Two-photon microscopy analysis of leukocyte trafficking and motility

Abstract

During the last several years, live tissue imaging, in particular using two-photon laser microscopy, has advanced our understanding of leukocyte trafficking mechanisms. Studies using this technique are revealing distinct molecular requirements for leukocyte migration in different tissue environments. Also emerging from the studies are the ingenious infrastructures for leukocyte trafficking, which are produced by stromal cells. This review summarizes the recent imaging studies that provided novel mechanistic insights into in vivo leukocyte migration essential for immunosurveillance.

Fig. 1

Migration of the B cell-T cell conjugate in the stromal cell network in the lymph node. The time lapse images show dynamics of cognate antigen-specific B cells (green) and helper T cells (red). Relatively sessile structures in cyan are primarily radiation-resistant stromal cells. Rapidly migrating cyan cells are presumed to be radiation-resistant memory T cells based on flow cytometric analysis of cells from the lymph node. Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled, hen egg lysozyme (HEL)-specific B cells from Hy10 (also known as VDJ9/κ5) mice [109] and 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR)-labeled, ovalbumin (OVA)-specific CD4 T cells from OT-II mice were adoptively transferred to a β-actin-CFP mouse [109] that had been lethally irradiated and reconstituted with bone marrow cells from wild type mice. One day after transfer, the recipient mouse was subcutaneously immunized with HEL-OVA conjugated protein emulsified in complete Freund’s adjuvant. An inguinal lymph node was excised 36 h after immunization and imaged by TPLM in the perfusion chamber as previously described [29, 109]. The excitation wavelength was 900 nm. The wavelengths of detected emission were 467–499 nm (for CFP⁺ cells), 520–550 nm (for CFSE-labeled cells), and 565–605 nm (for CMTMR-labeled cells). Arrowheads indicate the same B-T conjugate. Time elapsed from the far left image is shown as minutes/seconds. The images are presented as 60 μm z projections. The scale bars show 30 μm on the nearest x-y plane from the viewpoint. See also supplementary Video 1.