Plan apochromat 20x 0.8 m27

A confocal laser scanning microscope LSM 880, LSM 800 or LSM 700 (Carl Zeiss AG) was used for confocal imaging. The pinhole aperture was set to one Airy Unit and experiments were performed at room temperature. The images were acquired using a 20x objective (Plan-Apochromat 20x/0.8 M27, Carl Zeiss AG). Images were analyzed and processed with ImageJ (NIH, brightness and contrast adjusted) and Zen imaging software (Version 2.3). Confocal images were analyzed using a custom-written ImageJ macro (available here: 10.5281/zenodo.4738934). Cell-sized compartments were identified, their radius calculated and the intensity within the compartment center defined as mean inner intensity I_In. The peripheral intensity was determined by quantifying the maximum intensity along a line orthogonal to the compartment periphery. This was repeated every 18^∘ and the mean value taken as I_Peri. The resulting data was plotted with Prism 8 (Version 8.4.3) and figures were compiled with Inkscape (Version 1.0rc1).

In vivo dendrite morphology imaging: For assessing md CIII dendrite morphology, we crossed class III specific driver driving membrane tagged GFP (nompC^Gal4 > mCD4::tdGFP) to gene specific UAS-RNAi lines. Aged matched live third instar larvae were mounted on a microscope slide and anesthetized by using few drops of halocarbon and ether solution (1:5 v/v). Three-dimensional z-stacks were imaged using Zeiss LSM780 with Plan-Apochromat 20x/0.8 M27 objective and 488 nm laser. Image dimensions were 1,024 by 1,024 pixels (607.28 μm × 607.28 μm) with 2 μm step size. Quantitative dendrite morphology analysis was performed as previously described in Das et al. (2017) (link).
GABA_B-R2 expression imaging: For visualizing GABA_B-R2 expression in md neurons, we utilized a recently developed Drosophila reagent for CRISPR mediated insertion of red fluorescent protein (RFP) after the first exon in GABA_B-R2 (Bloomington Stock# 84504). We visualized expression of GABA_B-R2::RFP in md CIII neurons using nompC^Gal4 > mCD4::tdGFP. We imaged the third instar larvae similar to dendrite morphology imaging. We captured red fluorescence using 561 nm laser. We identified md CIII neurons using GFP signal, and we identified other md and es neurons based on cell body position. Red and green fluorescence intensity were analyzed using Zeiss Zen blue software.

FaDu cells (8 × 10⁵) treated with drugs (100 μM) or vehicle control for 24 h under normoxia or hypoxia (<0.1% O₂) were trypsinized, replated in 96-well plates (5000 cells/well), and allowed to grow for 3 days. Cells were then stained with LIVE/DEAD™ Cell Imaging Kit (R37601, Thermo Fisher Scientific) and imaged with a Plan-Apochromat 20x/0.8 M27 lens on a Zeiss 710 confocal microscope. Viable cells are identified by their esterase activity that converts non-fluorescent cell-permeant calcein AM to the intensely fluorescent calcein (green, labelled as “live”), while the compromised membranes of dead cells allow binding of BOBO-3 Iodide to the DNA (red). To measure cellular metabolic capacity, 50 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 11465007001, Millipore Sigma) was added to each well, incubated for 3 h, and imaged as mentioned before. Cellular capacity to form formazan crystals was analyzed as a readout of metabolic activity.

Samples were imaged on several Zeiss LSM 710 and 700 confocal scanning microscopes with either a Plan-Apochromat 20x/0.8 M27 or Plan-Apochromat 63x/1.40 oil immersion objective. All images are maximum intensity projections of captured confocal stacks. Imaging was performed using Zeiss ZEN software with a custom Multitime macro. Except as described below, the Multitime macro was allowed to automatically select appropriate laser power and gain for each sample. As a result, each image is independently scaled for intensity and was evaluated based on laser power and gain required in addition to image quality, with limited weight placed on raw intensity.

Live cell confocal imaging of SH-SY5Y cells was performed using an inverted confocal laser scanning microscope (LSM780; Carl Zeiss microimaging, Inc.) equipped with an incubation system and a Plan-Apochromat 20x/0.8M27 dry immersion objective. Live imaging was performed under an incubation system at 37°C and 5% CO₂, and two to five images (×20) per sample were taken using the phase-contrast and green fluorescence mode. For assessment of neurite length in SH-SY5Y cells, phase-contrast live cell imaging was used. The image scale was converted from pixel units into micrometers (µm) using ImageJ software (version 1.41, Rasband WS, ImageJ, National Institutes of Health, Bethesda, MD, USA). The length of neurites was traced and measured from the distal end of the neuron growth cone to the tip of the neurite as previously described (Lin et al., 2016 (link)). A total of 80–100 neurites were analyzed per group. The somatic GFP-tagged α-synuclein punctate signal represented α-synuclein aggregations from individual cells. A total of 100–150 cells were analyzed per group.