Figure 4: The effect of varying threshold on the binarization of a protein-aggregate image. Red pixels are particles, based
on the threshold. The green boxes show enclosed particles found after thresholding. Image 1 is the original protein aggregate
image. Image 2 shows the result of a threshold of 25 darker than background. Image 3 shows the result of a threshold of 15
darker. Image 4 shows the result of a threshold of 15 lighter. Image 5 shows the result of thresholding 15 lighter and darker.
Image 6 is the same as Image 5, but with the addition of neighborhood analysis.
To avoid this problem, the imaging particle analysis system should allow for thresholding based on pixels that are either
darker or lighter than the background. While this solution is certainly helpful, it still may allow the thresholding process
to form some image artifacts. Fortunately, common image-processing algorithms are available to overcome this problem by using
neighborhood analysis to group disparate clusters of thresholded pixels into logical whole images. Figure 4 shows the differences
obtained for a thresholded image of a single protein aggregate image using a dark-only pixel threshold, a dark-and-light pixel
threshold, and finally, a dark-and-light pixel threshold plus neighborhood analysis.
Figure 5: Thresholded images of National Institute of Standards and Technology traceable 10-µm spheres. Sphere images in sharp
focus are on the left, and less sharp images are at right. The table shows variance in measured diameter based on different
The third factor to consider when using dynamic imaging particle analysis is the effect of image quality. Although the overall
topic of image quality is quite broad and well beyond the scope of this article, a basic tenet of the subject is that image
sharpness is the most critical measurement of image quality (6). Indeed, in imaging particle analysis, the sharpness of the
particle images is directly proportional to the accuracy of the measurements obtained.
Table II: Equivalent spherical diameters (ESD).
Figure 5 demonstrates this by showing images and measurements obtained in an imaging particle analysis system for National
Institute of Standards and Technology traceable size bead standards. The images at left are beads in sharp focus, and the
images at right are beads in less sharp focus. The variation in size and shape caused when the bead images are less sharp
is easy to see, and Table II shows the variation in size measurements that would be obtained by the system with different
thresholds. For 10-µm calibrated spheres, the variation in measurement for the blurry images is more than 12 µm for a difference
of 100 in threshold value, whereas the variation in measurement for the sharp images is only 1.67 µm over the same threshold
range. It is clear that image sharpness greatly affects the accuracy and precision of particle measurements.