Flicker Vignette - How do I measure spot intensity?

This vignette shows how do you can measure spot intensity under the circular mask. The background corrected measurement estimate is computed under the circular mask. Note the separate background measurement (C-B) is used for computing background corrected density for measurements made by circular mask (C-M). The background estimate will be used for multiple measurements until you change it. If you are measuring a series of spots by circular mask, then if you change the background intensity measurement, it will be associated with all spots measured since then and saved in the spot list until (or if) you change it again. You can number the spots if the you have enabled the (Quantify | List-of-spots else trial-spot measurement-mode (C-J)) option should be enabled. You may also list the saved spots in the report window. Note that if you save the flicker state, then the spot list will be saved in the .flk file state and an associated .spt spot list file in the spt/ directory. You can restore the spot list if you start flicker on the saved .flk startup file.

Method

  1. To measure the total integrated gray-value, (Quantify | Use sum density else mean density) is enabled.
  2. To measure the mean integrated gray-value, (Quantify | Use sum density else mean density) is disabled.
  3. To show the circular mask, set (View | |Set view measurement options | View measurement circle) to enabled.
  4. Select the new circular mask size in the measure circle (diameter) slider. Note: the NxN is one of 1x1, 3x3, 5x5, ..., 51x51 options which is the size of the rectangle enclosing the circular mask.
  5. To sequentially number and capture a series of spots, enable list-of-spots mode by using the (Quantify | List-of-spots else trial-spot measurement-mode (C-J)) checkbox. Then it will number a series of (C-M) spot measurements as as "+1", "+2", "+3", etc. overlays. Otherwise, the measurement will be numbered as a "+M" overlay and data is not saved in the spot list.
  6. If you want to do background correct, capture the backround estimate using the (Quantify | Measure by circle | Capture background (C-B)). This is valid for multiple (C-M) measurements.
  7. Select the spot you want to use to estimate the background by clicking on it in either the left or right image.
  8. Capture the background corrected estimate using the (Quantify | Measure by circle | Capture measurement (C-M)).
  9. To measure a set of spots, just repeat subset of these steps for each new spot.
  10. To list the set of saved spots in the popup report window, use the (Quantify | Measure by circle | List spots in the spot list). Hint: clear the popup report window first. You can save this list from the popup report window using its SaveAs button.
  11. To export a spot list to Excel, after clearing the report window, use the (Quantify | Measure by circle | List spots in the spot list (tab-delimited)). Then cut and paste the data into Excel.

Setup for the demo

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Contact us     Flicker is a contributed program available at open2dprot.sourceforge.net/Flicker          Revised: 01/19/2004