2. Depending on the exact target and/or brain region of interest,
any other genetic background strain can be used. Expression of
a fluorescent protein in a target structure facilitates accurate
localization for in vivo electroporation.
3. Since current distribution along the pipette tip critically
depends on the exact geometry of the cone, shallow cone
angles are preferred for a more even distribution. This is
achieved more easily with a thin-walled glass capillary as compared to a thick-walled one.
4. Also, a wider tip opening will favor a more even distribution.
This, however, may come at the cost of more tissue damage so
that we found a tip opening of 3.5–4.5 μm to be a reasonable
compromise.
5. For simplification, a uniform flow of electric current, a constant
specific resistance ρ and no conductivity through the wall of the
glass are assumed in this description.
6. In theory, hole resistance could also be changed by the depth
parameter d. This, however, would either require the deposition of material or to carve out a shallow around a hole which
would make the fabrication process more complicated and with
longer time.
7. During tissue insertion, permanent low positive pressure
(10–20 mbar) is applied to the pipette to allow constant dye
perfusion of the tissue and to prevent tissue from extending
into the holes of the pipette.
References
1. Haas K, Sin WC, Javaherian A, Li Z, Cline HT
(2001) Single-cell electroporation for gene
transfer in vivo. Neuron 29(3):583–591. [pii]:
S0896-6273(01)00235-5
2. Nagayama S, Zeng S, Xiong W, Fletcher ML,
Masurkar AV, Davis DJ, Pieribone VA, Chen
WR (2007) In vivo simultaneous tracing and
Ca(2+) imaging of local neuronal circuits.
Neuron 53(6):789–803. https://doi.org/10.
1016/j.neuron.2007.02.018; [pii]: S08966273(07)00138-9
3. Nevian T, Helmchen F (2007) Calcium indicator
loading of neurons using single-cell electroporation. Pflugers Arch 454(4):675–688. https://
doi.org/10.1007/s00424-007-0234-2
4. Hovis KR, Padmanabhan K, Urban NN
(2010) A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits. J
Neurosci Methods 191(1):1–10. https://doi.
org/10.1016/j.jneumeth.2010.05.017
5. Ke MT, Fujimoto S, Imai T (2013) SeeDB: a
simple and morphology-preserving optical
clearing agent for neuronal circuit reconstruction. Nat Neurosci 16(8):1154–1161. https://
doi.org/10.1038/nn.3447
6. Schwarz D, Kollo M, Bosch C, Feinauer C,
Whiteley I, Margrie TW, Cutforth T, Schaefer
AT (2018) Architecture of a mammalian glomerular domain revealed by novel volume electroporation using nanoengineered microelectrodes.
Nat Commun 9(1):183. https://doi.org/10.
1038/s41467-017-02560-7
7. Sale AJH, Hamilton WA (1968) Effects of high
electric fields on micro-organisms. 3. Lysis of
erythrocytes and protoplasts. Biochim Biophys
Acta 163(1):37–43
8. Carraro T, Dorsam S, Frei S, Schwarz D
(2018) An adaptive Newton algorithm for
optimal control problems with application to
optimal electrode design. J Optim Theory Appl
177(2):498–534. https://doi.org/10.1007/
s10957-018-1242-4
Targeted In Vivo Electroporation Using Nanoengineered Microelectrodes
119
any other genetic background strain can be used. Expression of
a fluorescent protein in a target structure facilitates accurate
localization for in vivo electroporation.
3. Since current distribution along the pipette tip critically
depends on the exact geometry of the cone, shallow cone
angles are preferred for a more even distribution. This is
achieved more easily with a thin-walled glass capillary as compared to a thick-walled one.
4. Also, a wider tip opening will favor a more even distribution.
This, however, may come at the cost of more tissue damage so
that we found a tip opening of 3.5–4.5 μm to be a reasonable
compromise.
5. For simplification, a uniform flow of electric current, a constant
specific resistance ρ and no conductivity through the wall of the
glass are assumed in this description.
6. In theory, hole resistance could also be changed by the depth
parameter d. This, however, would either require the deposition of material or to carve out a shallow around a hole which
would make the fabrication process more complicated and with
longer time.
7. During tissue insertion, permanent low positive pressure
(10–20 mbar) is applied to the pipette to allow constant dye
perfusion of the tissue and to prevent tissue from extending
into the holes of the pipette.
References
1. Haas K, Sin WC, Javaherian A, Li Z, Cline HT
(2001) Single-cell electroporation for gene
transfer in vivo. Neuron 29(3):583–591. [pii]:
S0896-6273(01)00235-5
2. Nagayama S, Zeng S, Xiong W, Fletcher ML,
Masurkar AV, Davis DJ, Pieribone VA, Chen
WR (2007) In vivo simultaneous tracing and
Ca(2+) imaging of local neuronal circuits.
Neuron 53(6):789–803. https://doi.org/10.
1016/j.neuron.2007.02.018; [pii]: S08966273(07)00138-9
3. Nevian T, Helmchen F (2007) Calcium indicator
loading of neurons using single-cell electroporation. Pflugers Arch 454(4):675–688. https://
doi.org/10.1007/s00424-007-0234-2
4. Hovis KR, Padmanabhan K, Urban NN
(2010) A simple method of in vitro electroporation allows visualization, recording, and calcium imaging of local neuronal circuits. J
Neurosci Methods 191(1):1–10. https://doi.
org/10.1016/j.jneumeth.2010.05.017
5. Ke MT, Fujimoto S, Imai T (2013) SeeDB: a
simple and morphology-preserving optical
clearing agent for neuronal circuit reconstruction. Nat Neurosci 16(8):1154–1161. https://
doi.org/10.1038/nn.3447
6. Schwarz D, Kollo M, Bosch C, Feinauer C,
Whiteley I, Margrie TW, Cutforth T, Schaefer
AT (2018) Architecture of a mammalian glomerular domain revealed by novel volume electroporation using nanoengineered microelectrodes.
Nat Commun 9(1):183. https://doi.org/10.
1038/s41467-017-02560-7
7. Sale AJH, Hamilton WA (1968) Effects of high
electric fields on micro-organisms. 3. Lysis of
erythrocytes and protoplasts. Biochim Biophys
Acta 163(1):37–43
8. Carraro T, Dorsam S, Frei S, Schwarz D
(2018) An adaptive Newton algorithm for
optimal control problems with application to
optimal electrode design. J Optim Theory Appl
177(2):498–534. https://doi.org/10.1007/
s10957-018-1242-4
Targeted In Vivo Electroporation Using Nanoengineered Microelectrodes
119