04 Feb 2020
University of Science and Technology of China project boosts focusing speed to see cardiovascular system.
Beyond imaging the extent of the animal's cardiovascular system for those applications, an ability to image the blood vessels of interest in 4D and capture the sub-second dynamics of the zebrafish metabolism will be a valuable additional tool in future.
Adding a temporal function to existing 3D imaging techniques has proven challenging, however, with techniques such as light-sheet fluorescence microscopy and confocal laser scanning microscopy usually having acquisition speeds that are too slow for the task.
A project at the University of Science and Technology of China in Hefei has now demonstrated the successful use of 4D-visualization, by employing structured illumination for the optical sectioning of the sample and an electrically tunable lens (ETL) for refocusing. The work was published in Biomedical Optics Express.
The team's ETL was a commercial unit from Optotune of Switzerland, in which the focal lens is controlled through the action of an actuator upon a container of optical fluid. The stabilization time of the liquid is 4 to 6 milliseconds, according to the team, said to represent a potential tripling of acquisition speed over a conventional piezoelectric mechanical component. A customized algorithm then analyzed the results.
To test the platform, the project used it to image the cardiovascular system of a live zebrafish, and found that it was able to image the beating heart of the animal using an exposure time of 5 milliseconds, rapid enough to catch the heartbeat pattern and overall mobility of blood vessels. Further trials were carried out on cerebral blood vessels, and on a region around the intestines.
"With structured illumination for optical sectioning and an ETL for axial refocusing, we realized fast 3D dynamic imaging with 25 layers per second," noted the project team. "However, the acquisition speed is still limited by the exposure and readout time of the camera. Using a higher intensity light source could effectively reduce the exposure time, and hence enable faster dynamic imaging."
Although 3D optical sectioning imaging with ETL significantly improves the speed of focusing and so provides opportunities to image rapidly moving samples, an ETL can be inherently prone to generating aberrations in the imaging system when the focus is changed. Although this was not found to be a problem in its reported study, the project team intends to investigate the use of suitable countermeasures, perhaps such as adaptive optics, to improve performance further.
Future work could also include expanding the work into a multicolor system with multi-camera combinations, perhaps allowing imaging of the 3D dynamic process of blood flow through both the blood vessels and heart, providing they are labelled with different types of fluorescent protein.
"This new 4D technique will facilitate in vivo imaging of organ function, generation, as well as drug responses in small-sized animals," commented the project. "The system also has the potential to image other thick fluorescent-labelled samples, such as brain tissue sections and organoids."
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