W plan apochromat

Embryos were fixed in 4% formaldehyde in Danieau’s solution at 4°C overnight. The next day, embryos were washed with Danieau’s solution and then permeabilized with 0.2% Triton X in Danieau’s solution for 30 min. Subsequently, embryos were incubated for 10 min in DAPI (1 µg/ml) and then washed several times with Danieau’s solution. Embryos were placed in an inverted agarose holder and covered with Danieau’s solution for imaging. An upright Zeiss LSM 780 NLO microscope equipped with a coherent Chameleon Vision II infrared laser was used for two-photon excitation (Carl Zeiss AG, Oberkochen, Germany). DAPI was excited with 780 nm and detected using a non-descanned GaAsP detector (BIG-Module) with BP450/60 or SP485. Samples were imaged with either a Zeiss W Plan-Apochromat 20 × 1.0 or 40 × 1.0 dipping objective. Images were acquired using a four tile scan of multiple z-sections (3–3.5 µm steps). Tiles were stitched with the ZEN software (RRID:SCR_013672, Zeiss). Images were imported into the Imaris software (RRID:SCR_007370, Bitplane, Belfast, Northern Ireland) and the spot tool was used to calculate cell number.

To immobilize giant
vesicles, calcium fluoride slides were pretreated with 10 mg/mL protamine
(Protamine sulfate salt from salmon, Sigma) for 10 min. After 5 washes
with DPBS, 20 μL of vesicle suspension was incubated on slides
for 1 h, before 20 washes with DPBS. Vesicles were imaged using the
alpha 300R+ confocal Raman microscope (Witec GmBH, Germany). A 35
mW, 532 nm laser light source was shone through a 63× 1.0 NA
water immersion objective lens (W Plan-Apochromat, Zeiss, Germany).
Raman scattering was collected through the same lens and directed
via a 100 μm diameter silica fiber to a 600 groove/mm spectrograph
(UHTS 300, WITec, GmbH, Germany) coupled to a back-illuminated charge-coupled
device camera, cooled to −60 °C. Area scans of vesicles
were imaged with 500 × 500 XY nm resolution. Spectral preprocessing
was performed with ProjectFIVE software (Witec GmBH). First, cosmic
rays were removed, and then the dark current background was subtracted,
followed by “shape” background correction. Spectra were
normalized to the maximum intensity of the water peak at 3000–3400
cm^–1. Finally, Raman images were reconstructed from
univariate analysis of the intensity of the NH₂ signal at 2208 cm^–1 and the SO₃ signal at 2220 cm^–1. Due to low intensity of the 708 cm^–1 polymer
peak with this instrumental setup, Raman images of polymer signal
were reconstructed from 2905 cm^–1 polymer peak.

To evaluate the effect of cross-linking (NXL,
DHT, EDAC) on the ability of the dermal CG scaffold layer to support
the iEC, iSC coculture and the forming of a vascular structure in
3D and the ability of the epidermal CCh film layer to support the
proliferation of keratinocytes, the CG scaffolds/CCh films were fluorescently
stained. Cell-seeded scaffolds were fixed overnight at 4 °C in
10% formalin (Sigma-Aldrich, Ireland) after 7 days of culture, and
CCh films were fixed for 30 min in 10% formalin after 3 days of culture.
For cytoskeleton staining, the scaffolds/films were incubated in Phalloidin-Atto
488 (Sigma-Aldrich, Ireland) (1:600 in PBS) (1 h, RT). Nuclei staining
was carried out using DAPI (500 μL per scaffold, 1 μg/mL
in PBS) (20 min, RT). Following staining, the scaffolds/films were
imaged using a Carl Zeiss LSM 710 confocal microscope, with an N-Achroplan
10× (N.A. 0.3) and W Plan-Apochromat 20× (N.A. 1.0) objective.
Image analysis was carried out using ImageJ.

BALB/c mice (n = 3) were injected s.c. between the scapulae with EcN OMVs (100 µg) and/or splenic DCs (1×10⁶ cells), stained with DiO and DiD respectively (Invitrogen), then euthanized after 4 days. After euthanasia, intact draining cervical lymph nodes were resected, placed in PBS, and immediately imaged via two-photon excited fluorescence microscopy on a custom setup. Imaging was conducted on a custom-designed microscope using a train of 830-nm, 90-MHz, 140-fs pulses from a Ti:sapphire laser oscillator (Chameleon; Coherent) for excitation. Laser scanning and data acquisition were controlled by ScanImage software [34] (link). For high-resolution imaging of labeled cells and particles respectively a 20X (numerical aperture: 1.0) water-immersion objective (Zeiss; W Plan-APO Chromat). Second harmonic generation from the extracellular matrix in lymph node was detected using a 417-nm centered wavelength filter. Fluorescence was detected using emission filters with 510-nm and 641-nm center wavelength with 65-nm bandwidth to image. Images shown are 20 µm stacks that were compiled using a maximum projection and a median filter was applied using Image J software (http://imagej.nih.gov/ij/).

A round cranial window (4 mm diameter) was drilled under general anesthesia (fentanyl, 0.05 mg/kg; midazolam, 5 mg/kg; medetomidine, 0.50 mg/kg) as previously described^{13 (link)}. The mice were kept under general anesthesia for the entire imaging period and were directly transferred to the microscope after the completion of the surgery. Body temperature was controlled and kept steady at 37 °C. The craniotomy was enclosed by a custom-made head fixation ring which allowed for the head fixation of the mouse in the two-photon microscopy setup. In vivo imaging was performed using a custom-built, fully motorized, two-photon microscope equipped with a Coherent Chameleon Ultra II laser and a Zeiss W Plan-Apochromat 40×/numerical aperture 1.0 objective. The microscope is motorized and controlled by a Sutter MP285 via ScanImage (version 3.8). Microglia were imaged using a 900 nm excitation wavelength and were detected via non-descanned detectors and an ET525/50m-2P emission filter (Chroma Technology). 40–50 µm-thick z-stacks were collected using ScanImage at 1024 × 1024 pixels at a depth of approximately 100–200 µm below the cortical surface. Laser power was kept constant for each experiment and did not exceed 45 mW.