Tsc sp8 system

Image acquisition was performed using an LSM710 system (×40, NA 1.2 water immersion objective or ×63, NA 1.4 oil immersion objective; Zeiss), an LSM5 PASCAL system (×63 NA 1.2 and ×20 NA 0.60; Zeiss), an A1Rsi system (×63 NA 1.3 water immersion objective; Nikon) or a TSC SP8 system (×40, NA 1.10 water immersion objective; Leica). Bright field images were acquired by detecting transmitted light (488 or 552 nm). For live-imaging, Xenopus gastrula embryos were embedded with 1.5% LMP agarose (#16520-050; Invitrogen) gel in 1/10× Steinberg’s solution on 35 mm glass-based dishes (Iwaki) or mounted in a house-made imaging chamber. For confocal imaging of immunostaining, stained embryos were mounted in shallow wells on 2% agarose plate, flattened with a coverslip. Images were cropped and/or processed with Photoshop CS4 (Adobe) or ImageJ (NIH). Fluorescent intensity was measured using Image J (NIH) or Zen (Zeiss).

A laser scanning confocal microscope (Leica TSC SP8 system, Germany) was used with the excitation wavelengths 405 nm, 488 nm and 561 nm for CFP, GFP and mCherry, respectively. The detection wavelengths were in the range of 450–500 nm for CFP, 500–535 nm for GFP and 580–630 nm for mCherry. For ER-Tracker Blue-White DPX staining, observations were made with excitation wavelength 405 nm and detection wavelengths in the range of 420–500 nm. For FM4–64 and propidium iodide staining, observations were made with excitation wavelength 561 nm and detection wavelengths in the range of 580–630 nm. Images were taken at a region ~ 0.5–1 cm from the root tips. Images were captured in a z-stack of 0.5 μm intervals for subcellular localization and a time lapse for mobility of protein observation.
Multiphoton microscope (Zeiss LSM 7MP OPO, Germany) was used to observe ClearSee-prepared tissue with the excitation wavelength 836 nm and signals were collected in detection range of 500–550 nm.

The viability of encapsulated cells was tested with a live-dead assay at designated times. The cell-gel constructs were washed three times with PBS buffer for the purpose of buffer exchange. After removing the PBS buffer for the last time, 100 µL of live/dead solution containing 4 µM EthD-1 (ethidium homodimer-1) and 2 µM calcein AM was added onto each cell-gel construct. After 30 minutes of incubation at 37°C with 5% CO₂, the staining solution was removed. The constructs were observed by using a Nikon Eclipse TE2000-U inverted fluorescence microscope with excitation filters of 450–490 nm (green, calcein AM) and 510–560 nm (red, EthD-1). The confocal analysis was performed on a Leica TSC SP8 system (Germany).