Trimscope

Deep tissue imaging was performed on a customized up-right two-photon platform (TrimScope, LaVision BioTec). Two-photon probe excitation and tissue second-harmonic generation (SHG) were obtained with a set of two tunable Ti:sapphire lasers (Chamaleon Ultra I, Chamaleon Ultra II, Coherent) and an optical parametric oscillator that emits in the range of 1,010–1,340 nm (Chamaleon Compact OPO, Coherent), with output wavelength in the range of 690–1,080 nm.
Imaging was performed in the PLN as previously described (98 (link)).

Splenic B cells were isolated from bone marrow chimeras (MojoSort Mouse Pan B Cell Isolation Kit; BioLegend). Control and WNK1-deficient B cells were labeled with CellTracker blue (CMAC, 20 µM) and CellTracker orange (CMTMR, 10 µM; both Invitrogen), respectively, for 20 min at 37°C. CD4⁺ T cells were isolated from WT C57BL/6J mice and from GFP⁺ OTII mice (EasySep Mouse CD4⁺ T cell Isolation Kit; Stemcell). WT polyclonal CD4⁺ T cells were labeled with CPD eFluor670 (10 µM; eBioscience) for 20 min at 37°C. Control and WNK1-deficient B cells (5 × 10⁶ each) were transferred i.v. in a 1:1 ratio together with GFP⁺ OT-II TCR transgenic CD4⁺ T cells and polyclonal CD4⁺ T cells (3 × 10⁶ each) into recipient mice that had received 15 µg HEL-OVA, 0.2 µg LPS in Alum (total volume of 20 µl) s.c. in the foot hock 18 h earlier. At 24-h after cell transfer, intravital imaging of reactive popliteal LNs was performed on a TrimScope (LaVision Biotec) equipped with a MaiTai NIR Laser (Spectraphysics) tuned to 780 nm and a Nikon 25× objective with NA 1.0 (Ficht et al., 2019 (link)). For analysis of cell migration and interactions, z-stacks of 11 images from 150 to 250 µm² areas were recorded every 30 s for 30 min at the B cell follicle border. Image sequences were analyzed by semi-automated tracking using Imaris (Bitplane) and by visual inspection for interaction time.

Fluorescein isothiocyanate- (FITC-) labeled dextran (order number 46947; molecular weight 500 kDa; 0.05 to 0.1 mL of a 5% solution in 0.9% NaCl; Sigma, Deisenhofen, Germany) or Texas red-labeled dextran (order number D1830; molecular weight 70 kDa; 1.0 mL of a 5% solution in 0.9% NaCl; Life Technologies, Carlsbad, CA, USA) was injected intravenously as a plasma marker to visualize cochlear microcirculation. Multiphoton microscopy was performed on a TriMScope (LaVision BioTec, Bielefeld, Germany) described elsewhere [35 (link), 36 (link)].
Two water immersion objectives were used for image acquisition, either 20x (numerical aperture 0.95, working distance 2 mm, field number 22 mm, and field of view in current study 0.5 mm × 0.5 mm) or 10x magnification (numerical aperture 0.3, working distance 3.5 mm, field number 26.5 mm, and field of view in current study 1 mm × 1 mm). 0.9% NaCl or ultrasound gel was applied as immersion liquid. Excitation was achieved with 800, 860, or 1180 nm.

For ex vivo imaging, extracted testes of TcrdH2BeGFP reporter mice were immobilised in an imaging chamber, which was flushed with oxygenated (95% O₂/5% CO₂) RPMI-1640 medium (Invitrogen) containing 1% penicillin/streptomycin, 25 mM HEPES and 5 g/litre glucose. The TriM Scope (LaVision BioTec) equipped with an upright Olympus BX51 microscope with a 203/0.95 water-immersion objective and a pulsed Ti sapphire-infrared laser (Mai Tai, SpectraPhysics) turned to 920 nm was used for imaging. The Imaris software 7.7.2 (Bitplane) was used for data analysis.