Associate Prof. Dr. Klazina Kooiman and Dr. Ines Beekers
Associate Prof. Dr. Klazina Kooiman, Head of Therapeutic Ultrasound Contrast Agent Group, and Dr. Ines Beekers, Postdoctoral Researcher in the Department of Biomedical Engineering of the Thoraxcenter, Erasmus MC, Rotterdam, the Netherlands
Please tell me more about your research
The focus of our group is to develop a local drug delivery system that can target specific cells or enable transport through the vessel wall to specific tissues using ultrasound contrast agents. These agents 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, the microbubble changes in size and it is this oscillation that can stimulate cells to take up the drug.
Much of our work examines the mechanisms of how these oscillating microbubbles affect cellular behaviour. This has been made possible by our modified system of an ultra-high-speed Brandaris 128 camera combined with Nikon’s high-sensitivity and high-resolution live-cell confocal microscope arrangement .
We now use this system to further expand our research into drug delivery through the cardiovascular system and to study the use of these microbubbles to treat bacterial infections (sonobactericide).
How did you decide to collaborate with Nikon Instruments? What were the deciding factors to choose a Nikon system?
After attending a talk Klazina gave at a workshop at Erasmus MC, a Nikon Netherlands representative approached her regarding imaging of the microbubbles we are working with.
Ideally, optically studying the microbubble oscillation behaviour and the cellular response simultaneously during ultrasound exposure would enable us to understand the mechanism of drug delivery far better. We had an ultra-high-speed camera to image the oscillating microbubble at nanosecond timescales, but the cellular response in the same field-of-view was being imaged at poor resolution, using a conventional wide-field microscope.
The Nikon specialists proposed a novel approach of combining their Eclipse Ni-E microscope and A1R+ confocal, to increase the spatial resolution of live-cell imaging, with our Brandaris 128 camera, which provided the high temporal resolution (25 million frames/ sec) required. This ultra-high-speed camera was engineered and manufactured at Erasmus Medical Centre, Rotterdam, in collaboration with the University of Twente, Enschede, both in The Netherlands.
This had never been done before, but the Nikon team was confident that their microscope system could be customized to our system and would transform our imaging capabilities.
The determination and months of dedicated work from Nikon’s European Headquarters service engineer Rogier Verduyn Lunel was paramount to the success of this project and why we continued to work with Nikon to achieve our goal.
How easy was it to establish the Erasmus-Nikon collaboration?
Rogier spent a lot of time with us to establish our needs and understand the optical properties of the Brandaris 128 camera so that he could design a compatible Nikon system.
At Nikon he modified the Eclipse Ni-E microscope by adjusting it for access for the camera and removing the foot of the microscope. The foot needed to be removed because our setup includes a water bath that needs to fit under the objective to enable the use of ultrasound. There was still constant contact with us throughout this time which made the collaboration easy to manage – we always felt Nikon was very attentive to our needs.
How was the testing of the custom-made system implemented?
As soon as the modified confocal was delivered to our lab, the Nikonteam was here to set it up. The alignment to the Brandaris 128 camera took a few weeks. They explained the system to us and made adjustments when needed.
The service engineer worked full time in our lab testing and optimizing, step by step. For example, after capturing an initial image, we found it was not bright enough, so we brought the team (Erasmus MC and Nikon) together and brainstormed about the options of different components and lenses that could resolve this issue. It required some very practical troubleshooting utilizing our knowledge of the Brandaris 128 camera and Nikon’s skills in confocal microscopy- it was great teamwork and also a lot of fun!
As this project had never been done before, there was constant reviewing of the set-up and continuous troubleshooting over several months to achieve exactly what we needed. We were then able to image both the microbubble and cellular behavior before, during, and after the ultrasound had been applied.
What were the challenges you faced during this process?
Alignment was the biggest technical challenge. It took a lot of perseverance to obtain an image and achieve parfocality on both the confocal microscope and the ultra-high-speed camera. We worked on optimising the light path to create a high-resolution image but without losing too much light, which is required for the ultra-high-speed camera. Also, the switching between the Brandaris 128 camera and confocal microscope needed to be as quick as possible and was reduced to around 300 msec.
