Targeting visually identified neurons for electrophysiological recording is a fundamental neuroscience technique; however, its potential is hampered by poor visualization of pipette tips in deep brain tissue. We describe quantum dot–coated glass pipettes that provide strong two-photon contrast at deeper penetration depths than those achievable with current methods. We demonstrated the pipettes' utility in targeted patch-clamp recording experiments and single-cell electroporation of identified rat and mouse neurons in vitro and in vivo.
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The authors acknowledge the Defense Advanced Research Projects Agency, Naval Research Laboratory Nanosciences Institute, Defense Threat Reduction Agency Joint Science and Technology Office MIPR B112582M and Invitrogen for providing the 625-nm QDs. We thank G. Szabó and Z. Máté (Institute of Experimental Medicine) for providing the CCK/DsReDt3 BAC and PV/GFP BAC transgenic mice. We thank J. Veres, Zs. Kohus, Z. Péterfy, N. Lenkey and E. Papp (Institute of Experimental Medicine) for providing brain slices with fluorescently labeled neurons; A. Holtmaat (University of Geneva) for providing the Thy-1 EGFP-M mice; and J. Weber, A. Ráksai-Maár, M. Prsa and M. Cane for technical assistance. This work was supported in part by the Wellcome Trust (grant 090915/Z/09/Z, J.K.M. and B.K.A.), Hungarian Academy of Sciences (Lendület LP-2011-012, J.K.M.), Howard Hughes Medical Institute and Swiss National Science Foundation (D.H.).
B.K.A., M.B., J.J.M., K.S., J.B.D., A.L.H. and I.L.M. have filed a patent application for production of QD-coated probes based on the results reported in this paper.
Integrated supplementary information
Chemical structures of the methoxy-terminated and PEG-appended dihydrolipoic acid (DHLA-PEG-OCH3) and zwitterionic-compact ligand (DHLA-CL4) ligands used to make the QDs hydrophilic and biocompatible. Structures are also shown for the hydrophilic-native inorganic ligands used during QD synthesis. These include trioctylphosphine (TOP), trioctylphosphine oxide (TOPO), and hexadecylamine (HDA).
(a) Average gray value (10 frames) for Alexa Fluor 594 filled and QD 625–coated pipettes across different depths at a fixed laser power of 40 mW. Rank Sum test P < 0.01. (b) Laser power required to obtain an average gray value of 100, corresponding to a visibly discernible fluorescence value. Open circles: Alexa Fluor 594 filled pipettes, n = 6; red filled squares: QD 625–coated pipettes, n = 6. Black lines correspond to respective averages. Alexa Fluor 594 filled pipette imaged at different depths (D) within in vivo brain of anaesthetized mice at the indicated laser power (LP). Rank Sum test P < 0.05. (c) Average gray value (10 frames) of Alexa Fluor 594 filled pipettes across different depths at a fixed laser power of 40 mW imaged at 800 nm. (d) Laser power required to obtain an average gray value of 100, corresponding to a visibly discernible fluorescence value.
(a) Red QD 625–coated pipette with only the tip labeled as used for patching a GFP-PV positive interneuron from a PV/GFP BAC mouse. Representative of 11 cells in 6 animals. Panels from left: z-stack 2P images obtained in the green channel (left), red channel (center left), and merged (center right); voltage responses to positive and negative current injections (200 pA) in the cell shown on the left. (b) Z-stack 2P image of a rat hippocampal CA1 pyramidal neuron loaded with 100 µM OGB-1 (green) through QD 625–coated patch pipette in an acute slice. Inset shows 3 spines selected for 2P glutamate uncaging, along with the imaging line. Right, top: individual glutamate induced excitatory postsynaptic potentials (gluEPSPs) evoked at the indicated spines. Right, bottom: corresponding Ca2+ signals in the individual spines. Similar Ca2+ responses were evoked in 9 out of 11 spines in 3 dendrites from 2 neurons in one animal.
Supplementary Figure 4 Repeated steps of in vivo single-cell electroporation of fluorescent gene vector with a QD-coated pipette.
(a) Repeated steps of in vivo single cell electroporation of fluorescent gene vector with QD coated pipette, demonstrated on L2/3 cortical neurons at ~300 μm depth. 1. Approaching the cell. 2. Seal formation on cell membrane. 3. Filling the cell. 4. Withdrawing pipette after successful electroporation. (b) Montage of electroporated cells. (c) Z-stack image of cells n1-3 six days later (75% success rate).
Supplementary Figures 1–4 and Supplementary Protocol (PDF 944 kb)
Example video of a targeted (VGAT-CHR2 expressing neuron) single cell electroporation. Note that after dura penetration 530 QDs are still bright and visible at the tip of the pipette allowing precise targeting of the selected neuron. Internal solution contains Alexa Fluor 594 (50 μM). Due to the low fluorescence intensity of VGAT-CHR2, every visualized frame corresponds to a 10 frame rolling window average. Video speed is accelerated 2 times. (AVI 6986 kb)
Example video of a simultaneous whole cell recording of a layer 2/3 neuron under light anesthesia. (AVI 1501 kb)
Example video of 625 QD coated pipette approximating to a GCaMP6f expressing neuron in layer 2/3. Note the membrane “blow” when the tip of the pipette gets closer to the neuron and the membrane-pipette seal formation after positive pressure is released. Importantly, the exact location of the pipette tip is clearly visible during the entire process. (AVI 5728 kb)
760 μm deep, layer 5 single cell electroporation with 625 QD coated pipette in Tg(Thy1-EGFP)MJrs/J mouse. Laser power at 940 nm was 40 mW. To avoid QD adsorption to the dura during piercing and improve visibility of the coated pipette, the dura was initially pre-pierced with another QD coated pipette. The successfully electroporated neuron expressed DsRed and GFP after 2 days (see Figure 3g). Video speed is accelerated 6 times. (AVI 210793 kb)
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Andrásfalvy, B., Galiñanes, G., Huber, D. et al. Quantum dot–based multiphoton fluorescent pipettes for targeted neuronal electrophysiology. Nat Methods 11, 1237–1241 (2014). https://doi.org/10.1038/nmeth.3146
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