Assembly and Operation of the Autopatcher for Automated Intracellular Neural Recording in Vivo

© 2016 Nature America, Inc. All rights reserved. protocol article provides further practical details on setting up the .This manner automated an in performed when method our of heartbeat the from in signals the pipette fluctuations of ance or the appearance in resist jumps large (e.g., changes human-detectable use which detection neuron of methods previous from differs This in pipette resistance that takes place over several consecutive steps. lowered intois the it brain,as looking for pipette a small patch but the monotonic increase of resistance the of analysis series time 2 (e.g., small in pipette a lowering automate to simple was for method different a discovered We recently throughput. and yield low had has it therefore and automate, to difficult be to proven been has clamping patch cell performed when especially form, of art an something considered is it and technique, laborious a is ing animals moving of use the neurons identified of patch clamping for targeted imaging two-photon with combined dynamics of single neurons in intact brains intracellular of study the for technique gold-standard the been fection analysis transcriptomic for contents cell of extraction morphology, cell visualize to of dyes staining cell infusion allows clamping patch whole-cell addition, In cell. receptors or in ion specific of channels the allow characterization to or recorded being cell the silence to or drive to intracellularly events spiking threshold supra and potentials membrane subthreshold both of urement ratio and temporal resolution of the recordings enables the meas signal-to-noise high The brain. living the in neurons electrical of the activity of measurement high-fidelity enables that nique In vivo I and increasing experimental durations by handling mentally intensive tasks automatically. enables labs that are already doing should enable preparation of mice takes ~1 h, and each autopatching experiment can be carried out over periods lasting severalthis protocol, hours. a functional experimental rig for automated whole-cell patch clamping can be set up in 1 week. High-qualitywhich is surgicala robot for automatically obtaining high-yield and high-quality whole-cell patch clamp recordings spiking and subthreshold synaptic events of single neurons in the brain. Whole-cell patch clamping Published online 3 March 2016; Georgia, USA. Correspondence should be addressed to C.R.F. ( Mechanical School of Institute Georgia Technology,Engineering, of Atlanta, Georgia, USA. Cambridge, Massachusetts, USA. 1 Giovanni Talei Franzesi Suhasa B Kodandaramaiah automated intracellular neural recording Assembly and operation of the autopatcher for 634 brain. mammalian live the in recordings whole-cell obtain autopatcher, as well as to the for procedure the using autopatcher Media Lab, Massachusetts Institute Technology,of Cambridge, Massachusetts, USA. NTRO protocol

| VOL.11 NO.4VOL.11 whole-cell patch clamping is an electrophysiological tech is patch clamping an electrophysiological whole-cell D 6 . Since its early application for recording recording for application early its Since . UCT in vivo in I ON 7,2 in vivo 5 20–2 | ), and may contribute to the high performance performance high the to contribute may and ), 2016 patch clamping for recording in awake freely freely awake in recording for clamping patch 3 . However, manual whole-cell patch clamp patch . whole-cell However, manual intracellular investigations to be accessible by a substantial number of neuroscience laboratories, and it 17–1 |

2 natureprotocols 1– 4 doi:10.1038 9 . In our method, we detect neurons by by neurons detect we method, our .In 3 . Multiple groups have demonstrated demonstrated have groups Multiple . 4 Departments of Biological Engineering and Brain and Engineering Biological Cognitive of Sciences,Departments MIT, Cambridge, Massachusetts, USA. in vivo 1 . Moreover, current can be delivered delivered be can Moreover,current . , Michael L McKinnon 1 – is an important neuroscience technique that uniquely provides access to both suprathreshold 3 , L Gregory Holst /nprot.2016.00 in vivo µ in vivo in 5 m) steps, and we perform a perform we and steps, m) and single-cell gene trans gene single-cell and in vivo. in patch clamping to scale up their efforts by reducing training time for new lab members 1,4,8–1 patch clamping that that clamping patch 7 6 Manual whole- Manual . It has also been [email protected] in vivo in 5 4 , Craig R Forest , Ian R Wickersham 7 in vivo in , it has has it , ------) ) or E.S.B. ( , 2 McGovern Institute for Brain Research, Massachusetts Institute Technology,of of an installed pipette for patch clamping ( clamping patch for pipette installed an of suitability the of assessment initial an conducts autopatcher The cell patch clamping into six stages ( whole- of process the down breaks algorithm autopatching The Automating yielding good recordings in good recordings a craniotomy yielding opened freshly trials the of >60% with average, on trials the of ~32% in cessful suc is mice anesthetized in clamping trained patch Whole-cell by humans. obtained those to similar are robot our using when 1 Data ( software autopatcher the is protocol this with pressures to the pipette at various stages of negative autopatching. and Included positive apply can that system regulation pressure electronic an includes also box control The gigasealing. during process, and it controls the application of current hyperpolarizing autopatching the throughout measurements impedance pipette to the and acquire patch with log box amplifier control interfaces custom-built a in outputs, digital and analog as well as inputs, analog motor.with linear equipped board grammable A digitizer pro a using by achieved is ( pipette patch the pipette of control patch Position the of control electric and pneumatic 63). (Step configuration whole-cell the obtain to tip pipette the at membrane of patch the rupture to applied are voltage and/or pressure negative of pulses brief phase, ‘break-in’ the in Finally, membrane. cell the with seal a form to attempt to pipette the to are applied current and hyperpolarizing pressure negative which next ( the detected, been has a neuron Once changes. resistance pipette neuron is detected, as indicated by a specific temporal sequence of pressure while constantly monitoring the pipette lower resistance until a a at increments small in advanced is pipette the which in ( stage next the by followed is This clogging. tip prevent to pressure positive high at interior pipette ( area recording desired the to pipette patch the lowers rapidly 1). Next, autopatcher step the The autopatcher executes this algorithm by assuming position, position, by assuming executes this algorithm The autopatcher 5 Department of Physiology,of Department Medicine,School of University,Emory Atlanta, T [email protected] his article describes how to set up and use the autopatcher, 4 Box Box 2 & Edward S Boyden ), which executes the algorithm. The success rates seen seen rates success The algorithm. the executes which ), 1 – , autopatching , steps 4 and autopatching 5) is stage executed, during 3 in in vivo , C Annabelle Singer whole-cell patch whole-cell clamping ).

Box 2 Box 1 – 3 , autopatching step 2) with the the with 2) step ,autopatching Fig. 1 Box 2 Box 1 in vivo – in vivo 3 , a

; see also , autopatching step 3), step autopatching , Box 2 Box . By following 4 George George W. Woodruff Supplementary Supplementary Boxes 1 , autopatching autopatching , A utopatching Fig. 1 Fig. 2 4 and . Thus,

2 b ). ). - -

© 2016 Nature America, Inc. All rights reserved. onto a manually controlled three-axis linear stage (MPC285, (MPC285, stage linear Instrument). Sutter three-axis controlled manually a onto Thorlabs) controller, TDC001 with motor (PT1-Z8 motor ear ( implementation current the In autopatching. during axial the direction in movement pipette patch the of control grammatic noise; electrical and table optics an on vibrations from mechanical is that table isolated sturdy installed (or other base are fixation mouse, the head custom immobilizing and for headstage amplifier the with along actuator, pipette The pipette. patch the manipulating for and acquisition data control; a custom autopatcher control for box; and a actuator pipette outputs, digital and analog as well as inputs, analog with equipped board digitizer signal a amplifier; in shown is Figure equipment autopatcher the of layout general The autopatcher of hardware and Overview software experiments. lengthy for yield and robustness increase to seeking electrophysiologists clamp patch may use even and be they for thus trained of from fatigue, do suffer not Robots field. the into entry may facilitate robot our to do starting for investigators measurements. The control box also performs closed-loop pneumatic pressure control of the patch pipette. Adapted with permissionprogrammable from ref. linear motor and a custom control box for data acquisition to enable closed-loop control of the motor based upon a inseries vivo of pipette resistance 2012): the system consists of a conventional algorithm (adapted from Kodandaramaiah system capable of performing the autopatching protocol. ( explained in the corresponding steps in the are highlighted with the red arrows and cell-attached recordings. Such instances than acquisition of whole-cell patched or autopatcher trials results in end points other clamp state (Step 36). Some fraction of the applied to achieve the whole-cell patch which pulses of high negative pressure are and 5); and (vi) the break-in stage, during been established ( and pipette voltage after contact with a cell has by modulating the pressure inside the pipette autopatching step 3); (v) attempting gigasealing the autopatcher scans for neurons ( 2); (iv) the neuron-hunting stage, during which pipette tip fidelity ( of interest followed by a second assessment of (iii) lowering of the patch pipette to the region unsuitable pipettes ( the resistance of the patch pipette to eliminatethe remaining steps (ii) an initial assessment theof autopatcher will programmatically perform so on) that need to be performed after which installing a pipette, software initialization andfollowing: (i) all the manual steps (Steps 12–31:cell patch clamp recordings et al. for autopatching (adapted from Kodandaramaiah of the algorithm and schematic. ( whole-cell patch clamp recordings Figure 1 i. 2 Fig. , 2012): the six stages of obtaining whole- patch setup (i.e., pipette, headstage, three-axis linear actuator, patch amplifier and computer), equipped with a few additional modules: a

c | 2 , hs s civd y onig pormal lin programmable a mounting by achieved is this ), The autopatcher—a robot for automated . The core components of the setup include the patch patch the include setup the of components core The . b ) Schematic of the autopatcher Box Box Box 2 2 , autopatching steps 4 2 Fig. 2a, Fig. , autopatching step 1); , autopatching step in vivo a include the ) The algorithm in vivo c ). The pipette actuator allows pro allows actuator pipette The ). Box in in vivo : overview 2

et al.

