Grain counting using segmentation and differencing methods

• When counting grains, the grains must be black on a light background. Use ``Color..Invert colors'' if necessary to achieve this.
• Maximize contrast in image before starting. Ideally, all the grain pixels should be below 127 and all background pixels should be above 127 (assuming an 8-bit image and a threshold of 0.5).
• Any large dark objects in the image, such as labels or borders, must be removed before starting. Large areas of uneven background can be removed using ``Filter..Remove Low Frequencies'' and setting kernel to 3$\times$3 or 15$\times$15 and `Amount of filtering' to 100.
• If extraneous items are misidentified as occurrences of the pattern, the threshold should be increased to improve specificity.
• If noise pixels are counted as grains, filter the image with a low-pass filter or noise removal filter before starting.
• ``Min size'' discards any spots containing fewer than the specified total number of pixels. ``Min size'' and ``threshold'' can be adjusted to give a more accurate grain count without counting stray dots.
• If the `labels' box is set, the following marks will be added to the image:
  • A number indicating the measured size of the spot. Clicking on ``Show size'' opens a menu allowing you to specify what is printed on the labels. This can be size, spot number, signal, or no label.
  • An arrow pointing to the center of the spot
  • A rectangle indicating the bounding box of the spot. Note: the box only indicates the maximum x and y range of the spot. Calculations are done on the subset of pixels within this range that exceed the threshold value.
  Be sure to back up the original image (Ctrl-B) before using this feature. The marks can be removed by clicking ``Undo'' (or Ctrl-U). If `labels' is not checked, the spots will be marked with a small cross which will disappear when the mouse is clicked on the image.
• `Label color' sets the color for the labels.
• If any single grain contains more than 10,000 pixels, the counting is automatically aborted.
• If the image contains more than 10,000 grains, tnimage will need to be recompiled with a larger value for MAXGRAINS.
• The size distribution data can be saved to disk by clicking on the ``Save'' button in the Size Distribution graph.
• The differencing algorithm uses the ``Size'' parameter which determines the area over which differencing is to be performed. A larger value will difference over a larger area, which would be suitable for larger grains, but will take longer. Using too small a value will increase the chance that noise pixels will be counted as grains. The differencing method is also more sensitive than quick segmentation, so the threshold should be increased to compensate.
• If ``Sz Graph'' is checked, a graph of size distributions will be created.
• Checking ``Dens Graph'' creates a graph of signal distributions, i.e., the number of spots vs. densitometrically measured signal per spot. This is useful, for example, in analyzing spot sizes on 2D gels.
• If ``Save Data'' is checked, a list of all spots, together with their x and y coordinates, sizes, and signal per spot, will be saved into a disk file. This will be followed by the size distribution and the signal distribution data.
• On large images, if counting grains using the differencing method is too slow, the same effect can be achieved by filtering the image, setting ``Difference filter size'' to 10 in the Filter dialog, then counting grains using the Quick Segmentation method. This allows different thresholds and filter sizes to be more rapidly tested.