Heavy mineral oil

The microfluidic channels were etched on a 500 µm thin silicon wafer using deep reactive ion etching according to a standard protocol^{38 (link)}. Both the droplet generator channels and the acoustic focusing channel were 380 µm by 100 µm (width × height). The width was set to correspond to half wavelength lateral resonance at approximately 2 MHz in the channel. The inlet and outlet holes were manually drilled through the silicon wafer. The microfluidic channels were sealed by anodic bonding of a 700 µm thin Borofloat-33 glass wafer and the silicon-glass wafer was diced into individual chips. A piezoelectric transducer having a fundamental resonance at 2 MHz (1 mm thickness, APC-840, Americanpiezo) was glued on the silicon side of the chip using cyanoacrylate glue (Loctite 420, Henkel). As fluid connectors, 1 cm long pieces of silicone tubing (228-0701, VWR) were glued using silicone adhesive (Elastosil A07, Wacker). Prior to the experiments the channels were flushed with Repel-Silane (2% solution of dimethyldichlorosilane dissolved in octamethylcyclotetrasiloxane, Pharmacia Biotech) to make the channels hydrophobic followed by rinsing the channels with heavy mineral oil (Sigma Aldrich) to remove unbound Repel-Silane.

We used two different injection protocols. The first protocol was based on similar injection studies with cockroaches [34 (link)] and crickets [36 (link)], and requires the removal of parts of the head capsule cuticle. In the second, newly developed protocol, we punctured a small opening in the head cuticle without removing it. In both cases, we injected only into the right optic lobe, whereas the left lobe was left intact. Furthermore, in both cases the bees were anesthetized by chilling them down to a temperature of approximately 1°C. The whole procedure was performed under a dissecting microscope equipped with red light illumination (optic fibre system with red light filters; greater than 640 nm, peak at around 680). Care was taken not to expose the bees to white light.
The injections themselves were performed using an electric nanoliter injector (Nanoject II, Drummond, cat. no. 3-000-205) loaded with a glass electrode (Drummond 3.5″, cat. no. 3-00-203-G/X) pulled with a micropipette puller P-97 (Sutter Instruments Co.). The electrode was first filled with heavy mineral oil (Sigma) that is not compressible, enabling a more accurate volume injection and then loaded with approximately 50 µl of injection solution. The micro-injector was directed with a mechanical micromanipulator (Right 3-000-024-R) to the desired position.