OT-I–GFP fluorescence can be differentiated from tissue autofluorescence. (a, b, and d–f) CMTMR (red)-labeled OT-I or OT-I–GFP Rag2^−/− cells (green) were transferred into WT recipients, and LNs were visualized by two-photon microscopy. Data are representative of more than three experiments. (a) Green fluorescence in a single z plane. Arrows indicate the location of OT-I–GFP Rag2^−/− T cells, as determined by CMTMR fluorescence (not depicted). (b) Pixel intensities in the green channel from the image represented in panel a. (c) Flow cytometric analysis of green fluorescence and autofluorescence in WT LN containing OT-I–GFP Rag2^−/− cells. (d) Analysis of green fluorescence contributed by CMTMR bleed-through when imaged using 525/50- versus 510/20-nm filters. Background measurements were taken in regions that did not contain tissue. Fluorescence values in the red and green channels were measured in regions that were drawn around individual OT-I or OT-I–GFP Rag2^−/− cells based on CMTMR. Lines represent linear equations fit to the data. See Materials and methods for the equations. (e) Representative images of maximum intensity z projections showing unprocessed and processed images. (f) Representative maximum intensity z projection at various stages of image processing. Image was acquired with the 510/20-nm filter. See Video 4 for three-dimensional reconstruction of image processing. The region shown represents a tissue volume of 150 (x) × 150 (y) × 80 µm (z). Bars, 25 µm.