SMAPpy help pages

What each window and plugin does, how it works, and every control and setting – the pages the ? button opens in the program. The settings tables are read off the plugins themselves, and the figures are drawn by the plugins' own code on simulated data.

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The program's windows and tabs

• 3D view – A box of the data, the slab, shown from any angle, with the Render tab's layers, filters and colours.
• Batch window – Runs one chain of plugins over many files, each on its own, and writes every file's table, numbers and figures to an output folder with a summary of all of them.
• Bead calibration – Turns z-stacks of fluorescent beads into the measured 3D PSF model the Spline 3D fitters use, and for a split camera into two models and the transformation between the halves.
• ROI manager – The window where sites are found, looked at one by one, kept or excluded, and evaluated.
• ROI tab – The shape every analysis ROI shares, and the way into the ROI manager and the evaluation of the sites.
• Render tab – The layers of the picture – which localizations each one shows, and how it is drawn.

Analysis › Drift

• COMET – Estimate drift from the selected localizations with COMET and subtract it from all of them.
• RCC – Estimate drift by cross-correlating rendered time windows (RCC) and subtract it from all localizations.

Analysis › Dual-Color

• AssignColors – Colour two-channel localizations by their photon split, r = (ch1 - ch2) / (ch1 + ch2): cut the histogram of r at its minima, or give each localization the probability of every colour from its own photon counts. Writes those probabilities, and channel: the colour decided on, or 0 where the probabilities are too close to choose between and where no colour fits at all.

Analysis › Measure

• Ground Truth – A fit of a simulation against where the molecules really were.
• Line Profile – Profiles along or across a line ROI, fitted without binning them.
• Localization Precision – The precision the data shows, beside the precision the fitter expected.
• Localization Statistics – Photons, localization precision and on-time, each with its law fitted.

Analysis › Process

• History – What has been done to these localizations, with the settings used.
• Image Tags – The per-frame image tags of the acquisition (piezo z, UV pulse, ...)
• Math Parser – A new localization field from an expression in the existing ones.
• Remove Localizations – Remove, or hide, the localizations inside or outside the ROI.

Analysis › Register

• Calibrate transform – Measure the transformation between the two halves of a split frame from localizations fitted over the whole frame: no initial shift, no magnification and no split position needed.

Chain

• Layers – The layers' bounds and grouping, as the Render tab would set them.

File › Export

• Image – Render the localizations to a picture file, with no window.

File › Load

• Auto – Read any known format, choosing the reader by the file name.
• MINFLUX – Read a MINFLUX .npy, .zip or .json export.
• SMAP – Read a SMAP _sml.mat file.
• csv – Read a csv, tsv or plain text table, guessing its columns.
• smappy HDF5 – Read a file smappy wrote.

File › Save

• smappy HDF5 – Write the current table, its history and its ROIs.

File › Simulate

• Blinking Structure – A labelled structure, blinking: localizations to try, or camera frames

Localize

• Gaussian 2D – Detect and fit with a free-width Gaussian PSF: x, y, photons, sigma.
• Gaussian 2D 2C – Detect and fit both halves of a split frame as one emitter with a Gaussian PSF, sharing x and y: adds the photon ratio that tells the two colours apart. No PSF calibration; the registration can be measured from the movie itself.
• Spline 3D – Detect and fit with an experimental spline PSF from a bead calibration: adds z.
• Spline 3D 2C – Detect and fit both halves of a split frame as one emitter, sharing x, y and z: adds the photon ratio that tells the two colours apart.

ROIManager › Analyze

• Histograms – Distributions of the evaluation results, one histogram per column.
• NPC Labeling Efficiency – The labelling efficiency of nuclear pores, from the corners each shows

ROIManager › Evaluate

• NPC Corners – Counts the corners a nuclear pore shows and the localizations it has.
• Statistics – Counts a site's localizations and averages their precision and photons.

ROIManager › Segment

• Density Peaks – Proposes ROIs where localizations pile up, after a Gaussian blur.
• NPC – Finds nuclear pores with a ring filter, fits a circle to each and keeps

ROIManager › Workflow

• NPC Analysis – Finds the nuclear pores of the file, counts their corners and localizations, and fits the labelling efficiency, in one run.