6. Combined focused-ion-beam (FIB)—Scanning electron
microscope (SEM) workstation (Neon 40EsB, Zeiss, Oberkochen, Germany).
2.2 In Vivo
Electroporation
Experiments
1. 6- to 8-week-old MOR174-9 mice [9] are used for this procedure, maintained under appropriate animal welfare guidelines
and approved by the responsible Institutional Animal Care and
Use Committee (see Note 2)
2. Microsurgical equipment including a surgical microscope
(Leica microsystems, Wetzlar, Germany).
3. Heating pad connected to a feedback circuit provided by a
rectal probe (FHC-40908, BASi Corporation, West Lafayette,
IN, USA).
4. Instant adhesive Loctite 4011 (Loctite Corporation, Rocky
Hill, CT, Mississauga, Ontario).
5. Paladur dental acrylic (Heraeus Holding GmbH, Hanau,
Germany).
6. Dental drill.
7. Sharp and small needle tip.
8. Flamed tungsten wire.
9. Two-photon microscope setup equipped with a Ti-Sapphire
laser (Coherent, Dieburg, Germany) including a 16Â water
immersion objective (0.8 NA, Nikon).
10. Extracellular Ringer’s solution (NaCl 135.0 mM, KCl
5.4 mM, Hepes 5.0 mM, MgCl 2 1.0 mM, CaCl 2 2.0 mM,
pH 7.2, 280 mOsm/kg).
11. Tetramethylrhodamine (TMR) or Fluorescein 3000 MW
dextran-conjugated
Dyes
(both
Invitrogen,
Life
Technologies GmbH).
12. Micromanipulator (Luigs & Neumann, Ratingen, Germany).
13. Stimulus isolation unit (ISO-STIM 01D, npi electronic
GmbH, Tamm, Germany).
14. Paraformaldehyde solution (4%, pH adjusted to 8.9).
3 Methods
3.1 Fabrication
of NEMs
1. Pull glass capillaries to a long shank so as to ensure better
applicability in later in vivo experiments. The final tip size
should be 3.5–4.5 μm in diameter (see Notes 3 and 4).
2. Place glass pipettes in the high-vacuum sputtering and coating
system in an upright manner and heat-coat twice for 40 s with a
piece of gold wire. The pipette needs to be placed close to the
heating coil. This procedure ensures an even deposition of the
gold layer on the surface of the pipette tip. Finally, a layer
Targeted In Vivo Electroporation Using Nanoengineered Microelectrodes
115
microscope (SEM) workstation (Neon 40EsB, Zeiss, Oberkochen, Germany).
2.2 In Vivo
Electroporation
Experiments
1. 6- to 8-week-old MOR174-9 mice [9] are used for this procedure, maintained under appropriate animal welfare guidelines
and approved by the responsible Institutional Animal Care and
Use Committee (see Note 2)
2. Microsurgical equipment including a surgical microscope
(Leica microsystems, Wetzlar, Germany).
3. Heating pad connected to a feedback circuit provided by a
rectal probe (FHC-40908, BASi Corporation, West Lafayette,
IN, USA).
4. Instant adhesive Loctite 4011 (Loctite Corporation, Rocky
Hill, CT, Mississauga, Ontario).
5. Paladur dental acrylic (Heraeus Holding GmbH, Hanau,
Germany).
6. Dental drill.
7. Sharp and small needle tip.
8. Flamed tungsten wire.
9. Two-photon microscope setup equipped with a Ti-Sapphire
laser (Coherent, Dieburg, Germany) including a 16Â water
immersion objective (0.8 NA, Nikon).
10. Extracellular Ringer’s solution (NaCl 135.0 mM, KCl
5.4 mM, Hepes 5.0 mM, MgCl 2 1.0 mM, CaCl 2 2.0 mM,
pH 7.2, 280 mOsm/kg).
11. Tetramethylrhodamine (TMR) or Fluorescein 3000 MW
dextran-conjugated
Dyes
(both
Invitrogen,
Life
Technologies GmbH).
12. Micromanipulator (Luigs & Neumann, Ratingen, Germany).
13. Stimulus isolation unit (ISO-STIM 01D, npi electronic
GmbH, Tamm, Germany).
14. Paraformaldehyde solution (4%, pH adjusted to 8.9).
3 Methods
3.1 Fabrication
of NEMs
1. Pull glass capillaries to a long shank so as to ensure better
applicability in later in vivo experiments. The final tip size
should be 3.5–4.5 μm in diameter (see Notes 3 and 4).
2. Place glass pipettes in the high-vacuum sputtering and coating
system in an upright manner and heat-coat twice for 40 s with a
piece of gold wire. The pipette needs to be placed close to the
heating coil. This procedure ensures an even deposition of the
gold layer on the surface of the pipette tip. Finally, a layer
Targeted In Vivo Electroporation Using Nanoengineered Microelectrodes
115