Evaluate 3D data with a bead calibration sample
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Evaluate 3D data with a bead calibration sample

Record a bead calibration data file

We assume that you have an appropriate cylindrical lens in detection path and are using an objective piezo (e.g. PIfoc, Physik Instrumente). The sample should be a thinly coated Tetraspeck bead sample. Beads should be preferred because of the superior brightness and - in case of tetraspec beads - of the sub-resoulution size. You should know the size of the input pixels.

  1. place Tetraspeck surface on microscope

  2. Set the piezo to a sawtooth or triangular pattern that scans your whole planned axial localization range. Exceeding the localization range is not critical, the excess measurements can be cropped later. For an example, we used the following settings:

    function
    triangular
    low position
    45 μm
    high position
    55 μm
    frequence
    0.02 Hz
  3. Set camera exposure time (0.02 s)
  4. Compute and remember piezo movement per frame ((55 μm - 45 μm) · 2 · 0.02 Hz · 0.02 s/fr = 8 nm/fr). This will later be needed as an evaluation input.
  5. focus on Tetraspeck
  6. start piezo
  7. record data

Produce calibration curve

We assume that you have recordad a bead calibration data file and have some plotting software, e.g. gnuplot, Origin or Matlab, at your disposal.
  1. Start rapidSTORM 2 and set User level to Intermediate or higher
  2. Set Input file to calibration data file (in this example: foo.tif)
  3. Set Size of one input pixel to pre-calibrated values (e.g. 142 and 125 nm in X/Y, respectively)
  4. Set Intensity threshold to a high value (5000 ADC) in order to filter noise
  5. Set Fit window radius to a value considerably higher than the best-focused PSF FWHM in order to include the PSF tails, and remember the input. (1100 nm)
  6. Set Fit iteration limit to 100 (optional)
  7. Check boxes PSF width is free fit parameter and Store PSF width
  8. Go to the Expression filter output and set
    1. Set Number of expressions to 4
    2. First Value to assign to to posz
    3. First Expression to assign from to 8 nm/fr * frame
    4. Second Value to assign to to psffwhmx
    5. Second Expression to assign from to psffwhmx - 25 nm[1]
    6. Third Value to assign to to psffwhmy
    7. Third Expression to assign from to psffwhmy - 25 nm
    8. Choose new output to 3D PSF width calibration table
  9. Go to the 3D PSF width calibration table output and set Number of B spline breakpoints to 10
  10. Click Run
  11. Plot the PSF widths in the localization file foo.txt (columns 6 and 7) against the Z truth (column 3) and check for local maxima and outlier points
    • If local maxima exist, narrow the range of usable Z data by setting an appropriate filter expression in the fourth Expression to assign from field in the Expression filter output. In our example, we used posz > 2000 nm && posz < 6000 nm . Go back to Step 11.
    • If outliers are present, raise Intensity threshold and go back to Step 11
    • Otherwise continue with Step 12
  12. You are done with the generation of the calibration file. The generated calibration curves are stored in foo-sigma-table.txt (see the Fundamental the section called “Z calibration file” for details about the file format).

Make 3D super-resolved image colour-coded by Z

We assume that you have a correct bead calibration data file file for your setup.
  1. (Re-)Start rapidSTORM
  2. Set 3D PSF model to Interpolated 3D
  3. Set Z calibration file to the file name of the calibration curve file.
  4. Set Intensity threshold to a similar value as in 2D (e.g. 1000)
  5. Set Fit window radius to the same value you used for producing the calibration curve (1100 nm)
  6. Go to the Image Display output and
    1. Set Colour palette for display to Vary hue with sample coordinate
    2. Set Coordinate to vary hue with to position in sample space z
  7. Run evaluation and wait for job to finish

Make 3D super-resolved Z stack

We assume that you have a correct calibration curve file for your setup.
  1. (Re-)Start rapidSTORM
  2. Follow Step 2 to Step 5 of Make 3D super-resolved image colour-coded by Z.
  3. Set user level to Intermediate
  4. Go to the Image Display output and
    1. Set Resolution in X direction to 20 nm
    2. Set Resolution in Y direction to 20 nm
    3. Set Resolution in Z direction to 50 nm
    4. Check Make 3D image
    5. Change extension of Save image to to .tif
  5. Run evaluation and wait for job to finish

[1] This value is a wild guess. It should denote how much wider a fairly large object like a Tetraspec looks than a fluorophore.

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