Image processing pipelines in Image Analyst MKII
Image Analyst MKIIPipelines Glossary
HomeWorkflowImage Processing BasicsFunctions GlossaryPipelines GlossaryProtocolsQuick How ToSearch

MitoSOX MitoTracker Deep Red Ratio End Point

Parameters:
Name # Type Description
Channel Number for MitoSOX 1 integer The first linked image window with matching channel number will be invoked.
Channel Number for MitoTracker Deep Red 2 integer The first linked image window with matching channel number will be invoked.
Channel Number for Hoechst 3 integer The first linked image window with matching channel number will be invoked.
Background level (percentile) 4 real Background is calculated as frame-by-frame subtraction of the given percentile of the image intensity histogram.
Spectral unmix coefficient between MitoSOX and Mitotracker Deep Red 5 string Bleedthrough of MTDR (Ch2) into the MitoSOX (Ch1) channel. To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix.
Approximate nucleus diameter (pixels) 6 integer Diameter of the nucleus in pixels
Dead cell detection threshold (percentile of Hoechst reference image) 7 real Higher percentile will make dead cell detection less sensitive. Typically 99.0-99.99.
Maximal diameter of MitoSOX debris to mask 8 integer Maximal cross section area in 3D, area=volume in 2D. 0 for not checking
Minimum cell size (area, pixels) 9 integer Maximal cross section area in 3D, area=volume in 2D. 0 for not checking
Maximum cell size (area, pixels) 10 integer Maximal cross section area in 3D, area=volume in 2D. 0 for not checking
MitoTracker detection threshold (% of maximal fluorescence) 11 real Cells brighter than this value will be accepted for analysis. Typically 10-25%.
Export file name for overlay image 12 string
Export file name for ratio image 13 string
Description:
Measurement of MitoSOX:MitoTracker Deep Red fluorescence ratio in an end point assay.
The MitoSOX:MitoTracker Deep Red fluorescence ratio is measured in live cells only, while dead cells and debris are suppressed. The pipeline analyses triple staining of MitoSOX, Mitotracker Deep Red and Hoechst 33342 using image segmentation and intensity measurements.
Excel Data Window output: Calculate MitoSOX:MitoTracker Deep Red ratio by dividing values Ch1_ROI1 column with values in Ch2_ROI1 column.

Staining and imaging protocol:
1) Incubate cell in culture medium with MitoSOX 2uM + MitoTracker Deep Red 10nM for 30-60min.
2) Replace culture medium with a clear medium containing no MitoSOX and MitoTracker and supplemented with MnTMPyP 20uM, Hoechst 33342 5ug/ml, BSA 0.4%
3) Capture three-channel live cell images within 30min (the MitoSOX fluorescence is relatively stable in the presence of MnTMPyP).

Notes on pipeline parameters:
“Spectral Unmixing Coefficient Matrix:” This corrects for the bleedthrough of MTDR into the MitoSOX channel, that is usually very low. To determine spectral crossbleed coefficients use the Tools/Calculate Crossbleed Correction Factor main menu point, or the Math/Blind Spectral Unmix with NMF function. See more on the layout of the Spectral Unmix Coefficient Matrix in the description of Math/Spectral Unmix. Enter MitoSOX as probe/channel #1 and MTDR as probe/channel #2 into the spectral unmix coefficient matrix.

Version history
Based on Internal version: V12
V2:
Layout of data columns as channels 1,2,3 has been fixed