File Formats

File Formats

This page documents the input and output file structures the tool uses. Understanding the frame layout of the multi-frame TIFF stacks is the key to using the tool's outputs correctly.

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Input images

Type	Extension	Notes
Multi-frame TIFF	.tif, .tiff	Contains the FRET, Donor, and Acceptor channels as separate frames.
Zeiss CZI	.czi	Converted automatically on load to a multi-frame TIFF.

CZI conversion

When a .czi file is loaded, the tool extracts the channels into a 3-frame TIFF (FRET, Donor, Acceptor) and preserves the original raw intensity values (no rescaling). A 4th channel is included when present for S3/S4 calculations.

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Segmented stacks (Segmentation output)

Saved into a segmented/ folder next to the source image, named:

outline_segmented_<name>.tif      # when "Generate outlines only" is on
whole-cell_segmented_<name>.tif   # when filled masks are used

Frame layout:

Frame	Content
0	Segmentation label mask (0 = background, 1..N = cell labels)
1	FRET channel
2	Donor channel
3	Acceptor channel (present for 4-frame data)

This is exactly the layout the Bleed-Through and FRET tabs expect. Raw intensities of every channel are preserved so downstream calculations are accurate.

Membrane + whole-cell stacks (Segment both)

When Segment both (membrane + whole-cell) is enabled, the file is prefixed both_segmented_ and uses a different layout intended for the Intensity and Densitometry tab:

Frame	Content
0	Outline (membrane) label mask — a band drawn inward from each cell border
1	Filled (whole-cell) label mask
2…N	Raw image channels, in their original input order (not reordered)

Both masks share the same label ids, so each cell's membrane band and whole-cell body correspond. The Intensity tab's channel registry lets you declare which frame is which. These stacks are not meant for the Bleed-Through or FRET tabs.

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Bleed-through parameters (bt_params.json)

Written by Save Parameters in the Bleed-Through tab to the default location and copied into each input image directory. Structure:

{
  "s3_s4_enabled": false,
  "donor_params":    { "...": "model type, coefficients, sigma, sampling, selected_fit_model, fit_results" },
  "acceptor_params": { "..." },
  "s3_params":       { "..." },
  "s4_params":       { "..." }
}

Each channel block records the fitting model, its coefficients, the processing settings, and the selected model so the fit can be restored exactly. Load it with Load Parameters.

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FRET outputs

Output	Format	Location / how
Efficiency maps (one per formula)	32-bit float TIFF	FRET_Analysis_Results/<name>_<formula>_efficiency.tif, when efficiency saving is enabled
Aggregate statistics	CSV	Exported from the aggregate statistics table
Figures (maps, histograms, box plots)	PNG / PDF	Save button on any plot or its pop-out window

Efficiency maps store percentage values (0–100%) as floating-point pixels, with background and excluded pixels set to 0.

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Intensity / Densitometry outputs

Output	Format	Location / how
Per-cell measurements	CSV	Export CSV — one row per cell × channel with columns image, group, cell, channel and every metric (membrane/interior/whole mean, area, integrated density, CTCF, membrane enrichment, membrane fraction)
Figures (histogram, box plot, scatter, legend)	PNG / TIFF / PDF / SVG	Save Plot (300 DPI) in any plot pop-out

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Notes

• The label mask must be frame 0 of any stack you feed to the Bleed-Through or FRET tabs. If you build stacks outside the tool, follow the layout above.
• 3-frame stacks (mask, FRET, Donor/Acceptor) are sufficient for S1/S2; 4-frame stacks are required for S3/S4.
• See Workflows and Data Flow for how these files move between tabs.