Hc pl apo 20 0.75 imm corr cs2

Confocal imaging of single neuron morphologies was carried out either with a Zeiss LSM 510 equipped with a 25 × long distance objective (LD LCI Plan- Apochromat 25x/0.8 Imm Corr DIC, Zeiss) or with a Leica SP8 equipped with a 20 × long distance objective (HC PL APO 20 × /0.75 Imm Corr CS2). In the Leica microscope, the 552 nm laser line was used for excitation, while in the Zeiss LSM we used the 561 nm line. For the Leica microscope, the hybrid detector (HyD) was used to maximize photon catch, either in counting mode or in standard mode with smart gain set to the lowest possible value (10%). With line accumulation set to 2 or 3, laser intensity was set to the minimum value that yielded sufficiently bright images. Neurons were imaged at a voxel size of 0.3 × 0.3 × 0.88 µm or slightly larger. If several image stacks were required to cover the full extent of any one cell, these were aligned to a common reference frame (using Amira or FIJI) and used as input to the skeletonize plugin of Amira (Schmitt et al. 2004 (link); Evers et al. 2005 (link)). Neurons were traced manually and the resulting skeletons were finalized by automatic midline fitting and diameter adjustment (using local brightness information of the image data).
When describing brain anatomy, the terms right and left are always used from the perspective of the bee.

Brains were dissected and fixed in 4% paraformaldehyde, 0.25% glutaraldehyde, and 2% saturated picric acid diluted in 0.1 mol l⁻¹ phosphate buffered saline (PBS) at 4 °C overnight. Following rinses in PBS (4 × 15 min) brains were incubated in Cy3-conjugated streptavidin (1:1000) in PBS containing 0.3% Triton X-100 (PBT) at 4 °C for 3 days. Following rinses in PBT (2 × 20 min) and PBS (3 × 20 min) brains were dehydrated in an ascending ethanol series (30%, 50%, 70%, 90%, 95%, 100%, 15 min each) and cleared in a 1:1 mixture of 100% ethanol and methyl salicylate (Merck, Darmstadt, Germany) for 15 min and in pure methyl salicylate for 1 h. Finally, they were mounted in Permount (Fisher Scientific, Pittsburgh, PA) between two coverslips. Samples were scanned with a confocal laser scanning microscope (Leica, TCS SP5, Leica Microsystems, Wetzlar, Germany) with a 20 × immersion objective (HC PL APO 20 ×/0.75 Imm Corr CS2, Leica). A diode pumped solid state laser (561 nm) was used to excite Cy3. Scanning frequency was 400 Hz and the voxel size was 0.54 × 0.54 × 2.0 µm³.

The samples were mounted on glass slides using the same product we used as a clearing solution. Small samples were supported by a silicon sheet with holes (8 mm × 8 mm square). The anther was mounted with water. The samples were visualized with a confocal laser-scanning microscope (TCS SP8; Leica Microsystems, Tokyo, Japan) equipped with a 405 nm and pulsed white-light laser (WLL) sources and a 63× glycerol-immersion objective lens (HC PL APO 63×/1.30 GLYC CORR CS2; Leica Microsystems), a 40× water-immersion objective lens (PL APO CS2 40×/1.10 W CORR HCX; Leica Microsystems), and a 20× objective lens (PL APO CS2 20×/0.75 IMM CORR HC; Leica Microsystems). For SR2200 fluorescence, images were captured at 410–480 nm after excitation at 405 nm with a solid-state laser. For Venus fluorescence, images were captured at 520–600 nm after excitation at 515 nm with WLL. We set the Z step at 500 nm for SAMs and 1–3 µm for root tips. After image acquisition, which took approximately 2 h for 150 μm in depth, the images were processed using LASX software (Leica Microsystems, Tokyo, Japan).