Researcher Interviews

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Assistant Prof. Klazina Kooiman & Inés Beekers

Therapeutic Ultrasound Contrast Agent Group, Thoraxcenter, Department of Biomedical Engineering, Erasmus MC, Rotterdam

Equipo Nikon

Please tell us about your research.

To successfully treat diseases, administered therapeutic drugs need to overcome barriers in the human body that hinder efficient delivery. Currently, high dosages are required because only a fraction of the drug actually reaches the target site. This leads to high toxicity levels in healthy tissue, causing undesirable side effects. This is the case, for example, for some cardiovascular diseases, tumours, or even in treating bacterial infections. The research that our team is carrying out at the department of Biomedical Engineering of the Thoraxcenter, Erasmus MC, Rotterdam, is to make sure that these drugs reach their site of action, either by getting them into a cell or through the vessel wall to the tissue beneath. We are focusing on vascular drug delivery and treating bacterial biofilm infections, specifically on heart valves. Our principal interest is in the use of ultrasound contrast agents which consist of coated gas microbubbles (1-10 µm) dispersed in an aqueous suspension that are similar in size to red blood cells. When ultrasound is applied, they change in size and it is this oscillation that stimulates cells to take up the drug. However, if the oscillation is too violent this can cause cell death. This latter phenomenon is especially interesting with respect to treatment of bacterial infections.

Microbubble behaviour has been widely studied; however, the underlying physical and biological mechanisms are poorly understood. The difficulty is that the microbubbles oscillate in the order of nanoseconds while cells react in seconds, minutes, hours, or maybe even days after the event. Drug diffusion is also a factor in the process. The challenge is to take into account all these different timescales. Our goal is to link the microbubble behaviour to the cellular response and to the therapeutic drug action via ultra-high-speed imaging combined with high sensitivity and resolution live cell confocal microscopy.

What system are you using for this research?

We have the ultra-high-speed imaging camera Brandaris 128 that was built some 17 years ago within the department in collaboration with the University of Twente. This camera can record movies at 25 million frames per second, meaning that you can go down to nanosecond resolution to see the microbubble oscillate in the ultrasound field – i.e. in a 2 MHz ultrasound field microbubbles oscillate 2 million times per second. Initially we had a very standard widefield microscope coupled to the Brandaris 128, however we could not really observe the cellular response due to very poor imaging resolution.

Other researchers using confocal microscopy live imaging systems could see how the cells responded, but they were unable to visualize the vibration of the microbubble because they did not have an ultra-high-speed camera. We were really amazed by these images and realized the immense potential of coupling such a system to our ultra-high-speed camera – offering a powerful tool for insights into mechanisms, therapeutic delivery, and opening up the possibility of new clinical applications.

How did you achieve the Nikon A1R+ system combined with your ultra-high-speed camera?

Dr. Kooiman: At one point I gave a lecture at a workshop at the Erasmus MC and afterwards I was approached by a Nikon representative. Even though at that time I had no Nikon microscope and no funding to purchase one, we discussed the research. I told him that I had this dream to connect a confocal microscope to our ultra-high-speed camera. He then consulted Nikon specialists on what possibilities were available. That is how this all started.

We had various meetings with Nikon specialists, exchanging ideas on how the microscope could be coupled to the Brandaris 128. The Nikon team suggested getting the upright Eclipse Ni-E microscope with an A1R+ confocal scan head, but serious modifications really needed to be done. We needed to fit a water bath under the objective of the microscope so we could apply ultrasound. That meant that the foot of the microscope had to be removed. The switching was also hugely important – we wanted to switch within just below a second from the confocal microscope to the ultra-high-speed camera and back to the confocal. Now it takes 300 milliseconds for the mirror to switch.

Recently you published a Technical Note in the journal Ultrasound in Medicine and Biology about this system, can you please tell us briefly about this publication?

Inés Beekers: Because both the confocal microscope and Brandaris 128 were custom-built and a lot of technical thought and work went into this project, in this technical note we focused on the specifications and explanation of the technical side of the setup. We felt it would be important to describe how it was customized and what options are implemented in this setup. With three different data sets we also hint on the type of information you can acquire. The advantage is that one can see both the microbubble oscillation and the state of the cells before and after this event.

What is your impression about Nikon?

What we especially appreciate is that if you have an idea, the Nikon team is very willing to think outside the box. They consider how to help you realize your experiment and even how to improve it. Your service engineer is also not easily satisfied, so if something is not quite right, he will go the extra mile to make it perfect.

In the technical note we also wanted to show the success of the collaboration with Nikon. The support from Nikon continued after the purchase and customization – it was a very productive collaboration. That is why Nikon personnel are included as co-authors in this publication.

What are your plans for your future research?

Dr. Kooiman: We are now starting to explore the possibilities. We currently have six projects running on this setup – on vascular drug delivery, bacteria, and better understanding of the oscillation of the microbubbles. The custom-built confocal microscope is also opening up new possibilities for us; we can for example do a live ultrasound-activated microbubble experiment for vessel-on-a-chip research.

Beekers N. et al. “Combined Confocal Microscope and Brandaris 128 Ultra-High-Speed Camera” Ultrasound in Med. & Biol., Vol. 45, No. 9, pp. 25752582, 2019