In confocal laser scanning microscopy the delicate walls of the vessels excite only very weak autofluorescence and give only little contrast that is additionally outshined by surrounding collagen fibrils. In addition, the transparent lymph fluid does not contribute to the detection of these vessels. By combining injected fluorochrome-labeled antibodies into a stromal pocket with the detection of two-photon excited tissue autofluorescence, previously undetectable lymphatic vessels and adjacent tissue specific structures as well as individual cells became visible in vivo. In contrast to previously used fluorescing dextrans, this approach features long term labeling of the lymphatic vessel morphology in vivo, even after post-experimental tissue preparation. It also allows analyzing individual cells within the optical clear lumen of the vessels. In addition, injection of anti-LYVE1 antibody into a stromal pocket is not limited to a particular mouse strain but can be applied to any transgenic mouse model established for immunological research. Our setup enabled studying cellular dynamics within the model of suture induced neovascularization of the cornea repeatedly over several hours. Observations included migration of immune cells along restricted paths, which may be facilitated by preformed tunnels or partly along protrusions of resident dendritic cells. Cellular velocities of stromal immune cells measured in our experiments are comparable to T-cell velocities measured in isolated non-inflamed lymphnodes, T-cell velocities within an infiltrating tumor model or macrophage velocities in a wound healing model of medaka fish. As immunohistochemistry demonstrated only few T-cells in our model that were far outnumbered by macrophages, migrating cells are likely to be macrophages. As autofluorescence emitted by these cells is based on intrinsic fluorophores such as NADH and macrophages and also dendritic cells contain lysosomes that contribute to the autofluorescence signal, we selectively detected the different autofluorescence spectra of NADH and lysosomes in different channels in our setup. By analyzing such autofluorescence spectra and intensity differences, we were able to optical characterize cellular and also non-cellular structures as published previously e.g. for erythrocytes, connective tissue, macrophages and stem cells. The finding of normal cellular velocities in this study affirms the clinical appearance of corneae with only little edema and normal conjunctival blood flow at the time points used as inflammation was shown to increase velocities of immune cells significantly. Besides increasing cellular velocities inflammation also increases cellular migration from the inflamed tissue via lymphoid vessels by upregulating leukocyte adhesion factors such as ICAM-1, VCAM1 and Ruxolitinib molecular weight E-selectin. By this, an increased cellular turnover within the inflamed tissue is facilitated and accompanied by transmigration of immune cells via lymphatic vessel walls. In situ real-time dynamics of transmural migration of dendritic cells into lymphatic vessels have recently been recorded by Pflicke and Sixt for the first time. Within their ex vivo ear sheet model, injected isolated DCs migrated into lymphatics by preformed pores.