The two-photon microscope (Ultima IV, Bruker) is based on an ultra-short pulsed laser (Mai Tai, Deep See HP, Spectra-Physics). The laser was tuned to 780 nm for fura2 excitation. All images were acquired with a water-immersion objective (10 , NA 0.3, Olympus). The fluorescence was collected in epi-configuration, selected by a dichroic mirror, and filtered with a band-pass filter centred at 525 nm and with a 70 nm bandwidth (Chroma Technology Corp). Finally, it was detected by a photomultiplier tube (Hamamatsu Photonics). Laser powers of about 10 mW were used in order to balance signal-to-noise ratio (SNR) against photo-damage effects that reduced the bee life span.
The field of view of 280 × 280 µm2 was resolved by 128 × 128 pixels. The fluorescence intensity was recorded with a depth of 13 bits. The image acquisition at a frame rate of 10.1 Hz was synchronized to the stimulus protocol.
In addition to the functional images, a z-stack of the antennal lobe was acquired with a spatial resolution of 512 × 512 pixels and a z-layer distance of 2 µm to perform the morphological identification of glomeruli.
The field of view of 280 × 280 µm2 was resolved by 128 × 128 pixels. The fluorescence intensity was recorded with a depth of 13 bits. The image acquisition at a frame rate of 10.1 Hz was synchronized to the stimulus protocol.
In addition to the functional images, a z-stack of the antennal lobe was acquired with a spatial resolution of 512 × 512 pixels and a z-layer distance of 2 µm to perform the morphological identification of glomeruli.
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