Design and performance of telescoping micropipette arrays for high throughput in vivo patch clamping

Precise positioning (e.g., 1-100 μm) of an electrode or arrays of electrodes is a critical requirement for many in vivo electrophysiology studies. We are developing an instrument to manipulate arrays of micropipette electrodes utilizing a novel actuation strategy wherein flexible fused silica micropipettes telescope within teflon conduits configured in nonrectilinear configurations. This strategy circumvents the steric hindrances that limit the number micropipettes to just one in conventional setups. To prove this concept, we have designed a device that allows the simultaneous actuation of an array of four micropipettes. We have experimentally characterized the linear translation of micropipettes in this device and have achieved a minimum step resolution of 1 μm. Further in vivo electrophysiology experiments validate the efficacy of this device for in vivo patch clamping. INTRODUCTION In vivo patch clamping (IVPC) is an electrophysiology technique used to measure sub-threshold synaptic events, as well as intrinsic channel activities, with extremely high signal-to noise and fine temporal precision, during defined behavioral or pathological states [1,2]. Using IVPC to record from multiple neurons simultaneously in behaving animals would allow us to understand how neural dynamics emerge from the synaptic and electrical processes within neuronal networks. Despite the advantages, it has proven to be a very difficult technique to use and to date, no system has been proposed for recording arbitrarily large numbers of cells, in the fashion of tetrodes or multi-electrode arrays. The stereotaxic hindrance in a conventional IVPC setup limits the number of micropipettes that can be simultaneously controlled in the confined experimental space to just one. We are working to develop instrumentation for precise positioning of the tips of densely packed array of micropipettes to be used for patch clamping in vivo. It is based on a novel actuation strategy that allows micrometer resolution positioning of flexible fused silica micropipettes that telescope within sheaths in non-rectilinear configurations (Figure 1). FIGURE 1. Schematic of the micropipette actuation strategy. The teflon sheath forms a curved, rigid and low friction conduit for the translation of a flexible fused silica micropipette. The micropipette tips used for IVPC can be closely spaced in a 2-dimensional array (e.g., 1 mm apart), while the distal ends (e.g., the ends away from the brain, which connect to amplifiers and manipulators) can be spaced far apart, so that inexpensive motors and stages can be used to perform the actuation. The fused silica micropipettes are flexible laterally yet stiff axially for accurate translation and force feedback. Since fused silica is not typically used for IVPC, we have characterized its ability to subserve precision movement and electrophysiology. MATERIALS AND METHODS In order for IVPC to be scalable to multiple micropipettes, several key design criteria must be satisfied. First, the micropipettes must have a very consistent tip size and access resistance [3]. Second, the micropipettes must be packable in close proximity (e.g., within a millimeter of each other) so that they can record neurons in the same brain, and ideally the same microcircuit, without steric hindrance from the amplifiers and translation stages that are typically attached to the micropipettes. Third, the system must allow for positive and negative pressure to be delivered to each micropipette, in an automated fashion, as traditional singleelectrode patching methods will not scale easily to many electrodes. FIGURE 2: (a) Photograph of the prototype fourmicropipette array, (b) the configuration of the curved Teflon sheaths within it, and (c) headstage containing 4 parallel translating fused silica micropipettes. Accordingly, we set out to design the micropipette array that allows independent control of an array of 4 micropipettes spaced 1 mm apart with a travel range of 10 mm and minimum step resolution of 1 μm (Figure 2). The micropipettes were formed by pulling polyimide coated fused silica capillaries (300 μm ID, 650 μm OD, Polymicro Technologies Inc) using the P2000 pipette puller (Sutter Instrument Company). They were routed between the control hardware and dense, parallel packing of the tips in a 1 mm x 1 mm square area via Teflon sheaths (AWG 21, Zeus Inc) that act as conduits (See Figure 2b,c). This packing density is advantageous for introducing multiple micropipettes into a 3 mm x 3 mm craniotomy on a head-fixed mouse. The sheaths are routed in a curved configuration through a 3D printed ABS chassis. The curvature of the sheaths was empirically determined (see the following section) to be the most effective for telescoping fused silica capillaries within them. They are fixed in place by filling the chassis with epoxy adhesive and letting it cure, thus forming rigid and low-friction conduits for the micropipette translation. A spring loaded shaft translating in a linear-roller ball bearing acts as an inexpensive interface between a pipette holder (Axon instruments Inc) and a piezo-motor (Newport Corporation) for linear actuation. EXPERIMENTAL RESULTS Linear Translation Characterization FIGURE 3: Experimental setup used for screening geometrical parameters affecting micropipette translation. Using the setup shown in Figure 3, we tested the feasibility of actuating micropipettes telescoping in sheaths fixed in various configurations. We systematically varied two main geometric parameters: the bend radius (822 cm), and micropipette length (5-20 cm) as shown. We observed stick slip phenomenon in the lower bend radii configurations (8 and 10 cm). Further, the maximum length of the micropipettes in the device had to be limited to 10 cm to facilitate easy back filling. Using these criterions, we designed the micropipette array to telescope within 6cm long sheaths bent at 12cm radius. We characterized the displacement of successive components of the actuation system as a function of the piezo-motor micro-steps (Figure 3). Shown in figure 4 is the displacement of the micropipette tip with respect to piezomotor actuated at 1-μm steps. We have been successful in achieving the aimed minimum step resolution of 1+/-0.287 μm and the actuator exhibits good linearity (r=0.997) for a range of up to 10 millimeters. FIGURE 3: Displacements of various components of the actuation system as function of piezomotor microsteps. Accuracy of displacement increases with successive steps in the system. We conducted the metrology experiments using both contact (dial gauge, Mahr Inc) and noncontact (capacitive sensor, Capacitec Inc) methods. Thus, the actual penetrative motion of the micropipette in the brain tissue is an interpolation of the motion characteristic observed in these two methods. FIGURE 4: Displacement of the micropipette tip with respect to actuator input. Measured accuracy is +/0.287 μm, r=0.997 (n=5). The principle difference observed was the introduction of a settling time of 1.6 seconds for the micropipette to reached the desired displacement when the measurements were taken using the dial gauge. This is because of the force exerted on the micropipette tip by the dial gauge head. However, no dynamic effects were observed in the electrophysiology experiments described in the following section. In rare instances, it is advantageous in an IVPC experiment to be able to retract the micropipette to de-clogging the tip. We have measured the micropipette translation backlash error to be 12