Abstract
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.
Highlights
Observing the dynamics of acoustically active microbubbles has been of major interest in medical imaging and therapy, and in the hydrocarbon industry
A 3D object can be considered as a stack of infinitely thin two-dimensional layers and when 3D objects are imaged through a microscope, the image formed onto the charge coupled device (CCD) element consists of contributions from all layers
On the other hand, when an object is out of focus, it is weighted only by the portion of the light where the object is in focus. 4.3. 2D Image formation An optical image of a radially symmetric flat object in focus, observed through a microscope, is expressed by convolution of the PSF of the optical system and the object
Summary
Observing the dynamics of acoustically active microbubbles has been of major interest in medical imaging and therapy, and in the hydrocarbon industry. The microbubbles in so-called ultrasound contrast agents consist of perfluorocarbon gas bubbles surrounded by an encapsulating lipid or albumin shell. These microbubbles are typically between 1 μm and 7 μm in diameter. A three-dimensional object under a microscope can be considered as a stack of thin twodimensional layers [6]. When three-dimensional objects are observed through a microscope, the projection consists of contributions from all of these layers. A bubble is considered in focus if the middle layer of the sphere is in the focal plane. If a bubble is larger than the depth of focus, the part of the bubble above the focal plane influences the projection and 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
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