Platinum wire

Brains were cleared as previously described (Menegas et al., 2015 (link)) at 37°C for 2 days, with a constant current of 1.2 amps. A Niagra 120 V (Grey Beard Pumps #316, Mt Holly Springs, PA, United States) pump was used to circulate clearing solution. A Precision Adjustable 60 V/5A power supply (Korad Technology #KA6005D, Shenzhen, China) was used to provide current. A 5-gallon plastic container (US Plastic #97,028, Lima, Ohio, United States) was used as a clearing solution reservoir and tubing was run though a second 5-gallon plastic container filled with water to cool the solution flowing through it. Chambers were constructed as previously described (Chung and Deisseroth, 2013 (link)) using a Nalgene chamber (Nalgene 2118–0002, Rochester, NY, United States) and platinum wire (Sigma-Aldrich 267228, St. Louis, MO, United States). Clearing was done in a room held at 37°C.

Ruthenium chloride (III), bdqi (1,2 benzoquinonediimine), 2,2′:6′,2′′-terpiridine, silver chloride (99.99%), platinum wire, silver wire (0.5 cm diameter) obtained from Sigma-Aldrich, St Louis, MO, USA; Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) and methanol were obtained from J.T. Baker, Center Valley, PA, USA; NO detection full commercial kit (ADI-917-020) from Emzo Life Sciences, Farmingdale, NY, USA; ethanol was obtained from Vetec, Duque de Caxias, RJ, Brazil. All solutions used were prepared from a reverse osmosis water purification system. All other chemical reagents were of analytical grade.

High purity flexible graphite foil (FGF, d_{25 °C} = 1.00 g/cm³, C: 99.5%, S <300 ppm, Cl < 50 ppm, ash < 1%, thickness 0.1 mm) was provided by Beyond Materials, Inc. (Tucson, AZ, USA) and dried in an oven at 60 °C for 48 h before use. Powdered graphite flakes (SP-1, d₂₅ °C = 1.05 g/cm³, C: 99.9%, ash < 0.5%, average size 30–150 μm) were acquired from Bay Carbon, Inc. (Michigan, MI, USA) and subjected to identical treatment. KMnO₄, H₂SO₄, K₂S₂O₈, P₂O₅, H₂O₂ (30 wt % in water), and platinum wire (ø: 0.5 mm, 99.99% trace metals basis) were obtained from Sigma-Aldrich (Madrid, Spain) and used as received. Ultrapure water was purified by a Millipore Elix 15,824 Advantage 15 UV system (Millipore, Milford, MA, USA).

A homebuilt nano-DESI probe was used, and it is composed of two fused silica microcapillaries with 150 μm OD and 50 μm ID (Polymicro Technologies LLC, Phoenix, AZ, USA), as shown in Figure 1a,b. The primary capillary (Figure 1b, left) was connected to a solution feeding capillary (360 μm OD and 100 μm ID fused silica) and to a platinum wire (Sigma-Aldrich, St. Louis, MO, USA) for electrical contact by utilizing a Micro-TEE connector (IDEX Heath & Science LLC, Oak Harbor, WA, USA). One end of the secondary capillary (Figure 1b, right) was in contact with the primary capillary at about 90°, and the other end of this capillary was a nanoelectrospray emitter with less than 10 μm ID. As shown in Figure 1b, the polyimide coating of the secondary capillary was removed to obtain a more stable and smaller liquid junction between the two capillaries. The nano-DESI solvent was fed by a syringe pump (Fusion 100 Touch, Chemyx, Inc., Stafford, TX, USA) at a flowrate of 300–600 nL/min. Various nano-DESI solvents were tested, including methanol (MeOH)/water (H₂O)/CHCl₃ (5:1:0.1, v/v), MeOH/H₂O (5:1, v/v), EtOH, and EtOH/CHCl₃ (5:1, v/v), and EtOH was finally selected to record the nano-DESI mass spectral profiles against the plant samples. All the used solvents except for deionized water were purchased from Fisher Scientific (Fairlawn, NJ, USA).

Flexible graphite foil (FGF, d_{25 °C} = 1.00 g/cm³, C: 99.5%, S < 300 ppm, Cl < 50 ppm, ash < 1%, thickness 0.1 mm) was supplied by Beyond Materials, Inc. (Tucson, AZ, USA) and dried in an oven at 60 °C for 48 h before use. Powdered graphite flakes (SP-1, d_{25 °C} = 1.05 g/cm³, C: 99.9%, ash < 0.5%, average size 30–150 μm) were purchased from Bay Carbon, Inc. (Michigan, MI, USA) and dried under identical conditions. KMnO₄, H₂SO₄, K₂S₂O₈, P₂O₅, H₂O₂ (30 wt% in water), and platinum wire, (ø: 0.5 mm, 99.99% trace metals basis) were obtained from Sigma-Aldrich (Madrid, Spain) and used as received. Ultrapure water was purified by a Millipore Elix 15,824 Advantage 15 UV system (Millipore, Milford, CT, USA).

Te dioxide (99.9995%), sodium hydroxide (ACS reagent, ≥97.0%, pellets), PVP, average molecular weight (~55,000), ethylene glycol (ReagentPlus^®, ≥99%), hydrazine hydrate (78–82%, iodometric), platinum wire (99.9%), ammonium sulfide solution (40–48 wt% in H₂O), and methanol (reagent grade, 98%) were purchased from Sigma Aldrich. Acetone (J. T. Baker^®), dichloromethane (BDH^®), and isopropyl alcohol (ACS Grade) were purchased from VWR International. Sodium sulfide (anhydrous, min. 99.5%), sodium sulfide nonahydrate (ACS reagent, >95%), sodium hydrosulfide hydrate, potassium sulfide (anhydrous, min. 95%), and potassium hydrosulfide (anhydrous, min. 95%) were purchased from Strem Chemicals. 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([EMIM][TFSI]) was purchased from Solvent Innovation GmbH (Germany). All chemicals were used as delivered without further purification.
Glass substrates, 9.5 × 9.5 mm, and 0.2 mm thick, were purchased from Thin Film Devices. <100>-oriented, boron-doped Si wafers (resistivity = 0.005–0.01 Ω cm, thickness = 525 ± 25 μm) coated with 300 nm of thermal oxide (SiO₂) were purchased from Silicon Valley Microelectronics.