Plate and Grid Background

Plate and Grid Analysis Background Methods

The Plate, Plate Array, Grid, and Grid Array analyses are available with the Grid Analysis Key. The following background methods can be used:

Plate

  • None - Unless data are going to be exported for analysis using a third-party software, do not choose None.
  • User-Defined - A well or wells must be designated as background.

Plate Array and Grid Array

  • Array - Allows the same spots to be designated as background in each sub-grid of an array.
  • Average - Uses space surrounding a spot as background.
  • Median - Uses space surrounding a spot as background.
  • User-Defined - A spot or spots must be designated as background.
  • None - Unless data are going to be exported for analysis using a third-party software, do not choose None.

Grid

  • None - Unless data are going to be exported for analysis using a third-party software, do not choose None.
  • Average - Uses space surrounding a spot as background.
  • Median - Uses space surrounding a spot as background.
  • User-Defined - A spot or spots must be designated as background.

User-Defined Background for Plate and Grid Analysis

Spots or wells must be designated for background subtraction. Any choice of spots/wells can be designated.

  1. Ensure a Plate, Plate Array, Grid, or Grid Array analysis is applied to the image and that the spots/wells are inside the spots/wells in the image.

  2. Use the Display Channel Color buttons in the Display tab to turn on/off channels.

  3. Choose User-Defined from the Background group in the application specific analysis tab.

    Dismiss the dialog warning that no background shape has been assigned.

  4. Click the first spot/well to be assigned as background. Multiple background spots/wells can be selected by:

    • Using Ctrl+Click to select additional spots/wells

    • Clicking and dragging a box around spots/wells to be selected

      Image Studio click and drag to select multiple wells

  5. Click Image Studio Assign Shape icon Assign and all selected wells/spots will be used to calculate background.

To remove spots/wells from the background calculation, select the spots/wells to be removed and click Image Studio Remove Background iconUnassign.

Bkgnd Calculation: The average pixel intensity of each background spot will be calculated, and the background spots will be averaged to find the background value.

Signal: The signal for each spot will be calculated by taking the Total for that spot and subtracting the product of the Bkgnd and the Area.

Average and Median Background for Plate and Grid Analysis

The Average and Median background methods use space surrounding a feature as background.

Bkgnd Calculation: The average or median pixel intensity of a border region surrounding each spot will be used as the background subtracted from that spot.

Signal: The signal for each spot will be calculated by taking the Total for that spot and subtracting the product of the Bkgnd and the Area.

When using the Average or Median background methods, do not allow the border region designated as background to overlap a feature being quantified or autofluorescence from a plate.

Array Background Method for Plate and Grid Arrays

The Array background method allows the same spots to be designated as background in each sub-grid of an array.

Array Definition Dialog Corresponds to Cells in Array - Tiff

  1. Ensure a Plate Array or Grid Array analysis is applied to the image and that the spots are inside the spots in the image.
  2. Choose Array from the Background group in the Plate Array Analysis tab or Grid Array Analysis tab.

  3. In the Advanced Array Definition dialog, click the black Background button at the bottom of the dialog and then click the squares that correspond to background spots in each sub-grid of the array.

    Image Studio advanced array definition dialog

  4. Click OK once appropriate wells have been assigned.

The Advanced Array Definition dialog with additional options for sizing and naming spots can also be opened by clicking the dialog launch arrow at the bottom of the Spot Layout group or Array group.

Bkgnd Calculation: Background spots in each sub-grid are used to determine background for that sub-grid. The average pixel intensity of each background spot will be calculated, and the background spots will be averaged to find the background value.

Signal: The signal for each spot in a sub-grid will be calculated by taking the Total for that spot and subtracting the product of the background from the background spots in that sub-grid and the Area of the spot being quantified.