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 2D: split camera, fit and colours
Two dyes imaged at once, on one camera: a dichroic splits each molecule's light between the two halves of the chip.
Both dyes appear in both halves, in different proportions. The proportion says which dye a molecule is.
spoken: Two dyes on one camera: each molecule's light is split between two halves of the chip, and how it splits tells the dye.
2. card: Three steps
Transformation: where the second half sees what the first sees, a little shifted, turned and magnified.
Fit: both spots of a molecule at once, one position, photons for each half.
Colour: from how the photons split.
spoken: Three steps: map one half onto the other, fit both spots of a molecule together, and assign the colour.
3. Gaussian 2D 2C fits a split camera. The acquisition and the camera go in as for any fit.
4. Calibrate from this movie measures the transformation from the movie's own molecules, which appear in both halves. No beads needed.
5. It is saved beside the result, to use again here. A dual-colour bead calibration works too.
6. Each molecule is fitted in both halves at once: one position, photons for each half. The width is free per half, as the colours differ.
7. After the fit, assign colours is on: the fit ends by running the Assign colours plugin, so the table comes out with its colours.
8. Run.
9. The output box says how well the halves were matched, and how many localizations got a colour.
10. The plugin the fit ran is here, in the Analysis tab. Run it to see how it decided, or to decide again.
11. How the photons split: one peak per dye. Each localization gets the channel of its peak; the few between the peaks get none.
12. 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.
13. To draw the colours, filter layer 1 on channel: from 1 to 1, in red.
14. Add a layer for channel 2, in green. Here the ring is one dye and the lines the other.
15. card: What to remember
- A split camera sees each molecule twice; the photon split is its colour.
- Transformation: calibrate from the movie, or use a bead calibration.
- Gaussian 2D 2C fits both halves together.
- Assign colours runs at the end of the fit and gives each localization a channel; run it again to change the rule.
- A layer per channel shows the colours.
spoken: That is two colours in 2D. Next: two colours in 3D, with a dual-colour bead calibration.