Lsm 900 airyscan 2 microscope

Initial gross oviductal images were captured using a Nikon SMZ25 stereoscope. During organoid culture, organoid development images were taken by JuLiTM Stage Real-Time Cell History Recorder (NanoEnTek) every 3 days after media replenishment. As the organoids diffusely distribute within the Matrigel dome, images were taken on multiple focal planes through each dome at these timepoints. Immunofluorescence and H&E images were acquired using Olympus DP80 CCD (charge-coupled device) and cellSens software (Olympus) on the afore mentioned sections. Confocal laser scanning was carried out on a Zeiss LSM 900-Airyscan-2 microscope with a ×63 objective using the software ZEN (blue edition) 3.0 and were reconstructed by sequences (z-stack).

Pellets were imaged using a Zeiss Axiomicroscope equipped with an Axiocam 105 camera as described previously (33 (link)). β-(1-4) Glycans were stained with calcofluor white (Sigma) as described previously (9 (link), 11 (link)). Stack acquisition was done on a Zeiss LSM900 Airyscan 2 microscope. All fluorescent images were imaged with the same setting (laser intensity: 3.5%, pinhole: 47 μm, master gain: 750V, digital offset: −15, and digital gain: 1.0). For quantitatively comparing fluorescence, the measure region with the size of 15 μm by 15 μm squares at hyphal tips was used. Fluorescence was measured using ImageJ software (version 2.0.0/1.53c/Java 1.8.0_172/64-bit) (63 (link)).

Murine colon adenocarcinoma 26 (Colon 26) cells were purchased from Riken Cell Bank (RCB2657, Tsukuba, Japan), seeded in 35 mm glass bottom dish (Matsunami glass, Tokyo, Japan) at a density of 5 × 10³ cells per glass dish, and incubated at 37 °C under a 5% CO₂ atmosphere for 24 h. The medium was removed, 400 μL of fresh RPMI medium without FBS/phenol red containing HP or aHP (porphyrin concentration: 5 µM) was added, and the cells were incubated for 24 h at 37 °C under a 5% CO₂ atmosphere. As polycations consisting of quaternary ammonium salt groups in the aHP interact with negatively charged macromolecules in FBS such as albumin via an electrostatic interaction, forming a precipitate in the cell culture medium, the medium without FBS was used when the drugs were applied to the cell culture. To observe the intracellular localization of photosensitizer, the cells were incubated for 5 min at 37 °C with Lysotracker Green (Invitrogen, 100 nM, from 0.1 mM stock in DMSO). The cells were examined using a laser scanning confocal fluorescence microscope (Zeiss LSM 900-Airyscan-2 microscope) with a 20× objective lens.