Xinping Zhang, BMed, PhD

Who have been your mentors?
When I began my career about 25 years ago, I was fortunate to have two outstanding mentors, Dr. Regis O’Keefe and Dr. Edward Schwarz, who supported my journey to becoming an independent researcher and helped shape my career over the years. Their guidance and inspiration have been invaluable to me throughout my professional life. I also want to acknowledge Dr. Brendan Boyce, who introduced me to bone biology and bone histomorphometry, as well as all my collaborators in imaging, material science and orthopedic research. Each has contributed to my continued growth and success.

What is intravital imaging? Why is it important versus more conventional microscopy techniques to analyze cells and tissues?
Intravital imaging is a technique that allows the observation of biological processes in living organisms in real time. This method is particularly valuable for studying dynamic processes within tissues and organs.

Unlike conventional microscopy techniques, which often require fixed (non-living) samples, and are usually destructive and challenging to visualize the tissue or organ in three dimensions, intravital imaging enables researchers to observe living cells, tissues and their residing microenvironment in 3D, providing insights into dynamic physiological processes as they occur. In addition, intravital imaging using multiphoton or confocal microscopy can achieve high spatial and temporal resolution, allowing researchers to capture detailed images of fast and complex processes, such as immune responses, neuronal activity, or cell migration, and the interactions between different cell types in their natural environment. In comparison, while the conventional microscopy can provide high spatial resolution, it generally lacks the capability to capture rapid changes in real-time. Another advantage of intravital imaging is that it enables longitudinal studies where the same area or process can be observed over time, offering insights into progression or regression of conditions like tumor growth or wound healing. Conventional microscopy usually does not have the resolution or capacity to visualize cellular events over time in a complex tissue form.

What led you to be an early adopter of intravital imaging? What was your first intravital imaging project?
I was very interested in the coupling between osteoblasts and blood vessels during the bone healing process. At the time, all the available technologies were destructive and lacked the ability to visualize vascular ingrowth at high resolution and in 3D. Additionally, none of the methods allowed me to study the physiology of blood vessels during repair. This led me to explore intravital imaging using multiphoton microscopy. Fortunately, the University of Rochester has several outstanding imaging scientists and multiple multiphoton microscopes available. I initiated a collaboration with Dr. Edward Brown, a two-photon microscopy expert and a pioneer in second-harmonic generation (SHG) microscopy. Our first publication utilized intravital imaging to visualize bone healing and vascularization during cranial defect repair. To achieve this, we mounted a glass window on top of the mouse skull, which allowed us to perform a longitudinal study of bone healing and vascular ingrowth over a period of 12 weeks.

Have you ever been surprised by the cell populations revealed in an intravital experiment?
I am consistently surprised and get excited when I see the 3D organization of the healing bone and vessels.

How are you currently applying intravital imaging in your research program?
We are currently using intravital imaging to study bone tissue vascularization during repair, as detailed in our published articles. In addition to visualizing bone and vessel formation, we aim to probe the healing microenvironment using several advanced intravital imaging techniques. Among these, we use two-photon phosphorescence lifetime microscopy (2P-PLIM) for oxygen measurement and two-photon fluorescence lifetime microscopy (2P-FLIM) to study cellular metabolism. We are also developing and adopting additional techniques to further enhance our understanding of the bone tissue healing microenvironment.

What are advantages and challenges associated with using intravital imaging in biological research?
I have talked about the advantages above. Regarding challenges, I feel that one of the primary challenges associated with intravital imaging in biological research is the requirement for specialized microscopes, such as multi-photon or confocal microscopes. Additional difficulties include the complexities of animal handling and preparation, surgical procedures, and motion artifacts from natural physiological processes such as breathing. Phototoxicity and photobleaching also pose significant challenges, particularly in longitudinal studies. While intravital imaging provides high-resolution images of living tissues, achieving sufficient depth is challenging due to light scattering and absorption. Moreover, intravital imaging generates large datasets, requiring substantial computational resources for storage, processing, and analysis. The image analysis itself is complex and requires advanced tools and expertise, including algorithms for motion correction, segmentation, and quantitative analysis.

Is there a specific barrier that once addressed might bring intravital imaging technology to the next level or make it more commonly used by other research groups?
The high costs of purchasing and maintaining the advanced equipment, combined with the need for specialized personnel, limit the accessibility of the intravital imaging technology to well-funded institutions. The large data sets generated by intravital imaging also require advanced computational tools for storage, processing, and quantitative analysis, which can be a significant barrier for many research groups. To make intravital imaging more widely accessible, solutions that reduce costs, enhance imaging depth, and improve efficiency and speed without compromising image resolution are essential. Equally important are improved methods for the storage and management of large image data sets.

What advice would you give investigators who want to incorporate intravital imaging into their research program? What learning resources would you recommend?
My advice is to be patient and persistent, even if you don’t immediately see the results you’re aiming for. Intravital imaging at high resolution can reveal complex systems with significant variability, and it is important to be aware of potential artifacts. Mastering how to present imaging data and interpret statistics is crucial. It’s essential to understand the limitations of the imaging system and to avoid forcing interpretations onto the data. Remember, it’s a lengthy learning process, so approach it with careful consideration and an open mind.

Books, courses, as well as workshops can be very helpful and are available online and in various meetings and conferences. However, I think hands-on training and experience are crucial. It allows you to familiarize yourself with the intricacies of the equipment, troubleshoot issues in real-time, and develop practical skills that are difficult to gain from theoretical resources alone. Planning experiments thoroughly and gaining independent experience with imaging systems will help you navigate the challenges and make the most out of the technology.