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An ImageJ plugin for the sizing and counting of microbubbles

Accumulating evidence demonstrates a strong correlation between ultrasound contrast image enhancement and the size distribution of the microbubble ultrasound contrast agent used. Accurate determination of the size and concentration of microbubble ultrasound contrast agents is therefore important, not only for the experimental verification of theories on nonlinear bubble oscillations but also for the routine assessment of their quality control. In the present work, we have assembled an open-source ImageJ plugin designed to identify and determine the diameter, size distribution and concentration of microbubbles using bright field microscopy images of dispersed microbubbles. The plug-in named “BubblesizerJ” uses image segmentation combined with existing algorithms for particle size determination and spheroid distance transform to identify, count and size microbubbles. BubblesizerJ was validated using digital synthetic images of black circles on a white background and bright field microscopy images of nominally monodispersed microspheres of known modal diameter and concentration. Demonstrating the utility of the plugin, was achieved by repeatedly determining the size and concentration of SonoVue®, a commercial ultrasound contrast agent. We have applied the plugin to the analyses of microscopic images with differing resolution and quality and it consistently returned diameters and concentrations not statistically different from known values. We demonstrated that the plugin enables users to rapidly and thoroughly determine the concentration and diameter of microbubbles.