is split: here into an upper and a lower half, with the main channel above. Mirrored is for a splitter that flips one half.
5. Detect and calibrate pairs the beads across the halves, fits the map, and builds a PSF for each half.
6. Overview: each bead and its partner in the other half, and where the pairs lie on the chip.
7. Transformation: how far each pair lands from where the map puts it. A tight cloud around zero is a good map.
8. As in one colour, the table lists the beads; a pair left out of the PSF can still help the map.
9. Save it, then Use in the fitter puts it into Spline 3D 2C.
10. Spline 3D 2C has the calibration. It knows the split from it, so only the acquisition and the camera are left.
11. A molecule is at one place in both halves, so x, y and z are shared. Its photons are not: how they split is its colour.
12. After the fit, assign colours is on: the fit ends by running the Assign colours plugin, so the table comes out with its colours.
13. Run.
14. Each localization has a z, its photons in each half, and a colour.
15. The plugin the fit ran is here, in the Analysis tab. Run it to see how it decided, or to decide again.
16. How the photons split: one peak per dye. Each localization gets the channel of its peak; the few between the peaks get none.
17. Probabilistic is stricter: it gives a channel only to what is clearly one dye. Run it with all localizations shown, or it sees only one dye.
18. To draw the colours, filter layer 1 on channel: from 1 to 1, in red.
19. Add a layer for channel 2, in green. Here the ring is one dye and the lines the other.
20. card: What to remember
- Dual-colour calibration: the beads give the map between the halves and a PSF for each; say how the chip is split.
- Spline 3D 2C: x, y and z shared, the photons free.
- Assign colours runs at the end of the fit; a layer per channel shows the colours.
spoken: That is two colours in 3D: calibrate on beads, fit with Spline 3D 2C, and assign the colours.