Introduction
Foramen ovale is a physiological channel of the atrial septum during the embryonic period. After birth, the primary and secondary septum get close to each other, adhere to each other, and gradually fuse to form a permanent atrial septum(1) . Suppose the fusion is not complete after three years of age. In that case, the remaining fissure-like channel is called patent foramen ovale (PFO), the most common congenital heart abnormality in adults (2 - 4) . PFO patients may suffer from low blood oxygen levels and cryptogenic strokes (CS), leading to serious health issues. (5) . Neurologists and cardiologists have become increasingly aware of the symptoms in recent years, and many hospitals have launched different examination programs.
For detecting PFO, three ultrasound techniques have been developed: transesophageal echocardiography (TEE) (6, 7) , ”gold standard” contrast-transthoracic echocardiography (c-TTE) (8, 9) , and contrast-transcranial doppler (c-TCD) (6) . By contrast, c-TCD offers a sensitive, easy-to-perform, non-invasive method for assessing the presence of RLS in the patient’s heart (10, 11) . The c-TCD method produces less discomfort for the patients during the test, and it is a useful initial screening tool in determining whether a patient has RLS. Microbubbles are used as contrast agents in the c-TCD test, which are prepared by mixing the isotonic saline solution with air, which is injected into a cubital vein for the test. In the presence of a right-to-left shunt, the air-containing echo contrast agent will bypass pulmonary circulation and induces microembolic signals in the basal cerebral arteries (12) . Nowadays, saline contrast use has been applied to enhance intracardiac navigation and is recognized as an effective kind of contrast agent when applying c-TCD for diagnosing PFO(13 – 15) . Importantly, many problems in this detection process still lead to missed diagnoses, which still needs further in-depth research. For example: changing the contrast agent(16) , changing the intravenous catheters (17) , improving the Valsalva action (18) , and so on, but there are few studies on the generation of microbubbles.
The behavior of microbubbles in venous blood is of great importance for testing accuracy. The microbubbles’ size has been shown to affect their longevity and acoustic behavior significantly (19) . After infusion of contrast agent, microbubbles can circulate throughout the body, and the size of the bubbles determines their lifespan and performance after entering the blood circulation. Large bubbles cannot pass through capillaries, while small air bubbles have a poor scattering effect on ultrasound (20) . In addition, the size of the bubble diameter is also related to tissue penetration because the Laplace pressure and blood pressure effects will further affect the lifetime of the microbubbles (21) .
Therefore, the size of microbubbles plays a significant role in c-TCD as the contrast media for diagnosing PFO. The recommended manual procedure for generating MBs is mixing 9ml isotonic saline solution and 1ml air with a three-way stopcock by exchange of saline/air mixture between the syringes and injecting the generated MBs into the subject through the cubital vein as a bolus (22) . Relevant studies are less available on the MBs generated by this method, especially on the BSD and repeatability of the BSD.
Herein, we performed a detailed measurement of the BSD to provide reliable and useful data for the future enhancement of c-TCD. For the first time, a microscopic shadow imaging technique was used to observe and record the MBs generated with the manual methods, then MBs were subjected to detailed physical characterization. The overarching aim of the research was to obtain the BSD data and characterize how much variation there is in c-TCD practice.