BIO407 Group5 Image processing of live cells, treated with DFX

## Study:

This data is part of the practical course BIO407 2025 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.

The data is originally from the paper:

Gottwald EM, Schuh CD, Dr點ker P, Haenni D, Pearson A, Ghazi S, Bugarski M, Polesel M, Duss M, Landau EM, Kaech A, Ziegler U, Lundby AKM, Lundby C, Dittrich PS, Hall AM. The iron chelator Deferasirox causes severe mitochondrial swelling without depolarization due to a specific effect on inner membrane permeability. Sci Rep. 2020 Jan 31;10(1):1577. doi: 10.1038/s41598-020-58386-9. PMID: 32005861; PMCID: PMC6994599.

 

## Study Component:

Job: Image Processing

Task: 4) Timelapse of mitochondria in deferasirox (DFX) treated cells

Timelapse data was analyzed: Mitochondria were segmented, their morphology and signal intensity were measured over time, and plotted.

 

## Group:

- Noah Erni

- Stephan Kuster

- Raoul Klein

 

## Biosample:

Opossum kidney (OK) cells (kind gift from the group of Prof O. Devuyst (Physiology, University of Zurich))

 

## Specimen:

DFX treatment: 200uM

Dyes:

-Mitochondria-GFP BacMam 2.0

-TMRM (mitochondrial membrane potential dependent dye)

 

## Image Acquisition:

Images were acquired using a Leica SP8 inverse STED 3x.

Acquired channels:

Channel 1: Mitochondria-GFP, 488nm excitation, 493nm-548nm emission

Channel 2: TMRM, 553nm excitation, 564nm-650nm emission

Objective: HC PL APO CS2 100x/1.40 OIL

 

## Image Data:

Raw timelapse data:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy.lif

Metatdata corresponding to the raw data:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy.txt

Isolated channels:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_1.tif

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_2.tif

Segmentation masks of mitochondria:

MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel1.tif

MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel2.tif

FIJI macro, documenting the analysis steps:

image_prc_macro.ijm.ijm.ijm

Mean circularity of segmented mitochondria at each timepoint:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_Results_channel1.csv

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_Results_channel2.csv

Mean GFP signal (channel1) of segmented mitochondria at each timepoint:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_Results_channel1

Mean TMRM signal (channel2) of segmented mitochondria at each timepoint:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_Results_channel2

Plot of mean mitochondria circularity over time:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_Results_Mean_circularity_mitochondria_channel1.png

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_Results_Mean_circularity_mitochondria_channel2.png

Plot of mean GFP signal in mitochondria over time:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_GFP.png

Plot of mean TMRM signal in mitochondria over time:

151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_TMRM.png

 

## Image Correlation

MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel1.tif and MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel1.tif contain the segmentation masks of the raw data in 151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy.lif. 

The individual channesl have also been isolated in 151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_1.tif and 151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_2.tif

 

## Image Analysis

We used threshold segmentation in FIJI as described in the macro image_prc_macro.ijm.ijm.ijm. Otsu thresholding was appied as well as the function Open and Watershed the 'Analyze Particles' command was used to measure the circularity and mean signal intensity of all segmented mitochondria. The mean measurements were then plotted for each timepoint.