T cells or B cells, transferred from arthritic BALB/c mice to severe combined immunodeficient (SCID) mice, are detectable by in vivo imaging in the popliteal lymph nodes (LNs) but not in the joints of the recipient mice. (a) Two-photon microscopy (TPM) image of the ankle joint of a SCID mouse 2 days after transfer of CellTracker Red (CMTPX)-labeled T cells and unlabeled non-T cells (antigen-presenting cells, or APCs) from arthritic BALB/c donors. No red fluorescent cells are visible in the joint. Second harmonic generation (SHG) signals from collagen fibers (around blood vessels) are detected in the green fluorescence channel in two-color acquisition. (b) TPM image of the joint-draining (popliteal) LN from the same mouse shows numerous red fluorescent donor T cells that homed to the LN. SHG (green) signals are from collagen in the LN capsule and stroma. (c) TPM image of the inflamed ankle of a SCID mouse 12 days after transfer of CellTracker Red-labeled T cells and CellTracker Green (CMFDA)-labeled APCs (>85% B cells) from arthritic donors. Although this joint was heavily inflamed, no red fluorescent T cells (or green fluorescent B cells) were found by in vivo TPM imaging. SHG signals from collagen are detected in the blue channel in this three-color acquisition image. Endogenous (auto) fluorescence from macrophages appears in light green. (d) TPM image of the popliteal LN from the same mouse shows large numbers of red fluorescent T cells and green fluorescent non-T cells (mostly B cells), which occupy the T-cell and B-cell zones of the LN, respectively. The TPM images shown are representative samples of ankle and LN images from six SCID mice at each time point. Scale bars, 100 μm.