Fast airyscan

Immunofluorescent (IF) staining for brain tissue was performed as previously described [44 (link)]. Embryonic brains were collected at indicated time points after in utero electroporation (IUE). Brains were immersed in 4% paraformaldehyde for 1 day and incubated in 20% sucrose for 2 days at 4 °C. Sectioned brain at a thickness of 14 μm on a Microm cryostat microtome (Thermo Fisher Scientific, MA, USA) was permeabilized with 0.25% triton X-100 for 10 min, then incubated in 10% normal horse serum for 1 h for blocking. The blocked sections were incubated with mouse anti-GFP (diluted 1:400) overnight. Sections were thoroughly rinsed in PBS and incubated with Alex-488-conjugated anti-mouse IgG (1:200). All photomicrographs were taken using Zeiss LSM880 AxioObserver Z1 with AiryScan FAST (Oberkochen, Germany) or Zeiss Axoimager M2 (Oberkochen, Germany). Leading process of GFP-positive neuron on cortical plate was used for measurement of axonal length as described [6 (link)].
Axonal length from neuronal cell culture was measured at 3 days after plating and the GFP positive longest neurite of primary neurons was defined as the major axon as previously described [6 (link)]. The measurement of axonal length was carried out using Zeiss ZEN imaging software (Oberkochen, Germany).

The effect of interactions of hollow or various curcumin-loaded nano complexes on the structural integrity of zwitterionic GUV was visualized with confocal and widefield fluorescence microscopes. Each solution of calcein-loaded GUV (500 µL) was mixed with a solution of the nanoparticles (150 µL, 0.28 mg/mL) or 10% Triton X-100 (150 µL) prepared in a leakage buffer, vortexed for 1 min, and allowed to stand for 15 min before mounting on glass microscope slides. To image calcein-loaded and empty GUV in the solution after leakage, the morphology was visualized separately in the rhodamine B (λ_ex 540, λ_em 625) and calcein (λ_ex 494, λ_em 517) channels, with the Axio Imager 2 fluorescence microscope equipped with an Axiocam 506 monochromatic camera (Carl Zeiss, Oberkochen, Germany) and Zeiss LSM880 AxioObserver Z1 confocal microscope equipped with AiryScan FAST (Carl Zeiss, Oberkochen, Germany). Zen 2.3 pro and 3.4 software (Carl Zeiss, Oberkochen, Germany) were used for image processing and optimization.

In vitro time-lapse super-resolution imaging was performed as described previously^{25 (link)}. Cells were cultured in a stage top chamber at 37 °C in a 5% CO₂ incubation system (Tokai Hit STXG-WSBX-SET). Image stacks of fluorescence-labeled migrating neurons and elongating axons were acquired with an LSM880 confocal laser-scanning microscope equipped with Airyscan FAST (Carl Zeiss, Germany) in super-resolution mode with a 40× water-immersion (NA 1.2; optical zoom, 1.8×) or 63× oil-immersion (NA 1.4; optical zoom, 3.0×) objective lens, with an interval of 30 s (40× objective lens) or 3.0‒6.0 s (63× objective lens), respectively. Image acquisition resolutions were: pixel dwell, 0.74 µs/pixel; scaling X/Y, 0.050/0.050 µm/pixel; 1.0-µm z-step size for the 40× water-immersion objective lens; and pixel dwell, 0.99 µs/pixel; scaling X/Y, 0.040/0.040 µm/pixel; 0.189-µm z-step size for the 63× oil-immersion objective lens. To avoid focus drift, the Definite Focus 2 function was used in every imaging frame.

For 3D reconstruction of microglia, 50 μm vibratome brain sections were stained overnight with anti-IBA1 Ab at 4 °C, followed by secondary Abs for 2 h at RT. Z-stack images were taken with a Zeiss LSM 880 with Fast Airyscan (Zeiss, Germany), using a Plan-Apochromat 40 × 1.3 oil DIC UV-IR M27 objective. The 3D reconstructions and measurements were done by filament tracing algorithm from Imaris software (Bitplane).

Confocal z-stack of three to four quadrants in a total of three LC sections per rat were acquired with a 63× oil immersion objective (n = 4–5 rats per group) on a Zeiss LSM 880 Confocal microscope with FAST Airyscan (Carl Zeiss Microscopy GmbH, Germany). CC3⁺ cells were quantified manually in the LC region using the ImageJ Cell Counter plugin (National Institutes of Health, Bethesda, Maryland). The results were expressed as the mean number of CC3⁺ cells per animal in the whole LC region and along the dorso-ventral LC axis. The relative GFAP⁺ area was quantified using a threshold (14 on a 0–255 greyscale) for the signal intensity that maximized the selection of expressing cells while minimizing the background noise. A mean of three to four quadrants per section was represented and expressed as the percentage area occupied by GFAP.

Mounting of fixed Drosophila embryos was done in ProLong Gold + DAPI mounting media (Molecular Probes, Eugene, OR). Fixed embryos were imaged on a Zeiss LSM 880 confocal microscope with FastAiryscan (Carl Zeiss Microscopy, Jena, Germany). Excitation lasers with wavelengths of 405, 488, 561 and 633 nm were used as appropriate for the specific fluorescent dyes. For imaging in embryos carrying Df(X)svb¹⁰⁸, only embryos without svb mRNA expression in the T1 segment were imaged, following the same reason described in the section on ‘Cuticle preparations and trichome counting’. Unless otherwise stated, all images were processed with Fiji/ImageJ (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)) and Matlab (MathWorks, Natick, MA, USA).