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.

# 研究概况
本数据集隶属于苏黎世大学2025年BIO407实践课程，课程主题为「高级显微学：从样品制备、数据获取到可视化分析」。
本研究通过多种显微成像技术，探究了去铁司糖（Deferasirox, DFX）对线粒体形态的影响。
该数据集源自以下发表论文：
Gottwald EM, Schuh CD, Dräger 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. 铁螯合剂去铁司糖可通过特异性作用于线粒体内膜通透性，引发严重线粒体肿胀但不伴随膜电位去极化. 科学报告. 2020年1月31日;10(1):1577. DOI: 10.1038/s41598-020-58386-9. PMID: 32005861; PMCID: PMC6994599.

# 研究任务模块
## 任务分类：图像处理
任务4：经去铁司糖（DFX）处理的细胞的线粒体延时成像分析
对延时成像数据开展如下分析：完成线粒体分割，随时间推移量化其形态特征与信号强度，并绘制对应统计图表。

# 研究团队
- 诺亚·埃尔尼（Noah Erni）
- 斯蒂芬·库斯特（Stephan Kuster）
- 拉乌尔·克莱因（Raoul Klein）

# 生物样本
负鼠肾（Opossum kidney, OK）细胞（由苏黎世大学生理学系O. Devuyst教授课题组惠赠）

# 样品处理
干预方案：采用200μM去铁司糖进行细胞处理
染色剂：
- 线粒体-GFP杆状病毒转导载体2.0（Mitochondria-GFP BacMam 2.0）
- 四甲基罗丹明甲酯（TMRM，线粒体膜电位依赖性染料）

# 成像参数
成像设备：采用徕卡SP8倒置式受激发射损耗显微镜3x（Leica SP8 inverse STED 3x）
成像通道：
通道1：线粒体-GFP，激发波长488nm，发射波长范围493nm~548nm
通道2：TMRM，激发波长553nm，发射波长范围564nm~650nm
物镜：HC PL APO CS2 100×/1.40油浸物镜

# 成像数据
## 原始延时成像数据
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy.lif
## 对应原始数据的元数据
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy.txt
## 分离后的单通道数据
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_1.tif
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_2.tif
## 线粒体分割掩码文件
MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel1.tif
MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel2.tif
## 分析流程宏脚本
记录分析步骤的FIJI宏脚本：
image_prc_macro.ijm.ijm.ijm
## 各时间点线粒体平均圆度数据
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_Results_channel1.csv
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_Results_channel2.csv
## 各时间点线粒体平均GFP信号强度（通道1）数据
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_Results_channel1
## 各时间点线粒体平均TMRM信号强度（通道2）数据
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_Results_channel2
## 线粒体平均圆度随时间变化统计图
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
## 线粒体内平均GFP信号强度随时间变化统计图
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_GFP.png
## 线粒体内平均TMRM信号强度随时间变化统计图
151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_intensity_TMRM.png

# 成像数据关联说明
MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel1.tif与MASK_151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression_mask_channel1.tif包含了151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy.lif中原始数据的分割掩码。单通道成像数据已被单独提取并保存至151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_1.tif与151106_slide1_bacmam20_PPC_7473_Esther_DH_Progression - Copy_Channel_2.tif文件中。

# 成像数据分析
本研究采用FIJI软件开展阈值分割分析，具体流程详见宏脚本image_prc_macro.ijm.ijm.ijm。分析过程中采用了大津阈值法（Otsu thresholding），并结合开运算与分水岭算法，随后通过「分析粒子」命令量化所有分割线粒体的圆度与平均信号强度。最终针对每个时间点的平均测量值绘制统计图表。