Nonlinear optical microscopy for immunoimaging: a custom optimized system of high-speed, large-area, multicolor imaging

Hui Li, Quan Cui, Zhihong Zhang, Ling Fu, Qingming Luo


Background: The nonlinear optical microscopy has become the current state-of-the-art for intravital imaging. Due to its advantages of high resolution, superior tissue penetration, lower photodamage and photobleaching, as well as intrinsic z-sectioning ability, this technology has been widely applied in immunoimaging for a decade. However, in terms of monitoring immune events in native physiological environment, the conventional nonlinear optical microscope system has to be optimized for live animal imaging. Generally speaking, three crucial capabilities are desired, including high-speed, large-area and multicolor imaging. Among numerous high-speed scanning mechanisms used in nonlinear optical imaging, polygon scanning is not only linearly but also dispersion-freely with high stability and tunable rotation speed, which can overcome disadvantages of multifocal scanning, resonant scanner and acousto-optical deflector. However, low frame rate, lacking large-area or multicolor imaging ability make current polygonbased nonlinear optical microscopes unable to meet the requirements of immune event monitoring.
Methods: We built up a polygon-based nonlinear optical microscope system which was custom optimized for immunoimaging with high-speed, large-are and multicolor imaging abilities.
Results: Firstly, we validated the imaging performance of the system by standard methods. Then, to demonstrate the ability to monitor immune events, migration of immunocytes observed by the system based on typical immunological models such as lymph node, footpad and dorsal skinfold chamber are shown. Finally, we take an outlook for the possible advance of related technologies such as sample stabilization and optical clearing for more stable and deeper intravital immunoimaging.
Conclusions: This study will be helpful for optimizing nonlinear optical microscope to obtain more comprehensive and accurate information of immune events.