ggirelli / deconwolf-tissue-smFISH

deconwolf-smFISH-analysis

Tissue smFISH at different magnifications, either processed with deconwolf or not.

NOTE: fields 001 and 005 are swapped between publication and data/plots from this repository.

Details

• Voxel size (ZYX)
  • 60x: 300x108.3x108.3 nm
  • 20x: 300x325x325 nm
• Fluorophore emission lambda: 665 nm

Datasets

• 60x: deconwolfed and raw
• 20x: deconwolfed and raw

The images from 20x were previously registered on the 60x ones.

Similarly, undilated masks were also initially provided for both magnifications.

Analysis steps

1. Mask dilation with no overlap
2. Nuclear feature analysis and filtering
3. DOTTER analysis and dots export
   • Using DoG approach, center_of_mass refinement.
   • Extract all dots, 1 M dots, calculating SNR, nSNR, and FWHM.
   • Corrected SNR (i.e., SNR2 column) added by EW.
   • Re-scale dots intensity (i.e., Value column) based on deconwolf scaling factor.
   • Filter by FWHM and nuclei selection
4. Reference dots selection
   • Based on dots intensity threshold
   • Generated overlays of selected dots
5. Comparing different magnifications
   • Shift correction between datasets
   • Check overlap and percentage of selected dots