BIO407 Group 4: Segmentation of cells in screening Experiment of DFX treated cells

## Study: This data is part of the practical course BIO407 2026 at the University of Zurich, titled 'Advanced Microscopy: From preparation to data and visualization'. The effects of Deferasirox (DFX) on mitochondria morphology were examined using different microscopy techniques. ## Study Component: Job: Image Processing Task: 3) Segment cells using Cellpose & measure total/average Tomm20 intensity for all conditions ## Biosample: COS-7 cells ## Specimen: Four different conditions: -Control 1: No treatment -Control 2: 5h DMSO treatment -Condition 1: 3h 200uM DFX treatment (in duplicate) -Condition 2: 5h 200uM DFX treatment (in duplicate) Cells were fixed with 4% PFA, 0.05% GTA, 0.5% Saponin in PHEM. Cells were labeled with DAPI, TOMM20-Alexa488, Tubulin-Alexa568, SiR-Actin. ## Image Acquisition: Images were acquired using a LeicaTHUNDER Imager 3D Live Cell on the Leica DMi8 platform. Acquired channels: Channel 1: DAPI, 390nm excitation, 460/80nm emission Channel 2: Alexa488-TOMM20, 475nm excitation, 535/70nm emission Channel 3: Alexa568-Tubulin, 555nm excitation, 590/50nm emission Channel 4: SiR-actin, 635nm excitation, 642/80nm emission Objective: 40x LEICA HC PL APO CORR air objective, 0.95NA ## Image Data: Raw data: Upload to Zenodo\Raw Data\20260225_BIO407_G4_LeicaThunder_ExperimentalConditionsScreening.lif Upload to Zenodo\Raw Data\20260225_BIO407_G4_LeicaThunder_ExperimentalConditionsScreening.lifext Processed images: Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P1.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P2.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P3.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P4.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P5.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P6.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P7.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P8.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\1_Control_P9.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P1.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P2.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P3.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P4.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P5.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P6.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P7.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P8.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\2_DMSO_P9.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P1.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P2.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P3.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P4.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P5.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P6.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P7.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P8.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\3_3h_DFX_P9.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P1.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P2.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P3.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P4.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P5.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P6.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P7.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P8.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\4_3h_DFX_2_P9.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P1.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P2.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P3.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P4.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P5.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P6.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P7.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P8.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\5_5h_DFX_P9.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P1.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P2.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P3.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P4.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P5.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P6.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P7.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P8.tif Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images\6_5h_DFX_2_P9.tif Segmentation masks of cells, created by cellpose: Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P1_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P2_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P3_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P4_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P5_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P6_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P7_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P8_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\1_Control_P9_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P1_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P2_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P3_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P4_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P5_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P6_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P7_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P8_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\2_DMSO_P9_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P1_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P2_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P3_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P4_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P5_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P6_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P7_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P8_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\3_3h_DFX_P9_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P1_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P2_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P3_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P4_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P5_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P6_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P7_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P8_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\4_3h_DFX_2_P9_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P1_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P2_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P3_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P4_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P5_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P6_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P7_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P8_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\5_5h_DFX_P9_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P1_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P2_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P3_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P4_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P5_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P6_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P7_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P8_rois.zip Upload to Zenodo\Processed Images and Segmentation Masks\Segmentation Masks\6_5h_DFX_2_P9_rois.zip FIJI macro, used to prepare data for cellpose: Upload to Zenodo\Macro Files\20260227_BIO407_G4_LifToTif_Converter.ijm FIJI macro, used to analyze segmentations: Upload to Zenodo\Macro Files\20260228_BIO407_G4_ROImeasurements.ijm Result tables: Upload to Zenodo\Result Table\20260228_BIO407_G4_MeasurementsOverAllConditions.csv Analysis file: Upload to Zenodo\Analysis Files\20260228_BIO407_G4_DataAnalysis.R Upload to Zenodo\Analysis Files\.RData Upload to Zenodo\Analysis Files\.Rhistory ## Image Analysis A Fiji macro was used to prepare the data for cellpose: The DAPI, Tomm20, and Actin channels were extracted, merged and saved as .tif files. (Fiji macro: Upload to Zenodo\Macro Files\20260227_BIO407_G4_LifToTif_Converter.ijm) We used cellpose v3 to segment cells based on DAPI & actin signal. The command used was: cellpose --dir 'Upload to Zenodo\Processed Images and Segmentation Masks\Processed Images --use_gpu -- diameter 300 --chan 3 --chan2 1 --no_npy --save_rois –-verbose Fiji was used to measure area, mean & total Tomm20 intensity of the segmented cells. (Fiji macro: Upload to Zenodo\Macro Files\20260228_BIO407_G4_ROImeasurements.ijm) The population distributions were plotted for each condition using R. (R file: Upload to Zenodo\Analysis Files\20260228_BIO407_G4_DataAnalysis.R)