Tube pulling experiments were performed on a setup that comprises a Nikon C1 confocal microscope equipped with a X60 water immersion objective, micromanipulators for positioning micropipettes and optical tweezers as previously described (Sorre et al., 2009 (link)). To pull a tube, a GUV was held by a micropipette, brought into contact with a streptavidin-coated bead trapped by the optical tweezers, and then moved away from the bead. The tube radius was measured by using the ratio of lipid fluorescence intensity on the tube and on the GUV as , where nm is the previously obtained calibration factor for using BODIPY TR ceramide as lipid fluorescence reporter in the same setup by performing a linear fit of membrane fluorescence ratio and lipid radii measured by ), where is the force applied by the optical tweezers to sustain the tube and the membrane tension controlled by the micropipette holding the GUV (Sorre et al., 2009 (link)) (Prévost et al., 2015 (link)). For experiments where GM1* lipids were used as a lipid reporter, we obtained the calibration factor nm.
X60 water immersion objective
Manufactured by Nikon
The X60 water immersion objective is a high-performance microscope lens designed for use in aqueous environments. It is capable of delivering high-resolution imaging and is suitable for a variety of research and industrial applications involving the examination of samples in liquid media.
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2 protocols using x60 water immersion objective
1
Lipid tube radius measurement using optical tweezers
2
Electrophysiological Profiling of NPY Neurons
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Brain slices (250μm) were prepared from adult NPY-GFP mice (6-8 weeks old) as previously described (7) . Slices were incubated at room temperature (RT), in oxygenated extracellular medium containing (in mM): 118 NaCl, 3 KCl, 1 MgCl 2 , 25 NaHCO3, 1.2 NaH 2 PO 4 , 1.5 CaCl 2 , 5 Hepes, 2.5 D-glucose (osmolarity adjusted to 310mOsM with sucrose, pH 7.3) for a recovery period (at least 60minutes). Once in the recording chamber, slices were perfused at 2-3 ml/min with the same extracellular medium. Slices were viewed with a Nikon microscope (EF600) outfitted for fluorescence (fluorescein filter) and IR-DIC (Infrared-Differemcial interference contrast) videomicroscopy. Viable arcuate NPY neurons were visualized using a X60 water immersion objective (Nikon) with a fluorescence video camera (Nikon).
Borosilicate pipettes (4-6MΩ; 1.5mm OD, Sutter Instrument) were filled with filtered extracellular medium. Cell-attached recordings were made using a Multiclamp 700B amplifier, digitized using the Digidata 1440A interface and acquired at 3kHz using pClamp 10.3 software (Axon Instruments). Pipettes and cell capacitances were fully compensated.
After a stable baseline was established, BHB (5mM) was perfused for 10minutes. The firing activity was measured over the last minute of the BHB perfusion and compared with the firing rate measured 1 min before the perfusion.
Borosilicate pipettes (4-6MΩ; 1.5mm OD, Sutter Instrument) were filled with filtered extracellular medium. Cell-attached recordings were made using a Multiclamp 700B amplifier, digitized using the Digidata 1440A interface and acquired at 3kHz using pClamp 10.3 software (Axon Instruments). Pipettes and cell capacitances were fully compensated.
After a stable baseline was established, BHB (5mM) was perfused for 10minutes. The firing activity was measured over the last minute of the BHB perfusion and compared with the firing rate measured 1 min before the perfusion.
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