whole-cell patch clamping, patch clamping, whole-cell

, a Head-fixed mouse b Pipette holder Three-axis linear Pipette i installation Programmable Pipette holder Pipette linear motor actuato Headstage Unsuccessful gigasealformation Unsuccessful break-in autopatching Pipette unsuitablefor Pipette blockedduringinitialdescenttodepth r assessment in in vivo i the brain i outside Pipette Pipette joystick Control - -

localization (required for patch clamp amplifier operation; device/digitizer acquisition data external traditional the and fier ampli patch the with interfaces also box control This control. an control electronic box instead of a for manual syringe pressure version 2012 original the and version current the (ref. 2012 in described that over applied to the pipette during autopatching. during pipette the to is applied pressure which change to valves individual the switch board front box’s logic signals Digital (TTL) from transistor–transistor the digitizer control valves. three-way the of bank a to inputs are on pressures These panel. mounted potentiometers pressure) using negative high mBar 0 to −350 and pressure low negative mBar 0 to −25 pressure, positive low mBar 0–25 pressure, regula tors, which pressure can modulate the pressures (0–800 mBar electronic high positive using by achieved these then is of pressures control Finer generators. vacuum tube Venturi using input air high-pressure steady same the from generated also are −25 and mBar) mBar a of −350 of pressure low pressure negative of negative (a Two100 mBar). high states pressure pressure negative discrete positive low a and mBar 1,000 of pressure positive high (a pressures positive discrete two to it downregulates and mBar) (~2,580 air trol supply box takes in a high-pressure steady Regional i pipette ii The autopatcher equipment described here has improved been described equipment The autopatcher Ground Signal Linear motor controller Output pressuretopipette iv amplifier Patch hunting Neuron measuremen Datalink Datalink comman natureprotocols Patc Patc h h d t v formation Gigaseal 2 Autopatcher Computer Computer 4). The key difference between between difference key 4). The interface control board box

| VOL.11 NO.4VOL.11 v i Analog output Break-in Analog inpu Fig. 2b, Datalink Datalink protocol High-pressure inlet 2 2 4 4. t is the use of of use the is d (~2.5 bar) | ). The con Cell-attached 2016 Whole-cell recording recording |

635 - - -

© 2016 Nature America, Inc. All rights reserved. installed inside the autopatcher control box. on the front panel of the control box (potentiometers in the lower left corner). Vacuum pressures are generated using by Venturidownregulating tube vacuuma compressedgenerators air sourcealso of ~2,580 mBar using manual and electronic pressure regulators whose outputs can be controlled assemblyusing manual.pdf’ knobs in outputs on the same board are sent to a bank of pneumatic valves (described in Margrie the autopatcher control box sends command voltage signals to the patch amplifier and reads the patch measurements from the assembly.amplifier output. ( Digital focusing on the autopatcher control box and its interface with the patch amplifier and external digitizer. ( Figure 2 636 protocol

| VOL.11 NO.4VOL.11

| The autopatcher: equipment photographs. ( d d b a ) Schematic of the autopatcher control box. A central digitizer board equipped with analog inputs, as well as analog and digital outputs, in External digitizer High positivepressurecontroller High suctionpressurecontroller Low positivepressurecontroller Low suctionpressurecontroller Patch amplifier Negative pressuremanometer Positive pressuremanometer Autopatcher | 2016 from externaldigitizer control box to secondarydigitizer amplifier anddigitizer interfacing withpatch Patch measurement Patch measurement Pressure control potentiometers Command signal S Patch command Pressure output Manometers upplementary Data 4 BNC jacksfor from amplifier | natureprotocols to amplifier to pipette

FRONT PANEL ) to switch between different pressure states during autopatcher operation. The four pressures are generated Faraday cage table and V ibration isolation digitizer patch amplifier control box, Autopatcher a ) Photograph of the autopatcher showing the general layout of major equipment. ( Pneumatic valve bank Command switch signal High positivepressure High suctionpressure Low positivepressure Low suctionpressure 0 to–350mBar –25 to0mBar 0–800 mBar 0–25 mBar Digital Out Digital Out Digital Out Digital Out Analog In Analog Out Analog Out head-fix holder mous Custo holder Pipette headstage Amplifier linear motor Programmable DIGITIZER BOARD et al. 1 e 1 2 3 4 m 1 2 regulators Electronic pressure 7 and in the assembly manual ‘autopatcher control box c vacuum generator vacuum generator downregulator downregulator Pressure Pressure Venturi Venturi c ) Photograph focusing on the pipette actuator ~2.58 bar downregulator Pressure BACK PANEL to computer USB datalink ~2.5 bar High pressureair manipulator Manual three-axis b ) Photograph

© 2016 Nature America, Inc. All rights reserved. existing patch system to make it automated. it make to system patch existing any to added be could principle, in that, toolbox modular a on could ignore the motor additional one and digitizer. However,cases such here we focus in and users, end by modified directly be lator) or external digitizers that allow external software access may version the of Sutter manipu example, the software-controllable automated. Of course,it motors make that can to be directly rig, controlled by computerpatch (for existing an to easily added be enable modularity; that is, we wanted to design a system that could nal digitizer, in addition to the external traditional digitizer—is to to the manually controlled Sutter manipulator, as well as an inter site. this on hardware and website software the the at posted autopatcher.or previously were (specifically, protocol software) this in the included materials the of Some ling the amplifier software within the autopatcher program program autopatcher files; (.dll the libraries link within dynamic using software amplifier the ling control for scripts includes also program software current The the configuration. new hardware with to interface modified been paper 2012 our in described that as same the is autopatcher for used 2012 algorithm the the While to interface. software changes necessitated system delivery pressure the in improvements These one. internal the to as well as digitizer, external traditional the to routed and box the within split is put out is executed. being The amplifier algorithm the autopatching while resistances pipette the compute to used are measurements These amplifier. patch the from signal output voltage or current ( ibility flex experimental enable to digitizer external traditional the or sent digitizer from to the internal the signals amplifier command the switch to used is relay internal An autopatching. during fier ampli patch the to signals command sending chan to output dedicated nel analog one has It interface. USB a via computer dust inthe air, markedly increasing the chance thatthe pipettetipwill beclogged. longer durations, evenifthey havebeenkept insealedairtight containers, asthe surface properties may change becauseof pull pipetteswithinafewhours before anautopatching experiment. It isnot advisabletousepipettesthathavebeenstored for tolerances. Thus, the pullerneeds tobeprogrammed for eachtypeof glasscapillary being used. As afinal note, itisimportant to to the other) canbehaveverydifferently when pulledusing the same program becauseof differing melting points and geometrical electronic circuitry isatsteady state. Capillaryglasswiththe same dimensions from twodifferent vendors (orevenfrom one batch mended thatthe pullerbeswitched onwellinadvance (orevenleftoncontinuously during days of experimentation) sothatthe the electronics and heating components inthe pipettepullercandrift overaperiod of time. To account for these drifts, itisrecom user manual for instructions onadjusting pulling parameters toobtainpipettes withoptimum geometries. Further, the performance of in pipetteswithlonger tapersand smaller tipdiameters withcorresponding increases inpipetteresistance. Refertothe pipettepuller’s puller) canbeadjustedtoobtainpipettesof the desired resistance range of 5–7M to program ittopullpipettesthathaveresistances inthe range of 3–9M configuration isobtained. To optimizethe pipette geometry, a good starting point isto follow the pipettepuller’sinstruction manual autopatching. Finally, abroad cone angle assistsinobtaining stablegigaseals and lowaccessresistance when the whole-cell curvature. Pipetteswithconcave taperstypically havehigh variability intheir resistances and may givevariable results while (corresponding toarange of 5–7M recordings using pipetteswithcertaingeometrical characteristics. Ourpipettestypically exhibit tipdiameters of 0.8–0.9 recordings obtained bythe autopatcher. Inourlaboratory, wehaveconsistently obtained high-quality and high-yield autopatched The patchpipetteisanimportant component of the autopatcher, and itsquality canhaveamajor effectonthe yield and quality of The rationale for including an extra linear motor—in addition addition motor—in an linear for extra including The rationale the to connects box control the inside board digitizer The Box 1 Fig. 2 Fig. | d Idealpatchpipettesforautopatching ). One analog input channel is used to record the the record to used is channel input analog One ). g ; going forward, we will post periodic updates to updates periodic post will we forward, ; going Ω resistance; Supplementary Data 1 Data Supplementary 2 4 , the software has has software ,the Fig. 3 ). The general taperof the pipetteleading uptothe tiphasaconvex http:// ). ------

end point has been reached or when other user input is required. input user other or when reached been has end point through these stages, with pop-up dialog boxes appearing when an users interface guide to features user interactive incorporates GUI The (GUI). graphical software’s autopatcher by the autopatching of observing progress the follow can experimenter The in described is recordings clamp patch whole-cell obtain 25–31). (Steps trial autopatching each for autopatcher in to order up set the trial clamping for patch up autopatcher the V—setting Stage in steps the follow then can experimenter 21–24). The Steps autopatching, for programs software initializing the of IV—(Stage autopatcher software and zation configuration requires initiali experiment each of autopatching The beginning preparation: headplate attachment and craniotomy (Steps 12–20). III—surgical Stage in described is autopatching for mice of tion as (such service fabrication http://www.emachineshop.com commercial a to sent be can they 2–4, in Data provided are parts custom these (CAD) all of design files computer-aided and drawings parts. mechanical custom require The steps integration these of Some rig. ogy standard custom otherwise an and into components off-the-shelf integrating the involves of rig setup autopatcher The experiment. individual each for involve steps instructions subsequent The rig. autopatcher a up functional set fully that instructions installation one-time are 1–11 Steps 7–11). (Steps components hardware autopatcher the control to software autopatcher the configuring on user the guides which 1–6; (Steps hardware autopatcher-specific testing and calibrating structing, stages. for Stage I—hardware con setup has instructions detailed performing and clamping patch autopatcher whole-cell automated the up setting for protocol The design Experimental The procedure followed by the autopatcher to automatically automatically to autopatcher the by followed procedure The prepara surgical high-quality for protocol step-by-step The Ω . Then, the velocityand heat settings (for aFlaming-Brown and they can be used for in-house fabrication, or or fabrication, in-house for used be can they and Box Box Ω . Typically, higher velocityand heat settings result 1 ). This is followed by Stage II—software setup, setup, II—software Stage by followed is ).This natureprotocols / ). in vivo in

| VOL.11 NO.4VOL.11 in vivo in is divided into six six into divided is protocol Supplementary Supplementary electrophysiol µ m

| 2016

Box Box -

637 2 - - - - .

