title: Integration of autopatching with automated pipette and cell detection in vitro.
abstract: Patch clamp is the main technique for measuring electrical properties of individual cells. Since its discovery in 1976 by Neher and Sakmann, patch clamp has been instrumental in broadening our understanding of the fundamental properties of ion channels and synapses in neurons. The conventional patch-clamp method requires manual, precise positioning of a glass micropipette against the cell membrane of a visually identified target neuron. Subsequently, a tight "gigaseal" connection between the pipette and the cell membrane is established, and suction is applied to establish the whole cell patch configuration to perform electrophysiological recordings. This procedure is repeated manually for each individual cell, making it labor intensive and time consuming. In this article we describe the development of a new automatic patch-clamp system for brain slices, which integrates all steps of the patch-clamp process: image acquisition through a microscope, computer vision-based identification of a patch pipette and fluorescently labeled neurons, micromanipulator control, and automated patching. We validated our system in brain slices from wild-type and transgenic mice expressing channelrhodopsin 2 under the Thy1 promoter (line 18) or injected with a herpes simplex virus-expressing archaerhodopsin, ArchT. Our computer vision-based algorithm makes the fluorescent cell detection and targeting user independent. Compared with manual patching, our system is superior in both success rate and average trial duration. It provides more reliable trial-to-trial control of the patching process and improves reproducibility of experiments.
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana.
authors.forename: Qiuyu
authors.initials: Q
authors.lastname: Wu 吴秋雨
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia.
authors.forename: Ilya
authors.initials: I
authors.lastname: Kolb
authors.affiliation: Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Brendan M
authors.initials: BM
authors.lastname: Callahan
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana.
authors.forename: Zhaolun
authors.initials: Z
authors.lastname: Su
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia.
authors.forename: William
authors.initials: W
authors.lastname: Stoy
authors.affiliation: MIT Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts; Department of Mechanical Engineering, University of Minnesota-Twin Cities, Minneapolis, Minnesota; and.
authors.forename: Suhasa B
authors.initials: SB
authors.lastname: Kodandaramaiah
authors.affiliation: McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Rachael
authors.initials: R
authors.lastname: Neve
authors.affiliation: Allen Institute for Brain Science, Seattle, Washington.
authors.forename: Hongkui
authors.initials: H
authors.lastname: Zeng
authors.affiliation: MIT Media Lab, Massachusetts Institute of Technology, Cambridge, Massachusetts; McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
authors.forename: Edward S
authors.initials: ES
authors.lastname: Boyden
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia; George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia.
authors.forename: Craig R
authors.initials: CR
authors.lastname: Forest
authors.affiliation: Department of Biological Sciences, Purdue University, West Lafayette, Indiana; chubykin@purdue.edu.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Chubykin
mesh_terms: D000465:Algorithms; D000818:Animals; D057205:Automation, Laboratory / Q000295:instrumentation; D002138:Calibration; D003196:Computer Graphics; D005260:Female; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D008297:Male; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D009474:Neurons / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000295:instrumentation / Q000379:methods*; D013997:Time Factors; D046509:Tissue Culture Techniques; D014584:User-Computer Interface; D014793:Visual Cortex / Q000166:cytology / Q000502:physiology
chemical_list: D005456:Fluorescent Dyes
keywords: computer vision; fluorescent cell detection; in vitro slice electrophysiology; patch-clamp