Experiments were performed in the late morning or early afternoon Zeitgeber time (Z.T.), inside a dark imaging chamber. An adult female animal 2–3 days-post-eclosion (dpe), was mounted onto a custom stage (Chen et al., 2018 (link)) and allowed to acclimate for 5 min on an air-supported spherical treadmill (Chen et al., 2018 (link)). Optogenetic stimulation was performed using a 617 nm LED (Thorlabs, Newton, NJ) pointed at the dorsal thorax through a hole in the stage, and focused with a lens (LA1951, 01" f = 25.4 mm, Thorlabs, Newton, NJ). Tethered flies were otherwise allowed to behave spontaneously. Data were acquired in 9 s epochs: 2 s baseline, 5 s with optogenetic illumination, and 2 s without stimulation. Individual flies were recorded for five trials each, with one-minute intervals. Data were excluded from analysis if flies pushed their abdomens onto the spherical treadmill—interfering with limb movements—or if flies struggled during optogenetic stimulation, pushing their forelimbs onto the stage for prolonged periods of time.
617 nm led
Manufactured by Thorlabs
The 617 nm LED is a light-emitting diode that emits light at a wavelength of 617 nanometers. This LED is a core component used in various laboratory and research applications.
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3 protocols using 617 nm led
1
Optogenetic Stimulation of Tethered Flies
2
Two-photon Imaging with Implanted Lenses
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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted September 4, 2021. ; https://doi.org/10.1101/2021.09.03.458910 doi: bioRxiv preprint Two-photon imaging via implanted lenses was performed as previously described (Lutas et al., 2019) with the following adjustments. A 10x 0.5 NA air objective was used (ThorLabs). For optogenetic stimulation of Chrimson via the implanted lens, a 617 nm LED (ThorLabs) was used (5 -10 mW at the objective face).
The copyright holder for this preprint this version posted September 4, 2021. ; https://doi.org/10.1101/2021.09.03.458910 doi: bioRxiv preprint Two-photon imaging via implanted lenses was performed as previously described (Lutas et al., 2019) with the following adjustments. A 10x 0.5 NA air objective was used (ThorLabs). For optogenetic stimulation of Chrimson via the implanted lens, a 617 nm LED (ThorLabs) was used (5 -10 mW at the objective face).
3
Optogenetic Modulation of VIP Neurons
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Optogenetic stimulation through the objective was performed with a 617 nm LED (Thorlabs), filtered through a 632/22 nm single-band bandpass filter (Semrock), at an amplitude through the objective of either 6 mW (VIP silencing experiments) or 1.5 mW (VIP activation experiments). Electrical tape between the mouse's head and the objective served to mitigate direct visual stimulation by the optogenetic light. The PMT was not gated, but protected by a strict shortpass filter. Optogenetic illumination began 0.25 seconds prior to visual stimulus delivery, and ended 0.25 seconds after. Small square wave optogenetic artifacts visible on the PMT were subtracted in post-processing.
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