Poster R18 - Intravital Two-Photon Endomicroscopy of the Native Thymus

Introduction: The thymus is a primary lymphoid organ located above the heart and as the site of T cell maturation, plays a critical role in the functionality of the adaptive immune system. The recovery of the thymus after injury and the role that the thymus vasculature system plays in the thymus regeneration process is of significant medical interest. Previously, our lab described the first intravital thymus imaging method which was used to characterize changes in the thymus vasculature after sublethal total body irradiation. However, our technique couldn’t stably optically-section the thymus or be used to image the same thymus multiple times, preventing the study of thymocyte motility. As an alternative, we developed a vacuum-stabilized endomicroscope which will allow for longitudinal study of the thymus.

Materials and

Methods: mTmG (Dmp1-ROSAmT/mG) mice were anesthetized with isoflurane and intubated. Evans Blue was injected retro-orbitally to label the vasculature. A thoracotomy through the second intercostal space was performed to expose the thymus. The vacuum-stabilized endomicroscope was then inserted into the chest and attached to the thymus. The effect of vacuum pressure on the thymus image stability and vasculature was calculated by the displacement of blood vessels over time and the change in blood flow velocity at different pressures, respectively.

Results, Conclusions, and Discussions: Here we describe a novel endomicroscope system capable of two-photon video rate intravital microscopy of the native thymus in mice (Fig. 1a). This microendoscope contains two optical components, an air objective (Fig. 1a1) and GRIN lens (Fig. 1a2), along with additional components to enable z-sectioning. The FOV diameter, magnification, lateral and axial resolution, chromatic aberration, and focal shift of the endoscope was determined. The microendoscope system was used to visualize the thymus in mTmG mice in vivo (Fig. 1b) and thymus flow velocity at various suction pressures (Fig. 1c) was calculated to determine the effect of suction pressure on the vasculature system. The stabilization of the vacuum system on the thymus was found to sufficiently minimize tissue movement (±5 μm displacement) to enable optical sectioning of the thymus (Fig. 1d). These preliminary results indicate that the vacuum-stabilized endoscope will serve as a viable replacement for the adhesion stabilization method for intravital imaging of the native thymus.
Our description and characterization of a vacuum-stabilized endomicroscope capable of visualizing the thymus in mice will allow for novel discoveries related to changes to the thymus microenvironment. In particular, we anticipate the unprecedented ability to characterize thymocyte motility in vivo. Further development of the vacuum-stabilized endoscope imaging method will improve our ability to study the thymus vasculature and thymocyte motility longitudinal.

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