In addition, physical alignment is important because there is an open connection between the confocal microscope and the Brandaris 128 camera. This meant having to physically move large pieces of equipment in a submillimeter range.
Due to the novelty of this system, some issues were only identified after having used the system for a while, but Nikon continued to collaborate with us on resolving these in situ.
How did you overcome these challenges together with our team?
Nikon’s team never gave up! Solutions to some of the challenges sometimes took several days to achieve and created lots of frustrations, but the team continued to persevere no matter how impossible it seemed and how long it took. This was especially true when aligning the instruments, which required multiple millimeter changes.
In collaboration with Nikon, we also defined a troubleshooting and maintenance guide to help users on-site when Nikon engineers were no longer around. For example, we came up with an easy trick to help with alignment – a little laser pointer attached to the Brandaris 128 camera that pointed on a dot on the confocal microscope to use for alignment.
Nikon’s service engineer played a huge role in overcoming all the challenges with his positive attitude and determination.
Were there any compromises?
Due to the dedication of the Nikon team to satisfy all our needs, we did not have to make any compromises.
At first, we thought we would have liked to have a full field of view, but this proved difficult to attain without losing too much light and image quality. Often, we would crop the image, so it was not a compromise at all.
What do you value about this Nikon system (before and now)?
The benefit of the modified set-up has already been recognized with the publication of several papers in the last two years. The original technical note  describes the implementation of the modified system and a few initial results. In recognition of the contribution that the Nikon engineers made to our research, we added them as co-authors on this technical note. We have published two papers that investigate the mechanism of vascular drug delivery by microbubbles [2,3] and one that investigates the microbubbles themselves in response to ultrasound . We also have several manuscripts in progress with some interesting findings from these initial projects. None of these would have been possible without this system.
It has also brought global recognition to our group. After the set-up was completed, we organized a symposium which included a live demonstration to people from all over the world.
How has this solution helped your research?
For vascular drug delivery, there are three known drug delivery pathways for oscillating microbubbles: sonoporation, the opening of cell-cell contacts, and endocytosis. In our paper in the Journal of Controlled Release , we investigated sonoporation and the opening of cell-cell contacts. Both these mechanisms were always thought to be independent processes, but using the combined system we showed that the microbubble-mediated opening of cell-cell contacts occurred as a cellular response after sonoporation. In a separate publication, we were able to resolve how microbubble oscillations correlate with the spatiotemporal dynamics of intracellular calcium waves and sonoporation .
For the publication on the microbubbles themselves  we for the first time were able to correlate characteristics of the microbubble coating from confocal microscopy recordings with the acoustic response of the microbubble using the Brandaris 128 camera.
Currently, we are working on several other publications and studies that we would not have been able to do before having the system. We have four publications in progress, and we use the modified imaging system in each of those projects.
“We could never have acquired any of this data without the great one-of-a-kind system!” stated Klazina Kooiman.
In the future, we plan to expand on our vascular drug delivery work and the microbubble use in bacterial treatment.
 Beekers, I. et al, (2019) Combined confocal microscope and Brandaris 128 ultra-high-speed camera. Ultrasound in Med. and Biol., 45(9): 2575-2582
 Beekers, I. et al, (2020) Opening of endothelial cell-cell contacts due to sonoporation. J. Control. Rel., 322: 426-438
 Beekers, I. et al, (2020) High-resolution imaging of intracellular calcium fluctuations caused by oscillating microbubbles. Ultrasound in Med. and Biol., 46(8): 2017-2019
 S.A.G. Langeveld, I. Beekers, G. Collado-Lara, A.F.W. van der Steen, N. de Jong, K. Kooiman, The Impact of Lipid Handling and Phase Distribution on the Acoustic Behavior of Microbubbles, Pharmaceutics. 13 (2021). doi:10.3390/pharmaceutics13010119.