Trimscope 2 pm system

CD11c EYFP mice (18 (link)) or C57BL/6JRj mice received 2.5x10⁵ OT-I Rgs1^+/+ -tdT + 2.5x10⁵ OT-I Rgs1^-/- -GFP (16 (link)) and were orally infected with 2x10⁹Lm-OVA the day after as described (20 (link)). At day 8 and at day 30 p.i., the gut lumen was surgically exposed (kept in saline at 37°C), and 2-photon microscopy (2-PM) was performed with an Olympus BX50WI microscope and a TrimScope 2-PM system controlled by ImSpector software (LaVisionBiotec, Bielefeld, Germany). Before recording, Hoechst dye was injected i.v. to label nuclei. YFP or Hoechst signals were used as a reference channel for real-time offset correction to minimize tissue shift (30 (link)). Sequences of image stacks were transformed into volume-rendered four-dimensional videos using Imaris software (Bitplane, Zurich, Switzerland), which was also used for semi-automated tracking of cell motility in three dimensions. Cell centroid data were used to calculate key parameters of cell motility. Speed was defined as total track length divided by total track duration in µm/min. Instantaneous speed was defined as the speed at each time point. The arrest coefficient was derived from the percentage of time a cell is migrating below a motility threshold speed of 5 µm/min using Matlab script (R2019b, MathWorks, Natick). The meandering index was calculated by dividing displacement divided by track length.

(5-(and−6)-(((4-chloromethyl) benzoyl) amino) tetramethylrhodamine) (CMTMR, CellTracker orange) and Chloromethyl-coumarin (CMAC, CellTracker blue)-labeled WT, Itk^-/- or Tiam1^-/- CD4⁺ T_N (3 x 10⁶) were injected i.v. into sex-matched C57BL/6 mice 24–32 h prior to imaging. The right popliteal LN of recipient mice was surgically prepared as previously described (48 (link)). 2PM imaging was performed using a TrimScope 2PM system (LaVision Biotec) and a 20X objective (NA 0.95; Olympus). 16-slice z stacks with 4-µm spacing of 250 x 250 µm field of views were acquired every 20 s for 20 min. Imaging was performed in the T cell zone as identified by the presence of high endothelial venules (labeled with Alexa Fluor 633–coupled MECA-79; 10 µg/mouse). Volocity software (PerkinElmer) was used to generate volume-rendered 4D movies and for semiautomated tracking of cell motility. Mean single cell track speeds were calculated from the x,y,z coordinates of cell centroids using Matlab (The MathWorks) (49 (link)).