Na water immersion objective lens

Imaging was performed with a 2-photon (2P) laser-scanning microscope (TriMScope, LaVision, BioTec, Bielefeld, Germany) using a 20× (1.0 NA) water immersion objective lens (Zeiss, Oberkochen, Germany) and GaAsP photomultipliers for non-descanned detector (Hamamatsu Photonics K.K., Hamamatsu, Japan). Two photon excitation was achieved with a Ti:Sapphire Laser (MaiTai BB, SpectraPhysics, Santa Clara CA, USA). Before and after calcium imaging, reference images were taken for COFLOUR (tdTomato, EGFP, and OGB) and hGFAP-EGFP (EGFP and OGB) mice. For excitation three wavelengths were used: 720, 800, or 900 nm and emitted fluorescence was simultaneously detected through three kinds of band-pass emission filters: 641/75, 531/40, or 475/50 nm. Spectra overlapping of fluorescence was decomposed off-line (see section “2.7. Image processing”). For calcium imaging, OGB fluorescence was detected through a 531/40 nm band-pass emission filter with excitation at 800 nm wavelength or 915 nm for GCaMP6s (Aldh1l1-GCaMP6s mice). Calcium imaging recordings were performed at 6–8 Hz. Optical filters were obtained from AHF Analysentechnik AG (Tübingen, Germany). All settings were controlled by Imspector Software (RRID:SCR_015249).

Multiphoton imaging was carried out using a Zeiss LSM7 MP system equipped with a 20×/1.0 NA water-immersion objective lens (Zeiss UK, Cambridge, UK) and a tunable Titanium: sapphire solid-state two-photon excitation source (Chameleon Ultra II; Coherent Laser Group, Glasgow, UK) and optical parametric oscillator (OPO; Coherent Laser Group). Excised LNs were continuously bathed in warmed (37°C), oxygenated CO₂ independent medium. A laser output of 820 nm and OPO signal at 1060 nm provided excitation of YFP CD11c⁺ and DsRed OT-II cells. Videos were acquired for 20 to 30 min with an X-Y pixel resolution of 512 × 512 in 2 μm Z increments. 3D tracking was performed using Volocity 6.1.1 (Perkin Elmer, Cambridge, UK). Values representing the mean velocity, displacement, and meandering index were calculated for each object. The intersection of DsRed and YFP objects was used to determine interaction between T cells and DCs respectively.

In vivo images were acquired of awake mice with a custom-built, two-photon laser-scanning microscope controlled by ScanImage (Vidrio, Ashburn, VA) written in MATLAB (Pologruto et al., 2003 (link)). Mice were habituated under the microscope for one hour per day starting at one week before the beginning of imaging and subsequently imaged over a period of 10 days. Apical or basal dendrites of L2/3 or L5 pyramidal neurons of mouse visual cortex were imaged using a 20×/1.0 NA water-immersion objective lens (Zeiss). SEP-GluA1 and dsRed2 were excited at 910 nm with a Ti:sapphire laser (Coherent) with 10 ~ 150 mW of power delivered to the back-aperture of the objective. Green and red fluorescence signals were acquired simultaneously and separated by a set of dichroic mirrors (MOM system, Sutter Instrument) and filters (ET525/50 m for green channel, ET605/70 m for red channel, Chroma). Image stacks were acquired at 1,024 × 1024 pixels with a voxel size of 0.12 μm in x and y and a z-step of 1 μm. Representative images shown in figures were masked based on dendritic dsRed2 signal, median filtered, and contrast enhanced.

in vivo 2p images were acquired from lightly anesthetized mice (1.5% isoflurane) using a custom-built, 2p laser-scanning microscope controlled by ScanImage (Vidrio) and a ×20/1.0 NA water-immersion objective lens (Zeiss). SEP–GluA2 (green) and tdTomato cell fill (red) were both excited at 910 nm with a Ti:sapphire laser (SpectraPhysics, 20 mW power at objective back aperture). Green and red fluorescence signals were acquired simultaneously and separated by a set of dichroic mirrors and filters (ET525/50 m for green channel, ET605/70 m for red channel, Chroma). Image stacks were acquired by resonance scanning at 30-Hz, such that 60 images were captured over 2 s for each xy plane. These images were then rigidly aligned (using Stack GPS, https://github.com/ingiehong/StackGPS) to compensate for small movements due to breathing and averaged for each plane. The field of view contained 1,024 × 1,024 × 70 voxels with a lateral xy resolution of 0.096 μm per px and an axial resolution of 1 μm per px. Live, 300-μm thick acute slices of SEP–GluA2 brains were imaged using the same optical setup, except that the tissue was held in place with a platinum/nylon harp, as described for 1p imaging. Slices were maintained in HEPES-buffered artificial cerebrospinal fluid (ACSF), consisting of 140 mM NaCl, 5 mM KCl, 10 mM glucose, 10 mM HEPES, 2 mM CaCl₂ and 1 mM MgCl₂, with pH adjusted to 7.40.

Eight-week-old female C57BL/6 mice were euthanized by cervical dislocation, and sections of colon were ligated and then immediately removed and maintained in warmed Hanks balanced salt solution (HBSS) (Gibco). Approximately 5 × 10⁷ SL1344, ΔSipA, or ΔSipA/pSipA-phiLOV cells were injected intraluminally along with Image-iT Live Red caspase-3/-7 substrate (Molecular Probes). Multiphoton imaging through the intestinal wall was carried out using a Zeiss LSM7 MP system equipped with a 20×/1.0 NA water immersion objective lens (Zeiss UK, Cambridge, UK) and a tunable titanium-sapphire solid-state two-photon excitation source (Chameleon Ultra II; Coherent Laser Group, Glasgow, UK) and optical parametric oscillator (OPO) (Coherent Laser Group). Images were reconstructed postmicroscopy into 3D images using ZEN software (Zeiss).