© 2016 Nature America, Inc. All rights reserved. 638 protocol ? (Autopatching step3)until the criteria for neuron detection are met again. the average resistance measurement inthe first4s of thisstep):Incase, the autopatcher reverts backto neuron hunting before neuron detection inautopatching step3,which isdetected bycomparing the average resistance measured inthe last4sto (ii) The autopatcher detects afalse positiveinthe neuron detection (pipetteresistance goes backtothe baseline valuemeasured stage wasreached. typically form inthe first30–90s of gigasealing, and they occurinabout60% of the autopatcher trials inwhich the neuron-hunting measurements during asuccessful gigasealing attemptisshown in in the ‘GIGASEALING RESISTANCE MONITOR’ graph inthe autopatcher software GUI( ramps down to−70mV. Additional detail aboutthe algorithm canbefound inthe original paper to atmospheric pressure after10s. At the same moment the pressure isswitched, the holding voltage ischanged to−30mVand the neuron wasdetected inautopatching step3):inthiscase, the autopatcher proceeds tothe next stage of gigasealing and switches (i) The autopatcher confirms thata neuron hasbeencontacted (pipette resistance staysatthe elevatedvalueinthe 10safter played inthe ‘GIGASEALING RESISTANCE MONITOR’ graph. Afterthe pipetteresistance ismeasured for 10s, one of twocaseswilloccur: the autopatcher commences agigasealing attempt( Autopatching step4:gigasealing—if aneuron isdetected during neuron hunting, the advancement of the pipette isstoppedand ? during asuccessful neuron hunting attemptisshown in box (the default valueis0.25M when the totalincrease islarger thanthe neuron detection threshold setinthe ‘Neuron detection threshold (M-Ohms)’ numerical entry the neuron-hunting process ( ance. These resistance valuesateachposition are logged and displayedinthe ‘NEURONHUNTINGRESISTANCE MONITOR’ graph during Autopatching step3:neuron hunting—the autopatcher moves the pipettedown by2 ? software GUI,which indicates thatthe pipette tipisbroken and instructs the experimenter toreplace the pipettefor the next trial. will immediately haltpositivepressure, retract the pipetteoutof the brain and displayapop-updialog box inthe autopatcher the pipettetodepth islowerthanpipetteresistance measured outside the brain bymore than2.0M (iii) The autopatcher detects abreakage of the pipettetipduring the descent todepth—(pipette resistance measured afterlowering in the main protocol for required userintervention. indicates thatthe pipettetipisclogged orfouled and instructs the experimenter toreplace the pipettefor the next trial. SeeStep33 case, the autopatcher retracts the pipetteoutof the brain and displaysapop-updialog box inthe autopatcher software GUI,which lowering the pipettetodepth isgreater thanpipetteresistance measured outside the brain bymore than0.35M (ii) The autopatcher detects aclogging orfouling of the pipettetipduring the descent todepth (pipetteresistance measured after ‘Neuron Hunt’ ( proceeds toAutopatching step3—neuron hunting (‘iv’in (i) The autopatcher detects no clogging orbreakage of the pipettetipduring the descent todepth: inthiscase, the autopatcher cases willoccur: ‘Resistance atbeginning of neuron hunt (M-Ohms)’ report box inthe autopatcher software GUI( Fig. 1 (Step 24),and waits1sbefore lowering the pressure inside the pipettefrom high positivepressure tolowpositivepressure (‘iii’in Autopatching step2:regional pipettelocalization—the autopatcher lowersthe pipettetothe depth specifiedbythe experimenter ? but thatisbeyond the scopeof thispaper.) SeeStep32inthe main protocol for required userintervention. tions canof coursemodify the software toalterthese defaults, e.g., ifveryfine pipettesare being usedtoattempt dendritic patching, install anew pipetteinthe autopatcher for the next trial. (Advanced usersdesiring topushautopatching beyond the current specifica resistance measurements is>50K (ii) The pipetteisunsuitable for autopatching: ifthe pipetteresistance isnot withinthe range of 3–9M (i) The pipetteissuitablefor autopatching: the autopatcher willthen proceed toautopatching step2. box ( the variance inmeasured pipetteresistances isdisplayedinthe ‘Resistance variation during initial pipetteassessment (M-ohms)’ report of the measured pipetteresistances isdisplayedinthe ‘Average resistance during initial pipetteassessment (M-Ohms)’ report box, and values inthe ‘Initial PipetteResistance Assessment’ graph inthe autopatcher software GUI( patching (‘ii’in Autopatching step1:checking the pipetteoutside the brain—the autopatcher firstchecks whether the pipetteissuitable for auto Box 2

| TROU TROU TROU TROU VOL.11 NO.4VOL.11 Fig. a ). It waits5sand then measures the resistance of the pipetteatthisdepth. Thisresistance valueisdisplayedinthe B B B B 5 LES LES LES LES ). At the end of thischeck, one of twocasescanoccur: | Autopatching H H H H Fig. 6 | Fig. 1 OOT OOT OOT OOT 2016 I I I I a N N N N a | ).

) before insertion into the brain. It measures the pipetteresistance in1-sintervals for 20sand displaysthese G G G G natureprotocols Fig. 6 Ω Ω , asdescribed inKodandaramaiah a , the autopatcher software GUIwilldisplayapop-updialog box instructing the experimenter to ). Contact withaneuron isdetected when resistance increases monotonically overthree stepsand Fig. 7a, Figure 6 Fig. 1 b ). The pipetteresistance isnow measured inintervals of 500ms and dis a b ). Panel 4of the autopatcher software GUIautomatically switches to . Figure 7 et al. 2 4 ). Arepresentative screenshot of aresistance trace recorded b . Successful gigaseals (sealresistance >1,000M Fig. 7 µ m every2sand measures the pipetteresist a ). Arepresentative trace of resistance Fig. Fig. 2 5 4 . The pipetteresistances are plotted ). At the end of 20s, the average Ω 5 ). At thispoint, one of three ): inthiscase, the autopatcher Ω orifthe variation inthe Ω (ref. 2 4)): inthis Ω (continued)

)

- -

- - © 2016 Nature America, Inc. All rights reserved. • • • Electrophysiology supplies • • • • • • • • • Mice andsurgery REAGENTS M the in described is below) bath detailed Methods saline Supplementary 21–39 a in Steps run of lieu practice a (in doing for protocol equipment any alternate troubleshoot an to before and issues hardware systems, autopatcher software the with and familiar get to experimenter saline a in an for immersed proxy run a tip as bath pipette practice the a with perform autopatcher the first of experimenter the that mended 37–39). (Steps attempt successful autopatching a after recordings clamp current and voltage Stage obtain in to filling instructions biocytin after recovery the and recording follow VII—whole-cell then can experimenter The is in process shown autopatching entire and to bring the pipette back to the brain surface for retrieval. The the ‘START OVER’ to can button be trial pressed stop the current any At 32–34). point, to wishes the if a experimenter start new from trial Step (Steps 24, trial the restart to how on experimenter the guide will boxes dialog pop-up the cases, such In patching). auto for unsuitable be to found is pipette the if (e.g., input user requires that recording whole-cell a obtaining before point end an may reach 36). autopatcher However,the (Step trials, in some until a whole-cell recording or experimenter recording cell-attached is the obtained from needed is input no trial, successful a In • EGTA (Sigma-Aldrich, cat. no. E3889) MgCl CaCl Potassium gluconate (Sigma-Aldrich, cat. no. P1847) All animal use must comply with institutional and governmental regulations. male or female, although other strains mice of can also be used Animals: C57BL/6 mice, 8- to 12-week-old mice (Taconic, cat. no. B6-M), Sterilized cotton swabs(Covidien/Kendall, cat. no. 61541400) Ethanol, 70%vol/vol International, (VWR cat. no. 71001-654) (McKesson,Betadine cat. no. 521234) Puralube ophthalmic ointment(Dechra, cat. no. 17033-211-38) International,Lidocaine (VWR cat. no. 95033-980) International,Sterile saline(VWR cat. no. 101320-574) Dental cement (Stoelting, cat. no. 51458) approved anesthetic xylazine, or10 mg/kgof 1–2% (vol/vol) in isoflurane oxygen, or other Anesthesia cocktail. For ketamine and example, 100mg/kgof amixof ? <100 M ms and updates the ‘GIGASEALING RESISTANCE MONITOR’ graph. It ishighly unlikely thatagigaseal willform ifthe sealresistance is (ii) Gigaseal hasnot beenobtained (sealresistance <1,000M indicating thatasuccessful gigaseal hasbeenformed. Proceed toStep34. (i) Gigaseal hasbeenobtained (sealresistance >1,000M gigaseal hasbeensuccessfully formed. One of twocaseswillthen occur: Autopatching step5:confirming gigaseal formation—60 safterthe commencement of gigasealing, the autopatcher checks whether a ATER Before any any Before Box 2

TROU 2 2 (Sigma-Aldrich, cat. no. C1016) (Sigma-Aldrich, cat. no. M8266) I ALS Ω B after60sof gigasealing operation. Proceed toStep34of the main protocol. LES | (continued) in vivo in H OOT in vivo in I experiments are attempted, it is recom is it attempted, are experiments N in vivo in G experiment is attempted. A modified, modified, A attempted. is experiment . experiment. This will allow the the allow will This experiment. Supplementary Video 1 Supplementary !

CAUT Ω ; ‘vi’in I ON

Ω ): The autopatcher continues measuring the sealresistance every500

- - .

Fig. 1 to an optimized and robust protocol. robust and optimized an to on patch high-quality pipettes, which are freshly pulled according impair recording otherwise quality. Successful patching also relies or pipettes, clean extremely be should what clog can tamination con or dust dirt, any as area, working the of cleanliness proper the skull, excellent craniotomy quality). It is also to crucial ensure to attachment headplate solid (e.g., quality accuracy, and surgery and purity solution cleanliness, preparation—environmental tal quality with the autopatcher is contingent on experimen pristine patch and clamp high yield experiment, obtaining high recording consistent get to groups research other enabled have that practices best the of ing Margrie Margrie (see tocols selves the with existing them acquaint users autopatcher that recommended It is highly to a variety of species, brain regions and experimental contexts and experimental to regions brain species, of a variety written with the mouse in mind it can also be applied, in principle, hippocampus in anesthetized mice helpful. is in and the the cortex extensively has tested been The autopatcher recordings) clamp patch high-quality obtaining for (e.g., it denoising and rig electrophysiology experimental an up patch in setting surgery. experience and rodent Prior of physiology clamp principles basic the with familiarity some has menter • • • • • • • • • • • • • • • • • • • • Syringe filter,Syringe 0.20 Syringe, 1ml(Becton, Dickinson andCompany, cat. no. 301025) Bottle-top filter vacuum (Nalgene, cat. no. 292-4520) KOH (Sigma-Aldrich, cat. no.P5958) Deionized water (Life Technologies, cat. no. AM9937) International,Bleach (VWR cat. no. 66025-688) (Warner,Glass capillaries cat. no. 64-0790) Low-gelling-temperature agarose (Sigma-Aldrich, cat. no. A9414) Glucose (Sigma-Aldrich, cat. no. 49163) NaHCO MgSO NaH KCl (Sigma-Aldrich, cat. no. P9541) HCl (Sigma-Aldrich, cat. no. H1758) (Sigma-Aldrich,Biocytin cat. no. B4261) Sucrose (Sigma-Aldrich, cat. no. S0389) NaCl (Sigma-Aldrich, cat. no. S3014) Na-GTP (Sigma-Aldrich, cat. no. G8877) Mg-ATP (Sigma-Aldrich, cat. no. A9187) HEPES (Sigma-Aldrich, cat. no. H3375) This protocol is written with the assumption that the experi the that assumption the with written is protocol This a and 2 PO 4 (Sigma-Aldrich, cat. no. M2643) 3 Fig 7 4 et et al. (Sigma-Aldrich, cat. no. S5761) (Sigma-Aldrich, cat. no. S8282) b 7 and Schramm and Schramm ): The autopatcher displaysapop-updialog box µ m (VWR International,m (VWR cat. no. 97048-592) in vivo in et al. et natureprotocols in in vivo 1 electrophysiology results. As with any any with As results. electrophysiology 9 , Kodandaramaiah , Kodandaramaiah et et al. patch clamping literature and pro literature patch clamping 2 2 7 4 ) ) to a obtain full understand , and although the protocol is

