Automated Cell Patch Clamping Method and Apparatus
First Claim
1. A method for automated whole-cell patch clamping, comprising the steps of:
- in an automated apparatus for cell patch clamping,regionally localizing a recording electrode of a cell patch clamping device by causing the tip of the recording electrode to be lowered to an appropriate depth for neuron hunting;
iteratively lowering the tip of the recording electrode by a small amount;
measuring the resistance at the recording electrode tip after each iteration of the step of lowering;
determining whether or not a target neuron has been encountered by constructing a temporal series of the resistance measurements after each iteration of the steps of lowering and measuring;
iteratively continuing the steps of lowering, measuring, and determining until the temporal series of resistance measurements indicates monotonic increases in resistance over a threshold number of consecutive iterations and the increase in resistance over this measured temporal series is above a pre-set neuron detection threshold;
stopping the motion of the recording electrode;
initiating gigaseal formation;
assessing whether or not gigaseal formation has been achieved;
if gigaseal formation has been achieved, initiating break-in and formation of a whole-cell patch clamp; and
verifying formation of the whole-cell patch clamp.
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Accused Products
Abstract
In an automated methodology for carrying out in vivo cell patch clamping, a cell patch clamping device is automatically moved into position and targeted to a neuron. Neuron contact is determined by analyzing the temporal series of measured resistance levels at the cell patch clamping device as it is moved. The difference between successive resistance levels is computed and compared to a threshold, which must be exceeded for a minimum number of computations before neuron contact is assumed. Pneumatic control methods are used to achieve gigaseal formation and cell break-in, leading to whole-cell patch clamp formation. An automated robotic system capable of performing this methodology automatically performs patch clamping in vivo, automatically detecting cells by analyzing the temporal sequence of electrode impedance changes. By continuously monitoring the patching process and rapidly executing actions triggered by specific measurements, the robot can rapidly find neurons in the living brain and establish recordings.
111 Citations
20 Claims
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1. A method for automated whole-cell patch clamping, comprising the steps of:
in an automated apparatus for cell patch clamping, regionally localizing a recording electrode of a cell patch clamping device by causing the tip of the recording electrode to be lowered to an appropriate depth for neuron hunting; iteratively lowering the tip of the recording electrode by a small amount; measuring the resistance at the recording electrode tip after each iteration of the step of lowering; determining whether or not a target neuron has been encountered by constructing a temporal series of the resistance measurements after each iteration of the steps of lowering and measuring; iteratively continuing the steps of lowering, measuring, and determining until the temporal series of resistance measurements indicates monotonic increases in resistance over a threshold number of consecutive iterations and the increase in resistance over this measured temporal series is above a pre-set neuron detection threshold; stopping the motion of the recording electrode; initiating gigaseal formation; assessing whether or not gigaseal formation has been achieved; if gigaseal formation has been achieved, initiating break-in and formation of a whole-cell patch clamp; and verifying formation of the whole-cell patch clamp. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method for achieving and verifying neuron contact in an automated electrophysiology device, comprising the steps of:
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in an automated electrophysiology apparatus, regionally localizing a recording electrode of the electrophysiology apparatus by causing the tip of the recording electrode to be lowered to an appropriate depth for neuron hunting; iteratively lowering the tip of the recording electrode by a small amount; measuring the resistance at the recording electrode tip after each iteration of the step of lowering; determining whether or not a target neuron has been encountered by constructing a temporal series of the resistance measurements after each iteration of the steps of lowering and measuring; and iteratively continuing the steps of lowering, measuring, and determining until the temporal series of resistance measurements indicates monotonic increases in resistance over a threshold number of consecutive iterations and the increase in resistance over this measured temporal series is above a pre-set neuron detection threshold that indicates that neuron contact has been achieved.
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10. An apparatus for automated cell patch clamping, comprising:
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a cell patch formation apparatus, comprising; at least one cell patch clamping device having a recording electrode; a 3-axis linear actuator configured for positioning the cell patch clamping device; and a patch amplifier with computer interface; a programmable linear motor configured for moving the cell patch clamping device up and down in a temporally precise fashion; and a computer interface configured for automated closed-loop control of the programmable motor based upon a temporal series of resistance measurements made at the tip of the recording electrode. - View Dependent Claims (11, 12, 13, 14, 15, 16, 17, 18)
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19. A method for controlling an automated cell patch clamping device, comprising the steps of:
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in a cell patch formation apparatus, comprising; at least one cell patch clamping device having a recording electrode; a 3-axis linear actuator configured for positioning the cell patch clamping device; a patch amplifier with computer interface; a programmable linear motor configured for moving the cell patch clamping device up and down in a temporally precise fashion; and a computer interface configured for closed-loop control of the programmable motor based upon sequences of resistance measurements made at the tip of the recording electrode, regionally localizing the recording electrode by causing the linear motor to lower the tip of the recording electrode to an appropriate depth for neuron hunting; causing the linear motor to iteratively lower the tip of the recording electrode by a small amount; measuring the resistance at the recording electrode tip after each iteration of the step of lowering; determining whether or not a target neuron has been encountered by constructing a temporal series of the resistance measurements after each iteration of the steps of lowering and measuring; iteratively continuing the steps of lowering, measuring, and determining until the temporal series of resistance measurements indicates monotonic increases in resistance over a threshold number of consecutive iterations and the increase in resistance over this measured temporal series is above a pre-set neuron detection threshold; causing the linear motor to stop the motion of the recording electrode; initiating gigaseal formation; assessing whether or not gigaseal formation has been achieved; if gigaseal formation has been achieved, initiating break-in and formation of a whole-cell patch clamp; and verifying formation of the whole-cell patch clamp. - View Dependent Claims (20)
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Specification