Size determination of microbubbles in optical microscopy: a best-case scenario

 Opt. Express 25, 33588-33601 (2017)

Michiel Postema1,2,3 Habtamu Abraham4, Ondrej Krejcar5, and Dawit Assefa5,6,*


1 LE STUDIUM Loire Valley Institute for Advanced Studies, 1 Rue Dupanloup 45000, Orléans, France

2 INSERM Research Unit U930: Imaging and Brain, UFR Médecine, 10 Boulevard Tonnellé 37032, Tours, France

3 School of Electrical and Information Engineering, Chamber of Mines Building, University of the Witwatersrand, 1 Jan Smuts Avenue, Braamfontein, Johannesburg 2050, South Africa

4 School of Electrical and Computer Engineering, Biomedical Engineering Department, Hawassa Institute of Technology, Hawassa University, Hawassa, Ethiopia

5 Center for Basic and Applied Research, Faculty of Informatics and Management University of Hradec Kralove, Hradec Kralove, Czech Republic

6 Center of Biomedical Engineering, Division of Biomedical Computing, Addis Ababa Institute of Technology, Addis Ababa University, Addis Ababa, Ethiopia

*Corresponding author:


Microbubble-based ultrasound contrast agents are used in clinical settings to enhance backscattered ultrasound signals from blood during perfusion and blood flow measurements. The dynamics of microbubbles contained in ultrasound contrast agents are typically studied with a high-speed camera attached to a microscope. Such microbubbles, with resting diameters between 1 µm and 7 µm, are considered in optical focus if the bubble centers are in the focal plane of the objective lens. Nonetheless, when a three-dimensional object, a stack of infinitely thin two-dimensional layers, is imaged through a microscope, the image formed onto the charge coupled device element consists of contributions from all layers. If a bubble is larger than the depth of focus, the part of the bubble above the focal plane influences the image formation and therefore the bubble size measured. If a bubble is of a size in the order of the wavelengths of the light used, the system resolution and the segmentation method influence the bubble size measured. In this study, the projections of three dimensional microbubbles (hollow spheres) were computed with an ideal, weighted three-dimensional point spread function to find out under which circumstances the optical image formation leads to a significant deviation in measurement of the actual size. The artificial images were subjected to segmentation techniques for objectively comparing original microbubble sizes with measured microbubble sizes. Results showed that a systematic error was observed for objects in focus with radius ≤ 1.65µm. Also it was concluded that even though a three-dimensional object is in focus, there is discrepancy of up to 0.66% in size measurement. In addition, size measurement of an object for the same shift above and below focus could differ by up to 3.6%. Moreover, defocusing above 25% severely deviates size measurements while defocusing up to 90% could result in mean percentage error of up to 67.96.

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OSA Publishing