Anin VivoMethod for Visualizing Flow Dynamics of Cells within Corneal Lymphatics

Abstract

Background: Monitoring the trafficking of specific cell populations within lymphatics could improve our understanding of processes such as transplant rejection and cancer metastasis. Current methods, however, lack appropriate image resolution for single-cell analysis or are incompatible with in vivo and longitudinal monitoring of lymphatics in their native state. We therefore sought to achieve high-resolution live imaging of the dynamic behavior of cells within lymph vessels in the rat cornea. Methods/Results: Inflammatory angiogenesis was induced by suture placement in corneas of Wistar rats. Preand up to 3 weeks post-induction, corneas were noninvasively examined by laser-scanning in vivo corneal confocal microscopy (IVCM) using only endogenous contrast. Lymph vessels and the cells harbored therein were documented by still images, real-time video, and 3D confocal stack reconstruction of live tissue. In vivo, conjunctival and corneal lymphatics were morphologically distinct, those with corneal location being onequarter the diameter of those in the conjunctiva ( p < 0.001). Cells were recruited to initially empty pre-existing lymph vessels during the first day of inflammation and maintained a dense occupation of vessels for up to 7 days. A diverse population of cells (diameter range: 1.5-27.5 lm) with varying morphology was observed, and exhibited variable flow patterns and were transported singly and in clusters of at least 2-9 adherent cells. Conclusions: The in vivo microscopic technique presented enables lymph vessels and cell trafficking to be studied in high resolution in a minimally-perturbed physiologic milieu.

Age-Related Thinning of Bowman's Layer in the Human Cornea In Vivo

“…By adjusting frame acquisition rate to 30 frames/sec and the motorized depth adjustment to the slowest speed, manually scanning through a limited depth of the anterior cornea can yield images spaced 0.5 µm axially. 23 This approach, however, is technically challenging.…”

Label-Free In Vivo Imaging of Corneal Lymphatic Vessels Using Microscopic Optical Coherence Tomography

“…We observed most lymphatic vessels empty; in some cases, however, individual cells, flowing at slow speed, were found, which is in line with former reports. 14 Altogether, the above listed features typically are attributed to lymphatic vessels 14,19,20 and mOCT has the resolution to image them clearly.…”

“…[9][10][11][12][13] Label-free in vivo imaging is made feasible by microscopic optical methods, including confocal microscopy, which offers high lateral resolution and even enables single cell analysis in lymphatic vessels. 14 However, due to limitations on speed, the acquisition of three-dimensional (3D) volumes using confocal microscopy is time-consuming and vulnerable for axial motion artifacts, which are hard to correct. Alternatively, laser speckle imaging can visualize lymphatic vessels, 15 but has no depth resolution.…”

The Management of Corneal Neovascularisation – Update on New Clinical Data and Recommendations of Treatment

“…44 IVCM has also been applied to the examination of human corneal blood vessels and lymphatics, and provides useful realtime information about the presence or absence of blood cell traffic, cell densities and cell characteristics in vivo. 39,45 IVCM provides useful adjunctive information regarding the activity of corneal blood vessels, as well as the opportunity to perform serial in vivo assessments in the same patient, thereby enhancing our understanding of the processes involved in the corneal immune response.…”