07 Workflow Checklist

This checklist is an extra page for avoiding common mistakes before running or publishing an analysis.

Before Loading

  • Confirm the TIFF dimensionality.
  • Confirm expected axis order.
  • Keep wavelength.json in the same folder as the TIFF if automatic spectral metadata should be loaded.
  • Decide whether rolling-ball correction should be applied.
  • Decide whether binning should be used.

After Loading

  • Check image shape.
  • Check channel count.
  • For 4D data, verify the selected spectral and z/time axes.
  • Check the spectral axis length against the number of channels.
  • Check physical pixel size and unit.
  • Inspect the average image and several individual channels.

Before Analysis

  • Set the correct number of components.
  • Check ROI component assignments.
  • Check loaded external spectra.
  • Check Gaussian seed rows.
  • Check fixed W/background seeds.
  • Preview seeds before running NNMF or fixed-H NNLS.
  • For fixed-H NNLS, confirm that every component has a valid final H seed. If any seed was residual-filled, inspect it before trusting the result.

After Analysis

  • Inspect the composite image.
  • Inspect each individual W map.
  • Compare final H spectra with seed spectra.
  • Check labels and colors.
  • Check histogram ranges.
  • For 4D results, browse several z/time slices.

Before Export

  • Confirm physical units.
  • Confirm component labels.
  • Confirm LUTs and display ranges.
  • Save a preset.
  • Export H spectra as CSV if needed.
  • Export Fiji/ImageJ-compatible TIFF.
  • Save or record the software version and preset used.

Common Interpretation Warnings

  • A visually clean component is not automatically chemically correct.
  • Fixed-H NNLS is only as good as the supplied spectra.
  • Seeded NNMF can adapt spectra away from the initial seeds.
  • Component labels should be assigned after checking both W maps and H spectra.
  • If a dataset changes shape or channel number, old presets and custom axes may need adjustment.