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: 3D fitting: bead calibration and spline fitting
In 3D, z is read from the shape of each spot, so the fit has to know that shape at every z.
A real PSF is not the ideal one a formula gives: aberrations bend it. So SMAPpy measures it, on beads, and fits with that realistic model. The fit is more precise, and z comes out right.
spoken: In 3D, z is read from the shape of each spot. SMAPpy uses a realistic PSF model, measured on beads, not an ideal formula.
2. card: Astigmatism
A cylindrical lens in the detection path makes each spot elliptical: wide in one direction above the focus, in the other below it.
spoken: A cylindrical lens makes the spots elliptical, and their shape tells how high each molecule sits.
3. card: Bead stacks
Fluorescent beads on a coverslip, imaged while the objective steps through focus: each bead records the PSF at every z.
Many beads are aligned and averaged, and a spline makes the average smooth, so the model can be read at any z.
spoken: Beads imaged through focus record the PSF at every z. Averaged and smoothed, they become the model.
4. Tools, Bead calibration opens its window. The Localize tab has the same button.
5. Add the bead stacks: files, or a whole folder. All of them go into one calibration, so more beads make a smoother model.
6. The z step comes from the file's metadata. A plain TIFF has none, so type it in.
7. The ROI size is the square cut around each bead: large enough for the widest spot, far out of focus.
8. Detect and calibrate finds the beads, aligns them in x, y and z, and builds the model.
9. Overview: the beads it found, green if used, and the averaged PSF seen from the side, narrowing through focus.
10. Each bead is a row, with how well it matches the others. Outliers are left out by themselves; space excludes one by hand, then Recalculate.
11. Calculate fit quality fits the beads back with the spline fitter, as the data will be fitted.
12. The fitted z of each bead against where it was: on the line over the range the calibration can be trusted.
13. Save the calibration next to the beads; the name says what it is.
14. Use in the Spline 3D fitter puts it straight into the fitter.
15. Spline 3D now has the calibration. Give it the acquisition; the camera and the detection are as in 2D.
16. Run.
17. Colour by z, and the ring rises and falls as it was simulated.
18. Far from focus the spots are faint and wide, and z is less sure. The z filter keeps the range the calibration covers.
19. card: What to remember
- SMAPpy fits 3D with a measured PSF, from beads.
- Bead calibration: add the stacks, set the z step, calibrate, check the fit quality, save.
- Spline 3D: the acquisition and the calibration; the rest as in 2D.
- Trust z over the calibrated range, and filter to it.
spoken: That is 3D fitting: measure the PSF on beads, check it, and fit with it. Next: rendering the result.