| VOL.11 NO.4VOL.11 et al. et protocol 2 4 , DeWeese | 2016 |

639 2 2 5 6 ------, . © 2016 Nature America, Inc. All rights reserved. • • • • • • • • • • • • • • • • • • • • • • • Equipment andtools for autopatching • • • • • • • • • • • • • • • • • • Equipment andtools for surgery EQUIPMENT gigasealing, stable recordings and easier break-in attempts. as measured in the image at the very tip of the pipette, are ideal for rapid of an ideal patch pipette exhibiting broad cone angle. Larger tip angles, autopatching (left), versus concave tapered pipettes (right). ( (right) ( a 40× magnification objective (left) and 100× water-immersion objective pipette with 1.5- water-immersion objective (right) ( visualized with a 40× magnification objective (left) and a 100× focusing on the pipette tip with a 0.9- of an ideal patch pipette pulled using a Flaming-Brown pipette puller Figure 3 640 • Ag-AgCl (Warner ground pellet electrode Instruments, cat. no. 64-1305) Rodent temperature control system (FineScience Tools, TR200) Stereomicroscope station(Leica, for surgery cat. no. M60) Needles, (Becton 31gauge Dickinson andCompany cat. no. 328438) Needles, (Becton 27gauge Dickinson andCompany, cat. no. 305109) bit,Drill 500 (PearsonDental drill Dental, cat. no. G24-00-05) Hair (Wahl, trimmer cat. no. 8786) Micro curette (FineScience Tools, cat. no. 10080-05) No. blade (Swann 10surgical Morton, cat. no. 0501) ‘Head Plate CAD.sldprt’) supplier; thetemplate isin Custom headplate to (needs in-houseor fabricated by be acustom cat. no. F000CE094) screws skull Self-tapping (Morris Precision Screws andParts, sourceCold station(Leica, light for surgery L2) Stereotax (Kopf, Model 900) protocol Table clamps (Thorlabs, CL5) cat. no. 8573K31) sheet,Thick Delrin 1.59mm(1/16thinch; McMaster-Carr, Socket (McMaster-Carr, capscrew head assortment cat. no. 92085A224) Ag-AgCl ground (Warner electrodes Instruments, cat. no. 64-1304) Stereomicroscope for autopatcher (Leica setup cat. no. M60) sourceCold light for autopatcher (Leica, setup L2) Microfil(World Precision Instruments, cat. no. MF28G67-5) (SutterPipette puller Instrument, P97) Pipette holder (Molecular Devices, HL-U-1) Low-noise rodent temperature monitor andcontroller (FHC40-90-8D) assembly manual in Autopatcher pressure control box (Neuromatic Devices; alternatively, see the commercial 3D printing service such as ‘Head fixation base CAD.sldprt’ for 3D printing the part or procure from Custom head fixation holder (see entitled ‘Adaptor plate 1.SLDPRT’ and ‘Adaptor plate 2.SLDPRT’) Custom (see mechanical parts Assorted mechanical components (Thorlabs, DP14A, MB4, C1505) controllerLinear motor with (Thorlabs, PT1-Z8, TDC001, TPS001) Three-axis manipulator (Sutter Instrument, MP285) Vibration Faraday isolationtable with cage (TMC, cat. no. 63-531) LabVIEW software (National InstrumentsLabVIEW 2011or later version) autopatcher.or Autopatcher control software ( RAM, dual-core processor, 1024×768resolution display) Desktop computer (Microsoft Windows atleast4GB system operating with Commander) Amplifier control software (MolecularDevices, MultiClamp Data acquisition software (Molecular Devices, Clampex) (Molecular digitizer Signal Devices, 1440B) Digidata Patch amplifier (MolecularDevices, 700B) MultiClamp Scissors (FineScience Tools, cat. no. 14060-09) no. handle Scalpel 3(FineScience Tools, cat. no. 10003-12) Inline(VWR solution filterInternational, cat. no. 66064-826) cat. no. 504180) points(World paper Sterile absorbent Precision Instruments,

| VOL.11 NO.4VOL.11 b

| ). ( Optimum pipettes used for autopatching. ( c ) Comparison of a convex tapered pipette, which is ideal for µ g m (Pearson Dental, cat. no. P86-02-38) ) µ m tip diameter (3.3 M | 2016 Supplementary Data 4 |

Supplementary Data2 Supplementary natureprotocols Supplementary Data3 Supplementary Supplementary Data1 Supplementary Supplementary Data 2 a ) in comparison with a patch µ m tip diameter (6.2 M https://i.materi Ω resistance) visualized with ) and is entitled andisentitled a alise.com which contains files , or b and use ) Photomicrographs h ttp://www.

Ω d

) Illustration resistance)

/ )

geometry thatgives optimum autopatchinggeometry inourlaboratory. optimized.empirically Refer to thathavegeometries already proven been to work slices inbrain or otherwise ( the pipette puller Patch pipettes months; warm it to 37 °C in a clean water bath before use in the experiment. vacuum filter (0.2- (up to 150 mM final concentration). Filter the solution using abottle-top the toosmolarity ~300 mOsm by iteratively adding small NaClquantities of • • • • • • • • • 1 mM MgCl 135mMNaCl,consisting of 2.5mMKCl, 10mMHEPES, 2mM CaCl cerebrospinalArtificial fluid cycles freezing of and thawing the solution should be avoided. thawed solution kept on an ice bath during experiments. °C. stored−80 at be for °C a few months.can −80 at solution This Use freshly (0.2- filter vacuum bottle-top a using solution the Filter gluconate. potassium of tion to 290–295 mOsm by iteratively adding small increments, e.g., 25–50 mg, urements approach the desired value). Next, increase the osmolarity of the solu the concentration of the base stock solution (e.g., to 1 M KOH) as the pH meas adding increments of 10–30 ml,volume, 500 e.g., solution KOH larger M a 5 (for of addition dropwise by 0.4 mM Na-GTP, 8 mM NaCl and 0.5% (wt/vol) biocytin. Adjust the pH to 7.2 CaCl mM 0.1 gluconate, potassium mM 135 containing solution a Preparetime. a at ml 50 solution pipette Intracellular REAGENT SETUP d c b a Heat-shrink tubing, 2.4mm(3/32ndinch) diameter (Mouser, 5174-13321) (McMaster-Carr, cat. no. 5648K251) Nondeformable, 6.35mm(1/4thinch) outer diameter tubing (McMaster-Carr, cat. no. 5779K677) Nondeformable, 3.175mm(1/8thinch) outer diameter tubing Helping hands(Amazon, cat. no. SEMZ101B) Assorted Luer (Cole-Parmer, fittings cat. no. EW-45511-00) Manometer (Dwyer, cat. no. 475-5-FM) Gold-coated pins, 1mm(Warner Instruments, cat. no. 64-1325) Ag-AgCl (Warner pellets Instruments, cat. no. 64-1309) Silver wire, 100–150 20 20 20 µ m m pore) and make aliquots in 1- to 1.5-ml volume tubes and store them µ µ µ m m m 2 . Adjust the pH to 7.3 by dropwise NaOH, addition of and adjust 2 ,MgCl mM 0.6

Pull the pipettes with resistancePull thepipettes with between 5and 7M µ Fig. m pore), and store the solution at 4 °C for up to a few µ 3 m indiameter (Warner Instruments, cat. no. 64-1318) 10 ). pointisto usepatch starting A good pipette µ m µ l of 5 M KOH at a time may work well, by lowering

Prepare cerebrospinal artificial fluid (ACSF) ,EGTA, mM 1 HEPES, mM 10 Mg-ATP, mM 4 Box We typically use the following recipe, making making recipe, following the use We typically 1 for a description of theideal pipette of for adescription 20 10 10 µ µ m µ m m 

CR I T I

CAL

Ω Repeated using 2 and

- -

© 2016 Nature America, Inc. All rights reserved. the desired craniotomy location ( implantation of the skull screws (top) and afterby implantationapplying freshly of mixedthe dentalheadplate acrylic (bottom). cement Scalearound bars, circle).the skull~2 Use mm.screws a ( burr and drill around bit theto drillperiphery three of anchor the headplateholessterilizing (~500 window. the scalp. ( First, perform longitudinal incision duringof the autopatching. scalp ( Scale bar, 5 mm. ( Figure 4 to the gold pinatthe topof the pipetteholder. Install the pipetteholder withthe Ag-AgCl wire inthe amplifier headstage. make the Ag-AgCl pipette electrode. Clean it thoroughly with filtered deionized water, and then connect the nonbleached end 6| in the ACSF bathduring the patchexperiment using a‘helping-hand’ device (seeStep25). Plug the gold-coated pininto the backof the amplifier headstage. The Ag-AgCl pelletis kept ontop of the skull,submerged gold-coated pinonthe other end. Coversoldered sections withinsulating heat-shrink tubing and shrinkitusing aheat gun. 5| yield and recording stability. from the mouse orother disturbances couldcausethe skulltomove relative tothe pipette. Thiswillsubstantially reduce cement, and they are then fastened tothisbaseatthe beginning of the experiment. Ifthe base isnot secured, then motion  underneath the autopatcher pipette actuator, and then clamp it securely in position using bolts or table clamps ( be commissioned from a commercial 3D printing vendor (e.g., Protolabs). Install the head fixation base on the optics table fixation base in acrylonitrile butadiene styrene (ABS) plastic using a 3D printer. Alternately, the custom head fixation base can 4| window thatfitswiththe curvedsurface of the mouse skull( with acylindrical sanding attachment tosand the bottomof the headplate todefine acurvedcontour along the headplate from commercial fabrication service providers. Use ahigh-speed rotary tool(e.g., Dremel, Amazon, ASIN#:B002L3RUVG) 1.59-mm (1/16th-inch)-thick Delrinsheet using alasercutter. Alternately, the fabricated headplates canbecommissioned 3| amplifier and traditional external digitizer ( from the box tothe computer, and connect the Bayonet Neill–Concelman (BNC)connections onthe front panel tothe patch 3.175-mm (1/8th-inch) outer diameter nondeformable tubing and barbedLuerfittings as necessary. Connect the USB cable Connect the pneumatic pressure outputonthe front panel of the control box tothe pipetteholder’s pressure input using fabrication. Alternately, afullyassembledcontrol box canbeprocured from acommercial vendor (e.g., Neuromatic Devices). test the autopatcher control box. Assembling the autopatcher control box requires proficiency inelectronic and mechanical 2| by placing itonthe vibration isolation tableand Faraday cage ( the pipetteactuator onanoptics table( 1| Hardware setup PROCE institutional and governmental regulations. (e.g., Massachusettsdislodge theInstitute flaky ofbone Technology tissue with (MIT)a tip Department of a needle of Comparative( Medicine Committee on Animal Care) d a

