Axon multiclamp 700b amplifier

The double-barrel nanopipette was mounted on a custom-designed holder. An Ag/AgCl and an Ag electrode were inserted in the aqueous and organic barrels, respectively. Each electrode was connected to a headstage amplifier (Axopatch CV-7B) mounted on the SICM frame. The headstage amplifiers were connected to a patch-clamp amplifier (MultiClamp 700B) and to an analog-to-digital converter (Digidata 1550B). The ion current was sampled using a sampling frequency of 10 kHz. The SICM setup consisted of an Axon MultiClamp 700B amplifier, an MM-6 micropositioner (Sutter Instrument), and a P-753 Linear actuator (Physik Instrumente) to allow precise three-dimensional movement of the nanopipette. The SICM software was used to control the positioning and topographical scanning capabilities of the SICM (ICAPPIC, London, UK). The z-piezo actuator had a travel range of 38 μm, while the travel range for x and y piezo was 96 μm. An Eclipse Ti2 inverted microscope (Nikon Instruments) and LED illumination system (pE-4000 CoolLED) with filter sets for DAPI, FITC, and TxRed were used for bright-field and epifluorescence imaging. An ORCA-Flash4.0 V3 Digital CMOS camera (C13440-20CU, Hamamatsu) was used to acquire optical and fluorescent micrographs.

The SICM setup was comprised of an Axon MultiClamp 700B amplifier, MM-6 micropositioner (Sutter Instrument, Novato, CA) and a P-753 Linear actuator (Physik Instrumente, Irvine, CA) attached to the pipette holder to allow precise, three-dimensional movement of the micropipette (Fig. 1a). SICM software was used to control the positioning and topographical scanning capabilities of the SICM system (ICAPPIC, London, UK). An Eclipse Ti2 confocal microscope (Nikon Instruments Inc., Melville, NY) and broad-spectrum LED illumination system (pE-300 CoolLED, Andover, USA) were used for bright-field (BF) and immunofluorescence (IF) visualisation of mitochondria and myonuclei, in skeletal muscle fibres, to ensure efficient biopsy. The SICM system is used to automatically approach the skeletal muscle tissue, moving the micropipette just above the region of interest (Fig. 1b), which is then inserted into the skeletal muscle fibre through manual control (Fig. 1c). Following successful biopsy of mitochondria, the micropipette was retracted and the tip snapped into a 0.2-ml microfuge tubes containing lysis buffer (Fig. 1d) as described in the “Laser capture microdissection (LCM) and tissue lysis” section.