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.
To measure the total integrated gray-value, (Quantify | Use sum density else mean density)
To measure the mean integrated gray-value, (Quantify | Use sum density else mean density)
To show the circular mask, set (View | |Set view measurement
options | View measurement
circle) to enabled.
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
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.
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)
Select the spot you want to use to estimate the background by
clicking on it in either the left or right image.
Capture the background corrected estimate using the (Quantify
| Measure by circle | Capture measurement (C-M)).
To measure a set of spots, just repeat subset of these steps for
each new spot.
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.
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.