Appendix

Ratiometric colour settings

When processing ratiometric localisation data (having a gFrac column) the splitting ratios for each dye species can either be set in the series metadata, or by using the Colour tab (Fig. 17). To add a labelling right click in the Fluorophores list box and select Add. Enter a channel name and splitting ratio in the dialog that opens. Alternatively click Guess to attempt to automatically detect the channels using the K-means algorithm. Once added, you can click in the name or ratio columns to edit. The plot above is a scatter plot showing a subset of all localisations and updates to show the resulting channel assignments.

Thresholds used in the Bayesian assignment process are adjustable in the Channel Assignment panel. A dye is assigned to a given channel if both it’s probability of belonging to that channel is greater than p_dye and it’s probability of belonging to any other channel is less than p_other. The defaults assign fluorophores to the most likely channel and ensure that the chance of a false assignment is less than 10%. We find they seldom need tweaking. If adjustment is necessary, p_other, which controls the rejection of potentially mis-assigned localisations, is most useful. It is tempting to think that p_dye should be higher (i.e. we should have a high certainty that a dye belongs to a given channel), but this would be a mistake - p_dye = 0.1 will include 90% of the statistical spread of localisations belonging to that channel. p_dye = 0.5 by comparison would only capture 50% of a dye’s statistical spread and would needlessly discard a large fraction of the localisations.

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Fig. 17 Ratiometric splitting in the Colour tab.

Isolating a single channel for processing

To apply processing steps to a single channel (rather than to all channels at once), it needs to be isolated in the pipeline. To do this, navigate to the Pipeline Recipe tab and select Add Module, as in Fig. 18 b. Then select the ExtractTableChannel recipe from the localisations recipes and press Add. This will result in a dialog box as shown in Fig. 18 c, where here the first color channel, chan0, is selected. Returning to the View tab and selecting filtered as the output in the upper-left portion of the window shows only the localizations present in the color channel chan0 (Fig. 18 a, bottom). Additional data processing will only operate on this color channel as long as filtered is selected as the output.

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Fig. 18 Visualization and color channel selection of 3-color super-resolution image of cis, medial, and trans-Golgi. (a) Top. All three color channels visualized in a single layer. (b). Selection of the ExtractTableChannel recipe in the Pipeline Recipe tab and ExtractTableChannel dialog box, set to extract color channel chan0 from the original data.