Comprehensive assessment of quantum dots for multispectral twophoton imaging of dynamic leukocyte migration in lymph nodes

In recent years, intravital twophoton microscopy (2PM) has emerged as the appropriate technique for direct in situ imaging of immune cell dynamics inside peripheral lymph nodes (PLNs) of live, anesthetized mice, yielding important insights into the regulation of immune responses. However, most current 2PM approaches are limited by the scarce availability of near-infrared (NIR) probes for multispectral time-lapse imaging, and by the use of a single excitation wavelength for multiple fluorophores. The recent availability of quantum dots (QDs) nanoparticles displaying unique optical properties have the potential to overcome this limitation but their suitability has not been yet comprehensively tested for 2PM imaging in vivo. In this study, we explored the use and delivery of NIR-emitting QDs into dendritic cells. Furthermore, we functionalized the surface of these nanoparticles with antibodies that recognize specific antigens expressed on the endothelium of the PLN microvasculature or their use as NIR plasma markers and examined the homeostatic recirculation of lymphocytes. This approach allowed to simultaneously visualize up to six different cell populations and lymphoid structures and identified varying lymphocyte migration patterns in defined microenvironments. Yet, QDs were more difficult to reproducibly couple to antibodies and showed a tendency to cause clustering of targeted antigens. Our data provide an in-depth analysis of the usefulness and shortcomings of QDs as imaging tools for anatomical landmarking in 2PM studies.