CR Ag-AgCl pipetteelectrodewire Ground electrode. Head fixation base fabrication Headplate fabrication Autopatcher controlbox. Autopatcher pipetteactuator. I T D

| I Surgical procedure for headplate implantation. ( URE CAL

STEP Secure clamping isveryimportant. The customheadplates are surgically attached tothe skullwithdental e b Take aninsulated wire, 300–450mmlong, and solder aAg-AgCl pelletonone end and a1-mm h . See ), thin down a 1- to 2-mm-wide pit at the desired craniotomy location until the remaining bone is ~100 Followthe instructions inthe assemblymanual in b S – f upplementary Data2 c f ) Preparation and surgery of the anesthetized mouse after administering approved anesthetic, followed by shaving and Followthe instructions inthe assemblymanual in . See . Take a50-mm-long silverwire and immerse 25mmof itslength inbleachfor 5–10minto S upplementary Data 2 Fig. 2 j ) and lift off the bone tissue and clear any remaining bone fragments ( Fig. 2b, a µ ) top (left) and side (right) views of the Delrin headplate to be affixed to the skull for head stabilization m in diameter, three small red circles; c ). Isolate the optics tablefrom mechanical vibrations and electrical noise g d ). b , and usethe ‘Head PlateCAD.sldprt’filetocutthe headplates from c ) to expose the skull ( Fig. and use ‘Head fixation base CAD.sldprt’ to 3D-print the head Fig. 2 4 ). j h a ). d ). Clear the skull further around the desired recording region ( 31G needle e ) and implant skull screws. ( S 500- upplementary Data4 µ h m burrdrillbit – S k upplementary Data3 ) Illustration of the craniotomy procedure. Identify natureprotocols g k ) Photographs of the mouse skull after ). All animal use should comply with f k i ) Finally, implant the headplate

toassembleand | VOL.11 NO.4VOL.11 µ m thick ( and install ∼ 100- protocol Fig. 2 µ m-thick skull i ), carefully | 2016

c ).

| ; red

641 © 2016 Nature America, Inc. All rights reserved. motor, internal digitizer and amplifiersoftware. manual.pdf’ in 10| programmable linear motor. Ifyouare using the TDC001motor controller from Thorlabs, thisisthe APTcontroller software. 9| autopatcher ( the code. Openthe ‘Autopatcher 2000.vi’filewithin ‘Autopatcher 2000.llb,’ and youshould seethe GUIusedtocontrol the Open the ‘Autopatcher 2000.llb’filewithin code necessary torunthe autopatcher inLabVIEW. Updates for thissoftware canbefound at acquisition drivers (DAQmx, National Instruments). Next, download and open 2011 version orlater),asthe freely availableautopatcher code iswritteninthisenvironment. Alsoinstall the latestdata 8| respectively. clamps, current and voltage the for pA/V 400 and mV/V 20 to Sensitivity’ Command ‘External the Set clamps. current M 500 to resistor feedback the configure software, control amplifier the In splitter. or hub USB a through them connect not Do computer. the on ports USB the to directly digitizer external and amplifier the Connect Devices). Commander, Molecular (MultiClamp software control amplifier patch the and Devices) 7| S 642 scan scan for neurons, among other things (see panel depend on the stage of autopatching, and they include methods for setting the beginning and ending depth ranges within which the autopatcher will control the programmable motor. This displays the absolute position of the pipette in motor coordinates. (Panel 4) The interactive elements displayed in this during an experiment, as well as an entry box where the experimenter can log comments. (Panel 3) Includes control elements that allow the experimenter to autopatcher operation. (Panel 2) Text indicators that display the current status of the autopatcher trial, and a log of all autopatching trials attempted pipette position (in Figure 5 protocol oftware setup

| Configuring autopatchersoftware. VOL.11 NO.4VOL.11 Programmable motorsoftware. Autopatcher software installation. Patch amplifier and data acquisition software acquisition data and amplifier Patch

| Autopatcher software GUI. Red dotted lines outline different panels of the GUI. (Panel 1) Pipette status indicators that display the instantaneous Fig. S 1 | upplementary Data1 µ 2016 m) m) from the surface of the brain, pressure applied to the pipette (in kPa) and the holding voltage (in mV) applied to the pipette during 5 ). | natureprotocols Fig. Fig. Install the drivers and software provided bythe manufacturer for controlling the Followthe instructions inthe configuration manual ‘Autopatcher software configuration Firstinstall the LabVIEWprogramming environment (National Instruments, LabVIEW 6 toconfigure the autopatcher software to recognize and control the programmable and S S upplementary upplementary Video 1 upplementary Data1 . Install the data acquisition software (e.g., Clampex, Molecular Molecular Clampex, (e.g., software acquisition data the Install . 4 2 to see how this box changes throughout the protocol). withLabVIEWtoshow alist of allthe ‘.vi’ filesusedin S upplementary Data 1 Ω for both the voltage and and voltage the both for h 3 ttp://www.autopatcher.or . This contains all the

. © 2016 Nature America, Inc. All rights reserved. autopatching. Thishasbeenwelldocumented byother groups performing manual  off the bone.  debris before proceeding tothe next step. opening craniotomies has beenpreviously described byLee exposure ( desired point onthe thinned skullverygently, opening upasingle small hole ~500 the bone tissueisthinenough toappearflakyand translucent ( 18| is shown in my location. Thisshould cure in10–15min.Apicture of the skullsurface before and afterthe headplate attachment around the skullscrews and around the periphery of the headplate ( then lowerthe stereotaxic armuntil the headplate sitsonthe exposed skull. Apply freshly mixed dental acrylic cement 17| that when the headplate isfixed the skullscrews are justinside the ‘window’ of the headplate. Place twoposterior skullscrews and one anterior skull screw with~7mmbetween the anterior and posterior positions so screws should bemade such thatthe desired recording craniotomy location isroughly centered inside the headplate window. self-tapping skullscrews into the drilled holes (indicated bythe three small red dots in 16| the spottomark the location of the craniotomy. Donot drill allthe waythrough the skull. the location for the craniotomy using the stereotaxic coordinates ( posterior; 15| with atemperature probe toregulate body temperature during the surgical procedure. the midline isparallel tothe anterioposterior axisof the stereotax frame. Ensure thatthe mouse isplacedonaheating pad Adjust the earbarsand nose cone of the stereotax such thatbregma and lambda reference points are atthe same height and 14| required). Ensure thatallhairclippings are removed from the surgical area during thisprocedure. in Betadine, followed byscrubbing with70%(vol/vol)ethanol tocleanthe scalp (repeat approximately three times as trim hairontopof the head ( 13| (DCM) atthe Massachusetts Institute of Technology. concerning laboratory animals. The procedure thatwedescribe here wasapproved bythe Division of Comparative Medicine ! according tothe approved animal protocol. throughout surgery and recording byregularly testing the depth of anesthesia and byadjusting the anesthetic dosing of 100mg/kg ketamine and 10mg/kg xylazine, or1–2%isoflurane in oxygen. Maintain the animal’s anesthetic plane 12| S precipitates and dust accumulation. storage box and pipette puller bars with ethanol and filtered deionized water at least monthly to reduce clogging from the intact brain. Beyond this initial setup, it is also important to clean the pipette holder, silver wire, ground wire, pipette the for performing the  to register allthe changes. 11|

urgical preparation: headplate attachment and craniotomy CAUT

PAUSE CR CR

S Fix themouseinstereotaxicapparatus. Prepare themousefor surgery. Anesthetize themouse. Restart thecomputer. Open thecraniotomyfor recording. Attach theheadplate. Implant theskullscrews. Incise thescalpandexpose theskull. upplementary Methods I I T T I I I ON CAL CAL

PO Fig. 4 Fig. 4j, All procedures involving animals must be performed in strict compliance with institutional and federal regulations Figure 4

I STEP STEP NT This completes the setup of the autopatcher hardware and software. The following steps describe the protocol c ). Use small retractors topullapartthe skinoverthe skull.Use amicrocurette toremove fascia. Identify The sizeand quality of the craniotomy hasamajor influence onthe yield and success rate of Make sure thatthe surface of the brain isnot damaged when youare attempting todislodge and lift k in vivo ). Keep the brain surface wetwithACSF whileattempting toopenthe craniotomy. Goodtechniquefor g . experiment. Before this, it is recommended that the experimenter follow the steps described in Restartthe computerafter completing allthe software installations toallowthe operating system Clampthe headplate tothe stereotaxic armsothatitishorizontal inthe stereotax frame, and Anesthetize amouse using anapproved anesthesia protocol. We typically useacocktail Using adental drill, drill three anchor holes (~500 to test the autopatcher operation using a pipette navigating in a saline bath as a proxy for Fig. 4 Applyophthalmic ointment tothe mouse’s eyes, and usehairtrimmers and scissorsto b ) toprepare the scalpfor incision. Scrubthe head withasterilecottonswabsoaked Gently milldown the surface of the skull,inacircular area ~1mmindiameter, until Use ano.10 surgical blade tomake acleanlongitudinal incision (anterior to Fixthe mouse inthe stereotaxic apparatus using the earand mouth bars. et al. Fig. 4h, 2 0 Fig. 4 . Ensure thatthe craniotomy hasbeencleared of allbone Fig. 4 i d ). Next, usea31-gauge needle topick awayatthe f ). Use aburrdrill bittogently make aburrhole at ). Donot applyacrylic cement overthe cranioto µ m diameter) into the skull.Screw inthe in vivo µ m indiameter, inducing minimal brain natureprotocols Fig. 4 whole-cell patch clamping e ). The location of the skull

| VOL.11 NO.4VOL.11 protocol

| 2 2016

0 .

