P 2000 micropipette puller

Freshly prepared T. marneffei and A. fumigatus conidia stocks for these experiments were stored at 4 °C for <2 months. For inoculation, 52 hpf tricaine-anesthetized embryos were mounted on an agar mold with head/yolk within the well and tail laid flat on the agar. The fungal conidial suspension was inoculated intramuscularly into a somite aligned to the yolk extension tip for local infection [24 (link),45 (link)] using a standard microinjection apparatus (Pico-Injector Microinjection System; Harvard Apparatus, Holliston, MA, USA) and thin-wall filament borosilicate glass capillary microinjection needle (SDR Clinical Technology, prepared using a P-2000 micropipette puller; Sutter Instruments, Novato, CA, USA). Inoculated embryos were held at 28 °C. The delivered conidial dosage was verified by immediate CFU enumeration on a group of injected embryos [20 (link)]. It took approximately 10 min to commence imaging after inoculation; in this report, the zero time point (t = 0) is taken as the beginning of imaging.

Nanopipette fabrication
was carried out using a Sutter P-2000 micropipette puller with 5 tunable
parameters heat (H), filament (F), velocity (V), delay (D), and pull
(P). The following program was employed to fabricate 50 nm nanopipettes
(Line 1: H700, F4, V20, D170, and P0, Line 2: H680, F4, V50, D170,
and P200) from 0.7 mm quartz capillaries.

Cells were placed in a plexiglass chamber under an inverted microscope allowing for continuous superfusion with a modified Tyrode solution by gravity flow at a rate of 1–2 mL/min. The modified Tyrode solution contained (in mM): NaCl 121, KCl 4, CaCl₂ 1.3, MgCl₂ 1, HEPES 10, NaHCO₃ 25, glucose 10 at pH = 7.35, which was supplemented according to the actual experimental design. The osmolarity of this solution was adjusted to 300 ± 3 mOsm with the addition of NaCl or water as necessary. In all experiments, the bath temperature was set to 37 °C using a temperature controller (Cell MicroControls, Norfolk, VA, USA). Electrical signals were amplified and recorded using a MultiClamp 700A or 700B amplifier (Molecular Devices, Sunnyvale, CA, USA) under the control of a pClamp 10 software (Molecular Devices) following analog-digital conversion performed by a Digidata 1332A or 1440A converter (Molecular Devices). Microelectrodes were manufactured from borosilicate glass with a P-2000 micropipette puller (Sutter Instruments, Novato, CA, USA) and had tip resistances of 2–3 MΩ when filled with pipette solution. Transmembrane currents were recorded in whole-cell voltage clamp mode. The series resistance was typically 4-8 MΩ, and the measurement was discarded if it changed substantially during the experiment.

The quartz tubes were pulled using a Sutter Instrument P2000 micropipette puller to create tips with a diameter of ~160 or ~250 nm. The SOT was then fabricated using self-aligned three-step thermal deposition of Pb at cryogenic temperatures. In the first step, the pipette was pointed toward the source, and a thin film was deposited onto the apex ring of the pipette, forming the superconducting loop of the SQUID. The pipette was then rotated to a 100° orientation, and an electrode was deposited on one side of the pipette, connecting the apex ring and the gold contact. The third deposition was performed at a −100° orientation, forming the second electrode on the opposite side of the pipette^{44 (link),45 (link)}. The carefully adjusted deposition thicknesses resulted in SQUIDs with a critical current ranging from 60 to 120 μA at zero field. The relatively large diameter tip allows for high magnetic field sensitivity, and a slight asymmetry in the Josephson junctions shifts the interference pattern of the SQUID, resulting in finite magnetic field sensitivity at low applied fields^{44 (link)}. This is crucial in order to conduct the experiment at a low enough OOP field to avoid overcrowding the sample with vortices.