Cross/Line Analysis

Lining [Time]

Tool to measure intensities in a line shaped area. We use it to determine protein recruitment or mobility upon DNA damage induction via multi photon absorption. The stack is aligned to correct for cell movement between subsequent time frames

Usage

The macro works on any time series of images, in which an object segmentation is possible. After stack alignment the user is asked to adjust the position of the line and define a background ROI

Technical Info

Core segmentation is done via an automatic thresholding method. In the result table the evaluated ROI, the size of the ROIs, information about the intensity distribution in the respective ROIs for each frame and the change of the signal in the line area in relation to the first frame are given. The signal change is calculated as following:

Resulti = ((LineInti-BGDi) / (CoreInti-BGDi)) / ((LineInt1-BGD1) / (CoreInt1-BGD1))

  i frame number
  Result     signal change
  LineInt    mean line intensity
  CoreInt   mean object intensity
  BGD background intensity

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  • Signal change within the line area (ROI #4)
  • ROIs generated by the Lining Tool
  • DNA damage repair protein enrichment at irradiation site

Crossing [Channels]

Tool to measure intensities in a cross shaped area. Cross and background ROIs are selected manually.

Usage

The macro works on any images, in which an object segmentation is possible. The user is asked to adjust the position of the cross and define a background ROI

Technical Info

Core segmentation is done via an automatic thresholding method. In the result table the evaluated ROI, the size of the ROIs, information about the intensity distribution in the respective ROIs for each frame.

    >
  • Marker quantification at the irradiation site
  • Cells after DNA damage induction: Red: γH2Ax, Green: TUNEL, Blue: Hoechst

Crossing/lining Options

  • Cross width [unit]: Width of the cross/line in units. That is pixels if no metadata is present otherwise the unit given in the metadata.
  • Cross padding [unit]: Width of the free area around the cross in unit (excluded from analysis).
  • Cross fixed length [unit]: Length of the cross/line in units.
  • Delete slice: Excludes a slice from the analysis (e.g. Slice in which the illumination has taken place)
  • Threshold Method: Automatic threshold method for the core channel.
  • Core Channel: Color of the core channel.
  • Cross Channel: Color of the cross channel. The macro switches to this channel on startup.