643 - © 2016 Nature America, Inc. All rights reserved. 24| controls willappearin30–40 s. Data 1 23| to monitor and record the currents measured during the experiment. 22| automatically recognize the amplifier connected to the computer. Set the21| correct channel of the amplifier to voltage clamp mode. Initializing software programs for autopatching autopatching experiment.  head screws. fixing baseinthe autopatcher ( 20| should bedone withcaution ifthere are major bloodvesselspresent. an opening. Bleeding canbecontrolled withsterileabsorbent padsorbyperfusing the craniotomy withACSF. Thisstep either side of the incision tocreate fine forceps, slowly fold the dura to the brain. Byusing the needle or of the brain toprevent damaging pointing tangentially tothe surface keeping the tipof the needle 500- 31-gauge needle tocuta100- 19| resistance in the last data points. as indicated by the monotonic increase in pipette depth of 720 pipette to a depth of 650 recording. The autopatcher initially lowered the successful trial resulting in a whole-cell patch the resistance measurements logged during a HUNTING RESISTANCE MONITOR’ graph displaying ( numerical entry box (default, 0.25 M in the ‘Neuron detection threshold (M-Ohms)’ to gigasealing mode can be changed as needed the algorithm uses to switch from neuron hunting gigasealing stage. The resistance threshold that algorithm of the autopatcher and proceed to the the experimenter to override the neuron detection graph. The ‘SKIP TO GIGASEALING’ button allows on the ‘NEURON HUNTING RESISTANCE MONITOR’ step taken during neuron hunting are plotted this stage. The measured resistances after each 4 of the indicators and user controls in panel of autopatching: red dotted lines highlight hunting stage ( software GUI displayed during the neuron- interface: neuron hunting. ( Figure 6 644 searching for neurons inthe ‘Pipette depth tobeginneuron hunting (micrometers)’ numerical entry box, and setthe depth b protocol ) Representative screenshot of the ‘NEURON

Figure

| Start the amplifier control software. Perform adurotomy. Start anewtrial intheautopatchersoftware. Start theautopatchersoftware. Start thedataacquisitionsoftware. Head-fix themouseinautopatcher. VOL.11 NO.4VOL.11 µ I m slitinthe dura while T

). The autopatcher willinitialize the control box, and anew LabVIEWprogram window withthe motorized manipulator | I Autopatcher software graphical user CAL 5 that can be accessed during µ m before encountering a neuron, Box

STEP 2 | 2016 , autopatching step 3) Keep the mouse onaheating padtoensure thatitsbody temperature isregulated throughout the µ | m and scanned to a a natureprotocols ) The autopatcher Use a Ω

(ref. 24)).

Fig. 2

Openthe ‘Autopatcher 2000.vi’inLabVIEWand click the ‘RUN’button(

c Open the amplifier software (e.g., MultiClamp Commander). The amplifier software should ) byfastening the headplate tothe mouse head fixation baseusing no. 4–40socket Openand runthe data acquisition software (e.g., Clampex, Molecular Devices)

Once surgical preparation iscomplete, secure the mouse tothe customhead- b a Setthe recording depth atwhich youwant the autopatcher tostart S upplementary

© 2016 Nature America, Inc. All rights reserved. in either the whole-cell mode orthe cell-attached mode. These automatically performed stepsare described in The autopatcher willnow perform automated whole-cell patching until the point when the experimenter chooses torecord 31| ? measurement should be<40pA.Thiscompletesthe manual setupstage of anautopatching trial. also help. Allmetal foil shielding should beconnected tothe optic tableground. The peak-to-peaknoise levelinthe current ing them outside the Faraday cage. Shielding the headstage withmetal foil and the spacesurrounding the silver wire may sources of noise such asmicroscope lampsand powersupplies and either shielding them, powering them down orrelocat done bygrounding the Faraday cage and the vibration isolation tabletothe signal ground of the patchamplifier, identifying sition software. Eliminate any sources of 60Hz(or50depending ongeographical region) electrical noise. Thiscanbe 30| complete the circuit. After the tipispositioned, add ACSF until boththe tipof the patchpipetteand the ground pelletare submerged. Thiswill step. Measurements of depth traversed bythe autopatcher insubsequent stepsare referenced from thisstarting position. the brain toprevent the tissuefrom drying out.Use the stereomicroscope tovisualize the pipettetipand craniotomy inthis can serveasa‘scalebar’for thisprocess. During thisprocedure, maintain athin(5–30 using the manual three-axis manipulator. The sizeof the craniotomy and the pipette, aswellother known quantities, 29| pressure to−15mBarand high negative pressure to−300mBar. on the control box’s front panel. Setthe high positivepressure to800mBar, lowpositivepressure to25mBar, lownegative 28| the pipetteswithinafewhours of pulling. it isfiltered (0.2-  a portion of the Ag-AgCl wire when the pipetteisinstalled inthe pipetteholder.  previous trial.  and install itinthe pipetteholder. 27| residual blood). 26| the skullsurface and thatitisnot subjecttoany mechanical motion.  25| S the current stage ( change periodically toreport the most current information ortocollectinputfrom the userthatisappropriate for ? autopatching Step2. stop the autopatcher program. Restartanew trial from Step24.Thisstepisnot required ifthe autopatcher proceeds to 32| describe whattodo ifsuch conditions are encountered. (see also menter toinstall anew pipettefor autopatching. Throughout the operation of the software, panel 4of the GUI( numerical entry box ( at which youwant the autopatcher tostopsearching for neurons inthe ‘Pipettedepth tostopneuron hunting (micrometers)’ etting etting up the autopatcher for an autopatching trial

TROU TROU

CR CR CR CR

Set pressurestatesintheautopatchercontrolbox. Install apipetteintheautopatcher Inspect thecraniotomy. Install thegroundelectrode. User interventionifthepipette isfound tobeunsuitable for autopatching Begin anautopatchingtrial. Monitor currentsanddenoisetheautopatcher. Position thepipettefor autopatching I I I I T T T T I I I I B B CAL CAL CAL CAL LES LES Figs. 5

H H STEP STEP STEP STEP OOT OOT – µ Ensure thatthe internal pipettesolution iskept inanice bathfor the duration of the experiment and that To achieve aclosedcircuit connection, ensure thatthere issufficient solution inthe pipettetosubmerge Alwaysuseafresh, unused pipettefor everytrial. Donot attempttoreuse patchpipettesfrom a During the operation, ensure thatthe ground wire Ag/AgCl pelletissubmerged inthe ACSF solution above 7 I I S m pore) before backfilling the pipettetoprevent internal clogging of pipettes. It is recommended touse N N ). Some of these automatic stepsmay result inoutcomes thatrequire userintervention. Steps32and 33 upplementary Video1 G G Fig. 5 ). Click the ‘BEGINNEWTRIAL’ button.Apop-updialog box willappearinstructing the experi Perfuse the craniotomy withsterileACSF ifneeded tocleanthe surface (e.g., of any Click ‘OK’inthe pop-updialog box inthe autopatcher GUI(referred toinStep24). Placethe ground wire’s Ag/AgCl pelletontopof the mouse’s skullclosetothe craniotomy. . Fillapatchpipettewithinternal pipettesolution using asyringe filterand Microfil, . Carefully position the tipof the patchpipette20–30 ). Observethe currents being measured bythe amplifierinthe data acqui Adjust the autopatcher control box pressures byadjusting the knobs ( Box 2 , autopatching step1).Click ‘OK’to natureprotocols µ m) film of fluid over the surface of µ m abovethe brain surface

| VOL.11 NO.4VOL.11 protocol Box Fig.

| 2 2016

) will -

| - -

645

© 2016 Nature America, Inc. All rights reserved. current required tohold the cellat−65mVinvoltage clampaftereachbreak-in attemptisupdated inthe ‘Holding Current negative pressure pulse, followed byanassessment of the membrane resistance todetect asuccessful break-in. The holding 36| ? to startanew trial from Step24. If ithas, proceed toStep36.Ifithasnot and youwish tostartanew trial, click ‘START OVER’ to stopthe current trial and 35| ? proceed torecording, and then proceed toStep37.Ifwhole-cell recording isdesired, proceed toStep35. 34| ? patcher proceeds toautopatching step3. step 2) 33| whole-cell patched neuron. successful break-in attempt resulting in a graph in the autopatcher software GUI after a displayed in the ‘WHOLE CELL CURRENTS MONITOR’ point ‘ii’. ( Whole-cell configuration was obtained at time experimenter at The ‘ATTEMPT BREAK IN’ button was used by the attempt. A G software GUI during a successful gigasealing RESISTANCES MONITOR’ graph in the autopatcher measurements displayed in the ‘GIGASEALING ( has achieved the whole-cell configuration. attempt so that the user can assess whether it to injected voltage square waves after a break-in MONITOR’ graph displays the currents in response the ‘ZAP!’ button. The ‘WHOLE CELL CURRENTS can be attempted by applying voltage pulses using of selected time duration. Alternatively, break-in autopatcher to apply pulses of negative pressure ‘ATTEMPT BREAK IN’ button, which causes the formed, break-in can be attempted by using the patching for gigasealing used in and it allows exploration of alternate strategies negative pressure at any time during gigasealing, pressure application to manually apply additional over-ride the autopatcher algorithm’s negative OF SUCTION’ button allows the experimenter to of autopatching. The ‘MANUAL APPLICATION operation and return to the neuron-hunting stage the experimenter to over-ride the autopatcher The ‘RETURN TO NEURON HUNT’ button allows seal resistances during a gigasealing attempt. RESISTANCE MONITOR’ graph displays the recorded of the main PROCEDURE). The ‘GIGASEALING autopatching steps 4 and 5; Steps 34 and 35 and break-in stages of autopatching ( software GUI displayed during the gigasealing 4 in interface: gigasealing and break-in. ( Figure 7 646 (pA)’ report boxes. The current trace corresponding tothe injection of a10-mVvoltage square waveafter a break-in attempt b protocol

) Screen capture of seal resistance TROU TROU TROU

Figure

| Break-in. Confirming gigaseal formation. User inputsduringgigasealing User interventionifthepipettegetsclogged,fouled orbroken duringdescenttorecordingdepth VOL.11 NO.4VOL.11

