Fluorescence Image Histogram

Two cursors (Min and Max), defined in the bottom rightcorner of the histogram, can be used to define the image display range. They will be taken into account only if the Use Image Histogram for Contrast checkbox above the histogram is checked.

Any pixel in the original image with value smaller than Min (resp. larger than Max) will be represented with the color corresponding to the lowest (resp. highest) color, which can be selected individually at the bottom and top of the selected palette.

In the particular example shown above, the main histogram peak to the left corresponds to background pixels, which are set to green with the chosen position of the Min cursor (green). A few pixels are found above the Max cursor and will therefore be set to red. Note that this is only a display feature. Pixels below or above these values are still included in the phasor calculation, unless stated otherwise by the Reject Low count Pixels or Reject High Count Pixels checkboxes of the Settngs>>Source Image window (see below).

Intensity Thresholds

In order to use the information provided by the image intensity histogram to instruct the software to reject low or high intensity pixels, open up the Settings>>Source Image window:

Two groups of controls can be used: Low Count Pixels Rejection Options and High Count Pixels Rejection Options.

1. Low Count Pixels Rejection Options

• • Reject Low Count Pixels: if unchecked, no "low count" test is performed on any pixel. If checked, the next two controls are used for each pixels.

  • Background Threshold x Factor: when different from zero, any pixel in the sum image of a data set, with an intensity smaller than A x P, where A is the parameter's value and P is the location of the sum image histogram peak, will be ignored when computing the fluorescence decay and phasor. For instance, a value of A = 1 means that all pixels with value below the image histogram peak position will be ignored, while A = 0 means that all pixels will be retained. A nominal value to exclude background pixels appears to be A = 3, but it is recommended to experiment, as histogram shape can vary from sample to sample. In particular, it may happen that the histogram peak does not correspond to the most likely background value.

• Fixed Background Threshold: complements the previous criterion, which may fail in some particular cases. This parameter B simply sets the minimum value of a pixel in the sum image for its phasor to be computed and included in the Phasor Plot Image, Phasor Graph and Fluorescence Decay calculations.

The rejection criterion uses the maximum of A x P and B.

2. High Count Pixels Rejection Options

• • Reject HighCount Pixels: if unchecked, no "highcount" test is performed on any pixel. If checked, the next two controls are used for each pixels.

  • Peak Threshold x Factor: when different from zero, any pixel in the sum image of a data set, with an intensity larger than K x M, where K is the parameter's value and M is the location of the sum image maximum, will be ignored when computing the fluorescence decay and phasor. For instance, a value of K = 0.7 means that all pixels with value above 70% of the maximum pixel intensity will be ignored, while K = 1 means that all pixels will be retained.

• Fixed PeakThreshold: complements the previous criterion. This parameter C simply sets the maximum value of a pixel in the sum image for its phasor to be computed and included in the Phasor Plot Image, Phasor Graph and Fluorescence Decay calculations.

The rejection criterion uses the minimum of K x M and C.