Annotated DNA Double Strand Break Ionizing Radiation-Induced Foci (gH2AX 53BP1) Confocal Microscopy, pt. 2

Summary

Dataset of confocal microscopy data of cells exposed to gamma-irradiation and immunostained with gH2AX and 53BP1.

• Nuclei segmentation Head and neck primocultures immunostained with gH2AX/53BP1, Training/Testing/Validation dataset (nucleus_segmentation.zip) (available in part 1, https://dx.doi.org/10.5281/zenodo.4067741) 
• IRIF Foci: Head and neck primocultures immunostained with gH2AX/53BP1, Training/Testing/Validation dataset. (foci_detection.zip) (available in part 1, https://dx.doi.org/10.5281/zenodo.4067741)
• Cell lines: U-87 and NHDF Cells exposed to 0.5-8 Gy 30 min and 8h post irradiation - confocal microscopy data of gH2AX/53BP1/DAPI annotated for gH2AX, 53BP1 and colocalized foci separately (cell_lines_U87.zip and cell_lines_NHDF.zip, this part of dataset) 
• Code: the code is available at https://github.com/tomasvicar/DeepFoci
• Preprint: Vicar et al, DeepFoci: Deep Learning-Based Algorithm for Fast Automatic Analysis of DNA Double Strand Break Ionizing Radiation-Induced Foci, 10.1101/2020.10.07.321927
• Publication: Vicar et al, TBA

Materials and methods

Dataset

Following cells were used:

1) The training/validation/testing datasets was based on patient-derived primary cell cultures prepared from spinocellular tumors and morphologically normal tissues adjacent to the tumor taken from patients suffering from head and neck cancer. The dataset was divided into two subsets: one for training, validation and testing the nucleus segmentation (237/10/30 fields of view (FOVs), respectively) and one for training, validation and testing the focus segmentation (239/60/100 FOVs). The dataset consisted of several cell types: a) tumor cells, b) tumor-associated fibroblasts, and c) cells from morphologically normal tissues. All cell types were fixed at different periods of time (0 (non-irradiated control), 0.5, 8 or 24 h PI) after exposure to 2 Gy of gamma-rays. The representation of cells in two subsets with respect to the cell type and post-irradiation time (i.e., DSB repair duration) was random.

2) The evaluation dataset was used to assess the robustness of segmentation procedures. It was composed of multiple types of differently treated cells in order to represent a highly challenging dataset maximally reflecting high biological and technical variability between samples, as it may appear in research or clinical practice. The dataset contained  a) mesenchymal NHDF fibroblasts coming from a standard permanent cell line, b) radioresistant U-87 glioblastoma cells coming from a standard permanent cell line, c) tumor cells (CD90-) and tumor-associated fibroblasts (CD90+) prepared as a primary culture from a spinocellular tumors of patients (different from dataset 1) suffering from a head and neck cancer, and d) cells prepared as primary cultures from morphologically normal tissue adjacent to tumors of involved head and neck cancer patients. NHDF and U-87 cells received 0.5, 0, 1, 2,  4 and 8 Gy of gamma-rays and were fixed at 30 min and 8 h post-irradiation, while the primary cultures were only exposed to the dose of 2 Gy (for a limited amount of the cell material) and fixed at 0 (non-irradiated control), 0.5, 8 or 24 h post-irradiation times.

Gamma irradiation

The cells were irradiated at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic in a following schemes: (a) patient-derived primoculture was irradiated with a single dose of 2 Gy (D = 1 Gy/min) of gamma-rays (60Co, Chisostat, Chirana, CR) , (b) U-87 and NHDF cells were irradiated with doses 0.5-8 Gy (D = 1 Gy/min). Cells were irradiated in RPMI 1640 medium (37 °C, normal atmosphere). Confocal microscopy of gammaH2AX and 53BP1 foci immunodetection was consequently performed.

Fluorescent staining

DNA double strand breaks (DSBs) were quantified in different periods of time post-irradiation (30 min, 8h and 24h post irradiation) by means of $\gamma$H2AX and 53BP1 foci immunodetection combined with  confocal microscopy. For details see \cite{falk2007chromatin}.

Confocal microscopy

The microscopy of samples was performed at the Institute of Biophysics, Czech Academy of Sciences, Brno, Czech Republic. Leica DM RXA microscope (equipped with DMSTC motorized stage, Piezzo z-movement, MicroMax CCD camera, CSU-10 confocal unit and 488, 562, and 714 nm laser diodes with AOTF) was used for acquiring detailed cell images (100× oil immersion Plan Fluotar lens, NA 1.3). Total 50 Z slices was captured with Z step size 0.3 μm.

File description

all files are compressed hyperstack tiffs (50 Z slices and 3 fluorescent channels, XYCZ order), 100x magnification

• foci_detection.zip: IRIF Foci (available in part 1, https://dx.doi.org/10.5281/zenodo.4067741): Head and neck primocultures immunostained with gH2AX/53BP1, Training/Testing/Validation dataset: FOVs 240/100/120 files for training, testing, and validation, organisation:
  • data_001.tif – 3channel Z.stack tiff
  • mask_001.tif – respective Z stack mask with single points per IRIF focus (manual annotation, training and testing subsets only)
  • data_description.xlsx – description of sample type (2Gy post irradiation times and characteristics of primary culture of squamous cell cancer of patients)
  • data_001_pos.csv – manual annotation of gH2AX/53BPI IRIF foci by two experts – cordinates file (in 2D, validation only)
• nucleus_segmentation.zip (available in part 1, https://dx.doi.org/10.5281/zenodo.4067741) Nuclei segmentation Head and neck primocultures immunostained with gH2AX/53BP1, FOVs 237 Training/ 30 Testing/ 10 Validation dataset
  • data_001.tif – 3channel Z.stack tiff
  • mask_001.tif – respective Z stack mask with manually annotated nucleus mask (manual annotation, training and testing subsets only)
  • data_description.xlsx – description of sample type (2Gy post irradiation times and characteristics of primary culture of squamous cell cancer of patients)
• U87.zip and NHDF.zip: Annotated gH2AX/53BP1 foci in cell lines exposed to increasing dose, annotations performed for gH2AX, 53BP1 and colocalized focus separatelly. 679 annotated FOVs for both cell lines.
  • control.png - RGB control figure showing merge and annotated overlay
  • data_53BP1.tif - TIFF Z-stack, confocal microcopy, 53BP1 channel
  • data_DAPI.tif -  TIFF Z-stack, confocal microcopy, DAPI channel
  • data_gH2AX.tif  - TIFF Z-stack, confocal microcopy, gH2AX channel
  • labels.json - foci labels for individual channel
  • mask.tif - generated Z-stack of nucleus mask