25,3 . | B B B Click ‘OK’tostopthe autopatcher program. Restartanew trial from Step24.Thisstepisnot required ifthe auto c Autopatcher software graphical user ) Illustration of currents measured and LES LES LES 5 4 Ω . Once a successful gigaseal is , now showing the autopatcher seal was obtained at time point ‘i’. H H H t Press the ‘ATTEMPT BREAKIN’buttontobreak into the gigasealed neuron. The autopatcher applies ahigh = 105 s to break into the cell. OOT OOT OOT | 2016 I I I in vivo N N N G G G | natureprotocols and in vitro a Box ) Panel slice

( , Check thatasuccessful gigaseal hasbeenobtained ( Box

2 , autopatching step5).Ifacell-attached recording isdesired, click ‘NEXT’to a c b Box i 2 , autopatching step5(ii)). ( Box 2 , autopatching ii

© 2016 Nature America, Inc. All rights reserved. ? customized using the LabVIEWprogramming environment ifitisof interest (butthisisbeyond the scopeof thispaper). MultiClamp Commander toswitchmodes. The basic recording and stimulation capabilityof the software canbeextended and by switching the right toggleswitchwiththe ‘Current Clamp’and ‘Voltage Clamp’labels. Thiswillsend acommand to software GUIto‘Autopatcher Control’ and pressing ‘Record’; (The recording canalsobedone entirely inthe autopatcher software byswitching the lefttoggleswitchinthe autopatcher in Pak recording) may alsobecompensated using the amplifiercontrol software and the ‘Membrane Test’ procedure, as described At thisstage, pipetteseries resistance, pipettecapacitance and whole-cell capacitance (inthe caseof avoltage clamp record the cellparameters such asmembrane capacitance, accessresistance, membrane resistance and holding current. resistance compensation asneeded. Use the ‘Membrane test’function inthe data acquisition software (e.g., Clampex) to 38| ? Control’state. the‘External in ( required if states two these between switch to Control’toggle the‘External commercial use software; softwareor 37| (B) Whole-cellpatchclamprecording isobtained ( is shown in screenshot of the current trace plottedinthe ‘WHOLE CELLCURRENTS MONITOR’ graph afterasuccessful break-in attempt clamp the patched cellat−65mVof <200pA,which occursin>80%of successful gigaseals neuron, successful break-in isindicated byamembrane resistance <200M to option A.Ifawhole-cell patchclamprecording isobtained, proceed tooption B. Foratypical mouse cortical pyramidal is plottedinthe ‘WHOLE CELLCURRENTS MONITOR’ graph ( pipette back to the surface of the brain at a rate of 3 PIPETTE’ button, which will result in the autopatcher withdrawing the patch recorded cell, biocytin filling is attempted by pressing the ‘SLOWLY RETRACT and replacement) to start a new trial. To recover the morphology of the back to the surface in one quick step (lasting ~500 ms for pipette retrieval button ends the recording, and the autopatcher will retract the patch pipette and the ‘SAVE TO FILE’ button saves the file to disk. Pressing the ‘START OVER’graph indicator. The ‘REPEAT’ button can be used to acquire data continuously,measured voltage (if recording in current clamp mode) are displayed in the pressed, the measured currents (if recording in voltage clamp mode) or (in pA, if recording in current clamp mode). When the ‘RECORD’ button is voltage (in mV, if recording in voltage clamp mode) or holding current current clamp mode. A numerical entry box allows the user to set the holding a second toggle switch allows the user to switch between voltage clamp and software under ‘External Control’. If the ‘Autopatcher Control’ option is used, feature under ‘Autopatcher Control’ or to use an external data acquisition A toggle switch allows the user to use either the built-in data acquisition displayed during the recording stage after autopatching (Steps 37–39). ( Figure 8 a Fig. Fig. A

) Panel 4 in TROU TROU (ii) Proceed withStep37. (ii) ? ) Whole-cellpatchclamprecording isnotobtained (i) Click the ‘NEXT’buttoninthe autopatcher software GUI. (i)

TROU Assess cellquality. Record the whole-cell patched neuron. patched whole-cell the Record 8 Step 24. clamp the patched cellat−65mVof >−200pA),click ‘START OVER’tostopthe current trial and tostartanew trial from mouse cortical pyramidal neuron, byamembrane resistance <200M patch clamprecording isobtained, proceed toStep36B;otherwise, proceed tothe next step. 250-mV amplitude pulsefor 200ms whileapplying lowsuction pressure instead of high suction pulses. Ifwhole-cell (250-, 500-,750-and 1,000-ms durations). Alternatively, usethe ‘ZAP!’buttontoattemptbreak-in, which applies a If the gigasealed cellislostduring breaking in,orleakywhole-cell recording isobtained (indicated, for atypical We recommend thatyouattemptthe break-in process 1–5times, withincreasing negative pressure pulseduration et al.

). By default, the autopatcher holds the whole-cell patched cell in voltage clamp mode at −65 mV and starts andstarts mV mode −65 at clamp voltage in cell patched thewhole-cell holds default,theautopatcher By ). | B B Autopatcher software graphical user interface: recording. LES LES B LES Figure 2 Figure 7 H H 8 . The cellcanthen berecorded either involtage orcurrent clampusing the data acquisition software. OOT OOT H OOT 5 I I , now showing the autopatcher software GUI N N I G G c N . Use the MultiClamp Commander software controls toperform electrode compensation and access G µ Record in voltage or current clamp mode using either the autopatcher modetheautopatcher either using current clamp or voltage in Record m/s.

Fig. 7 Fig. c 8 ). Ifwhole-cell patchclamping isnot obtained, proceed .) The usercanswitchbetweencurrent and voltage clamp Ω Ω and current injection required tovoltage and current injection required tovoltage natureprotocols 2 4 . Arepresentative

| VOL.11 NO.4VOL.11 protocol

| 2016

647 © 2016 Nature America, Inc. All rights reserved. T Troubleshooting advice canbefound in ? preserve the morphology for postexperiment processing and reconstruction. resealing of the biocytin-filled cellaccompanied bythe formation of anoutside-out patchatthe pipettetip.Thiswill 150 button inthe autopatcher software GUItoretract the pipetteinstepsof 3 39| 648 a autopatcher) and denoise the the currents 30 (monitor S patching step 1) 32, protocol

TROU tep b

le le

| µ Pipette retractionfor recovering biocytinfilledcell. Box VOL.11 NO.4VOL.11 m, followed byafast retraction tothe surface of the brain ( 1 1 B | 2 LES

Troubleshooting table. (auto H OOT - | 2016 I (0.25–6 (0.25–6 Hz) frequencies modulated at low Current trace is level is >40 pA Peak-to-peak noise observed >100 pA/min are unstable and drifts Baseline current is P patching (3–9 M mal range for auto is not within the opti The pipette resistance N roblem G | natureprotocols

Ω

- ) - caused caused by improper air table Mechanical disturbance placement Improper ground wire Faraday cage noise sources inside the 50- or 60-Hz electrical the pipette holder Broken silver wire inside wire inside the pipette holder Poor conductivity of the silver Inadequate ACSF bath in the pipette Inadequate internal solution Bad ground electrode P program settings Suboptimal pipette pulling fixation owing to improper headplate Movement of the skull table is not rigidly attached to the flotation, or equipment that T able ossible reason 1 . Afterrecording from the cell,press the ‘SLOWLY RETRACT PIPETTE’

Fig. Ensure that both the ground electrode’s Ag-AgCl pellet and solution to submerge a portion of the Ag-AgCl wire Ensure that the pipette has been filled with enough internal their active surfaces wire. Older pellets also benefit from rechloriding to refresh that the Ag-AgCl pellet is soldered properly to the ground Ensure that the ground wire has proper connectivity, and S 5–7 5–7 M best results with pipettes with resistances in the range of in the range of 3–9 M Program the pipette puller to pull pipettes with resistances if the head-fixation is inadequate attempted if the dental acrylic has detached from the skull or the skull and the dental acrylic. Autopatching should not be stereomicroscope to observe any relative motion between tweezers, lightly touch the skull while looking through the mouse is head-fixed in the custom holder. Using a pair of and that there is no relative motion between them when the Ensure that the headplate is rigidly attached to the skull have been fastened rigidly to the air table pressurized air and that all components of the autopatcher Ensure that the vibration isolation air table is floating on is on the skull, surrounded by conductive saline checking using a multimeter. Ensure that the Ag-AgCl pellet the Ag-AgCl pellet is soldered properly to the ground wire, Ensure that the ground wire has proper connectivity and that as necessary loops are identified and eliminated. Add grounded shielding turned off or removed from the Faraday cage and all ground Ensure that all sources of 50- or 60-Hz noise have been on the pipette holder and the silver wire using a multimeter Check the electrical continuity between the gold-coated pin to correct this problem followed by thorough cleaning in distilled water, is sufficient properly chlorided. Soaking the wire in bleach for 3–5 min, Make sure that the silver wire in the pipette holder is the pipette tip are submerged in the ACSF bath olution 8 ). The initial slowretraction allowsthe gradual Ω µ . . Also, see m every1sfor uptoatotal distance of Figure Ω . . In our laboratory, we have obtained 3 for examples of pipette tips (continued)

