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Counting Features Per Unit Volume
The most straightforward way to count the number of features per unit volume requires the comparison of two section planes a known distance apart (and close enough together that features cannot “hide” in the space between them). Aligning images from serial sections is difficult, but the confocal light microscope simplifies this procedure by obtaining optical sections. Any feature that is seen in the bottom section and is absent in the upper section must have a unique uppermost point within the volume between the sections. Counting those tops provides an unambiguous and unbiased value for the number in the volume defined by the image area and the section spacing.
This interactive tutorial illustrates the unbiased counting of the number of features per unit volume by comparing two aligned, parallel images (in this case sections 6 µm apart obtained with a confocal microscope). In the tutorial, fluorescence images of oil droplets from two sections are thresholded, and the features separated with a watershed. Placing the resulting images into the red and green channels of a color image shows yellow wherever the two colors overlap. Since features can change size from one section to the other, the presence of yellow within a feature means that it continues through the two sections, and therefore should not be counted. Those features that are entirely green are ones that appear in the lower section but not the upper, and are isolated and counted to obtain the result.
The tutorial initializes with one of two section images appearing in the Specimen Image window. The Choose A Specimen pull-down menu selects either the upper or lower section image. The Original Image button shows the original grayscale image in the Specimen Image window, while the Binary Image button shows the result of thresholding and watershed separation. In the right-hand window, the Color Overlay button shows the two binary images from the two section planes superimposed in different color channels so that the presence of yellow within a feature indicates a match. The Unique Features button shows only those 47 features that appear in the lower section but not in the upper, and consequently have a top in the sampled volume.
Contributing Authors
John C. Russ - Materials Science and Engineering Dept., North Carolina State University, Raleigh, North Carolina, 27695.
Matthew Parry-Hill, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.
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