Hcx pl fluotar l40x 0.60 corr dry objective

Calcium imaging experiments in AFD and FLP were performed in a temperature-controlled CherryTemp microfluidic system (Cherry Biotech) and analysed as previously described^{54 (link),62 (link)}. Briefly, adult worms were glued and imaged using a Leica DMI6000B inverted epifluorescence microscope equipped with a HCX PL Fluotar L40x/0.60 CORR dry objective, a Leica DFC360FX CCD camera, an EL6000 light source, and equipped with fast filter wheels for FRET imaging (excitation filter: 427 nm (BP 427/10); emission filters 472 (BP 472/30) and 542 nm (BP 542/27)). YFP/CFP ratio of the YC2.3 cameleon indicator was used to compare resting calcium levels across conditions, as well as relative changes in response to short-lasting stimuli. When the impact of starvation and thermal shift were examined, recordings were made 6 h after the condition changes. A calcium binding-defective version of YC2.3 was created by gene synthesis to introduce the following four mutations affecting each of the four EF-hand of the YC2.3 calmodulin domain: D21A, D57A, D94A, D130A^{81 (link)}.

To assess intracellular calcium in AFD, we maintained [ttx-1p::YC2.3] worms at 15 or 25°C, overnight. Adult worms were prepared as described in Saro et al., 2020 (link) and imaged using an inverted epifluorescence microscope (Leica DMI6000B) equipped with a HCX PL Fluotar L40x/0.60 CORR dry objective, a Leica DFC360FX CCD camera (1.4 M pixels, 20 fps), an EL6000 Light Source, and a fast filter wheel for FRET imaging. The recording and calcium imaging analysis were performed as in Saro et al., 2020 (link). Data are reported as YFP/CFP ratios with no baseline normalization in order to enable the comparison between resting calcium levels across animals and conditions. Animals were selected based on the overall fluorescence level of the reporter in order to have similar expression levels across conditions, which we verified a posteriori. This verification was made by summing the CFP and YFP emission signals (after excitation at 405 nm) in order to obtain a metrics representing the total fluorescence independently of varying FRET levels. This metrics was not significantly different between the sets of traces recorded at 15 and 25°C, respectively (15°C: average = 517, sem = 93; 25°C, average = 552, sem = 80; arbitrary units; p=0.73 by Student’s t-test).