Electrophysiological recordings at the single cell level within complex matrices such as tissues, organs or even in vivo is a fundamental technique for neuroscience, cardiology and electrophysiology. One significant hurdle to accomplishing this is visualizing the patch clamp or pipette tip deep within these dense tissues. Currently, these studies are typically accomplished by patch clamp visualization using two-photon (2P) imaging of organic dyes such as fluorescein that are continuously expelled from the pipette tip. Limitations of this technique include their relatively low 2P action cross-section, their susceptibility to photobleaching, and dye accumulation causing increased background, especially after multiple descents.

To address this, NRL and its collaborators have developed a more robust fluorescent labeling of standard borosilicate glass pipettes allowing their 2P visualization far deeper within complex tissues. Recording pipettes are coated with luminescent semiconductor quantum dots (QDs) and used to visualize the probe under 2P microscopic illumination, enabling simultaneous electrophysical recordings and cellular binding measurements.

This approach has been successfully demonstrated for targeting cells deep within rat and mouse brain in vivo. Using a series of green, yellow and red emitting QDs, experiments were conducted on hippocampal DS-red-labeled cholecystokinin positive interneurons and GFP-expressing parvalbumin-positive interneurons. GFP expressing neurons were even successfully electroporated in vivo at > 750 µm using 625 QD coated peptides.

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Schematics of the (a) classic approach for pipette visualization during targeted manipulations of a labeled neuron (green) by means of infusion of Alexa Fluor dye (red) into the extracellular space for shadow patching and (b) the new approach based on QD-coated pipettes (red).
Composite Z-stack 2P image of a rat hippocampal CA1 pyramidal neuron loaded with Ca2+ sensor OGB-1 (100 μM, green) through 625 QD-coated patch pipette (red) in an acute brain slice.
2P monitoring of QD-coated pipettes during patching of hippocampal neurons in acute brain slices.