Troubleshooting table (continued). (auto - - the descent to depth gets broken during of pipettes (>5%) A significant fraction localization regional pipette clogged during gets blocked or (>20%) of pipettes A large fraction >50 K pipette resistance is The variation in P >100 >100 K and exhibit variance are not flat-lined Resistance traces roblem Ω Ω

removed Dura has not been fully pressure Insufficient high positive Condition of the craniotomy Age of the mice pipette AgCl debris inside the from the electrode holder Particulates are introduced during backfilling of pipettes Particulates are introduced Particulates in the pipette P improper headplate fixation Movement of skull from in the craniotomy Bone fragments and debris autopatching pipette tip at the start of Improper positioning of the ossible reason

the electrode wire might get dislodged and clog the pipette When installing the pipette into the holder, the AgCl on followed by a wash in filtered deionized water it by washing it in 0.2- Remove the electrode holder from the headstage and clean using deionized water, ethanol and lens paper ance measurements during autopatching. Clean the Microfil may end up clogging the pipettes internally, affecting imped accumulation of particulates at the tip of the Microfil that Leaving the Microfil outside overnight may result in the ethanol solution when not in use during an experiment. container. Store the Microfil in filtered 70% (vol/vol) or lint, by storing the Microfil and syringe in an enclosed During an experiment, keep the Microfil away from dust Use a new filter and a new syringe for every experiment filtered with a 0.2- Always ensure that the pipette internal solution is S See See TROUBLESHOOTING guidance for Step 30 surface. Wash with ACSF to clean fragments that might have been introduced at the brain Periodically check the craniotomy for any debris or bone as soon as possible to prevent tissue drying stereomicroscope. Once the pipette is positioned, add ACSF difficult to visualize the pipette tip and the brain using the ACSF temporarily during this step, as refraction can make it of the craniotomy. For proper visualization, you can drain the craniotomy and that it is not touching the skull at the edges Ensure that the pipette tip is positioned at the center of the entry of the pipettes Check whether the dura has been removed at the point of structures below the cortex to keep the pipette tip clean if targeting deeper brain clogging persists. Such higher pressures can also be used autopatcher control box to 1,000 mBar if blocking or Increase the high positive pressure setting on the and that there are no debris or blood on the brain surface Ensure that the craniotomy is clean and moist at all times during the descent to depth, affecting the yield 12 weeks. Older mice may result in a higher rate of clogging Best results are obtained with mice aged between 8 and replacing it (vol/vol) ethanol. Make sure that the wire is fully dry before tip. Clean the electrode wire using lens paper soaked in 70% olution natureprotocols µ m m filter before backfilling the pipette. µ m-filtered m-filtered 70% (vol/vol) ethanol,

| VOL.11 NO.4VOL.11 protocol

(continued) | 2016

649

le le | VOL.11 NO.4VOL.11 1 1 Box |

Troubleshooting table (continued). 2

| 2016 gigaseals (<30%) of successful Low percentage <100 M positives or in seals result in false (>60%) attempts of neuron hunting Significant fraction P roblem | natureprotocols Ω

eliminate false positives 0.25 M threshold (M-Ohms)’ of Default ‘Neuron detection brain relative to the skull Excessive movement of the improper headplate fixation Movement of skull from pipette Internal clogging of the brain relative to the skull Excess movement of the P the craniotomy Suboptimal condition of brain relative to the skull Excessive movement of the fixation owing to improper headplate Movement of the skull ossible reason Ω is insufficient to

pad pad beneath the mouse, and the heating pad should allow the top of the skull should be ~25 mm from the heating breathing can increase. For 8-week-old C57BL/6 mice, if the animal is resting its weight on the chest area, labored of the animal. If the head is too low relative to the body or Labored breathing could also be caused by improper posture as well. The breathing rate should be faster than ~0.5 Hz anesthetic, is not good for the animal, and it causes motion properly regulated. Labored breathing, from too much throughout the experiment and its body temperature is that the animal is maintained at proper anesthetic plane pipettes in the autopatcher during autopatching. Also ensure way while performing the craniotomy or while installing the 18. Ensure that the brain surface is not damaged in any to the brain tissue while performing the craniotomy in Step is unsuitable for autopatching. This can be caused by damage eomicroscope because of heartbeat or breathing, the tissue If there is visible motion of the brain tissue through the ster S Step Step 18, or when pipettes are inserted during autopatching accidentally damaged while performing the craniotomy in the tissue healthy. Ensure that the brain surface is not of ACSF at all times during the experiment so as to keep Ensure that the brain surface is kept moist with application (autopatching Step 3) See TROUBLESHOOTING guidance for See TROUBLESHOOTING guidance for Step 30 GUI to a value between 0.3 and 0.75 M detection threshold (M-Ohms)’ in the autopatcher software TROUBLESHOOTING for Step 32). Increase the ‘Neuron of high variance in pipette resistance (as described in insufficient to eliminate false positives arising because study to be optimal at 0.25 M The neuron detection threshold was found in our original See TROUBLESHOOTING for See TROUBLESHOOTING for Step 30 See TROUBLESHOOTING guidance for Step 30 the spine for lung expansion without causing excessive motion of olution Box Ω . . However, this might be 2 (autopatching step 3) Box Ω to mitigate this 2 ,

(continued)

© 2016 Nature America, Inc. All rights reserved. PROCEDURE Steps 32–36). The time duration of recording from each whole-cell patched neuron (Steps 37–39) is determined is determined 37–39) neuron (Steps patched The time of duration whole-cell from 32–36). each recording Steps PROCEDURE ( complete to min 3–7 takes itself autopatching the whereas 25–31), (Steps min 2–3 takes ~5 min (Steps 21–24). Installation of the pipette in the autopatcher and positioning it at the craniotomy typically takes of at the the software beginning experiment the autopatcher initializing Once the is mouse ready 12–20). for autopatching, 7–11). h (Steps 3–4 takes software the configuring and installing up, set been have components hardware 5 and the Once 6). (Steps min ~30 takes holder the pipette and preparing the electrode ground fabricate were 3 to and Fabricating 4). d (Steps ~1 take components the headplates and all base fixation that head custom assuming The d, prefabricated. 2–3 were take boards circuit should that and it procured and 2), (Step step setup involved most the is box control The autopatcher min. 30–60 takes 1) (Step actuator the pipette assembling of the at time setup, procured been have nents The reader should budget an initial setup time for and installing integrating all of components the autopatcher. If all compo ● T by the specific experimental goals. experimental specific the by a 36B(ii) 36B(ii) S 37,38

tep Each experiment commences with the of commences Each experiment preparation surgical a mouse, takes min (Steps 45–60 to which typically complete b T le le IMI 1 1 N | G

Troubleshooting table (continued). P patching with 5 min of auto or become too leaky patched cells are lost (>10%) of whole-cell A significant fraction breaking-in process cells is lost during the (>20%) of gigasealed A significant fraction roblem - prepared improperly The pipette solution is the craniotomy Suboptimal condition of brain relative to the skull Excessive movement of the headplate fixation owing to improper Movement of the skull harsh on the cell Break-in process is too geometry Suboptimal pipette tip P ossible reason

until break-in is achieved using short zap pulses (25 pulse to electroporate the membrane to break in. Start by Commander software can be used to send a sharp voltage break-in. Alternatively, the ‘zap’ function in the MultiClamp of high negative pressure (−150 to −200 mBar) during in vacuum pressure might be too high. Use a smaller value If the cell is lost when you attempt to break in, the break- program by 1–2 points be achieved by increasing the velocity setting in the pulling For instance, in the Sutter P97 puller, this can sometimes (with corresponding increase in resistance of 0.5–1 M changes to the program to get pipettes with smaller tips diameter. Refer to the pipette puller user manual to make it may be indicative of an excessively large pipette tip increasing to 40–100 M If a substantial fraction of trials result in resistance S This This will increase the effective duration of the recordings ice throughout the experiment to preserve the ATP and GTP. close to 290 mOsm as possible. Maintain the solution on Ensure that the osmolarity of the pipette solution is as is not observed during the autopatching experiment are inserted during autopatching and that excessive bleeding while performing the craniotomy in Step 6, or when pipettes Ensure that the brain surface is not accidentally damaged the tissue healthy of ACSF at all times during the experiment so as to keep Ensure that the brain surface is kept moist with application Step 3) See TROUBLESHOOTING guidance for See TROUBLESHOOTING guidance for Step 30 olution natureprotocols Box Box Ω , , without forming a gigaseal, 2 µ , autopatching steps 1–5; main main 1–5; steps autopatching , s) s) and increase the zap durations Box

| VOL.11 NO.4VOL.11 2 (autopatching protocol | 2016

Ω

). ).

651

- © 2016 Nature America, Inc. All rights reserved. from Chuong Medicine Committee on Animal Care). ( regulations (MIT Department of Comparative use complied with institutionaland governmental in response to 20-ms blue light pulses. All animal in a Thy1-ChR2 mouse, showing evoked spiking ( Reproduced with permission from ref hyperpolarization at onset of red light delivery. ( recording and optogenetic stimulation of the brain. ( 88 M bar). Access resistance, 37 M (periods of whisker movements indicated by black of membrane potential during active whisking fixed mouse showing persistent depolarization cortical neuron in barrel cortex of an awake head- fixed mice. Current clamp recording from a layer-4 recorded in ( fill of the autopatched cortical pyramidal neuron 544 23 M indicated by a black bar in ( synaptic event indicated by black arrow in potentials (EPSCs) ( showing spontaneous excitatory postsynaptic autopatched cortical neuron clamped at −80 mV the brain. ( M Access resistance, 44 M +90, +120, +150 and +180 pA current injection). injection (2-s-long pulses of −30, 0, +30, +60, autopatched cortical neuron during current autopatcher. ( Figure 9 patch clamping byskilledmanual The quality of neuronal recordings obtained withthe autopatcher iscomparable tothatobtained via manual whole-cell Quality of recordings and potentially to enable parallel computer control of multiple autopatchers with a substantial increase in throughputthat can automatically perform craniotomies duration, which can vary depending on the experiment. There is ongoing work to combine the autopatcher with timetechnologies taken to run each trial (3–7 min, including the time taken to install the pipette in the setup) and the desiredrion recording of resting membrane potential <−50 mV and spike amplitude >35mV. The throughput of the autopatcherpatched, is governed the cells by thecan be held for an average of ~45 min, with the longest recordings lasting up to 3 h, using a qualitytopatching crite in the more dorsal regions before targeting ventral regions in the pipette descent path. Onceattempts the made cells are in a autoparticular craniotomy. One strategy to overcome this (if allowed by the experiment) is to attemptpatching au declines with the time that the brain is exposed after the craniotomy is made, as well as with the numberor at ofdifferent patch depths or in different species may require different pipette geometries and software settings.our experience The yield of best autoresults were obtained using pipettes with resistances between 5 and 7 M accept pipettes with resistances between 3 and 9 M of the surgical preparation of the mouse and the quality of the craniotomy. Although the autopatcheropening has a beenfresh configured craniotomy. to Variability in yield might be observed owing to variations in the quality ofage pipettes, ( the quality experimental conditions (as described here), one can expect successful whole-cell recordings in ~32.9% of the recordedtrials, on neuronsaver can be filled with biocytin for morphological analysis ( The autopatcher can be used to obtain high-quality current and voltage clamp data from anesthetized mice ( T ANT 652 h g c hroughput andyield protocol Ω ), and zooming in on a synaptic barrage event ) Jaws-expressing neuron in the cortex showing ) Channelrhodopsin-2–expressing cortical neuron ; depth of cell 442

µ Ω Ω | I m from the surface of the brain. ( VOL.11 NO.4VOL.11 n C ; depth of cell, 468 ; input resistance, 124 M = 24 out of 73 trials) I

PATE | Example data acquired by the b a – et al. ). ( g d a , D ) Voltage clamp recordings from an ) Current clamp recording from h f ) Simultaneous whole-cell ) Autopatching in awake head- 2 RESULTS 6 b . | ), zooming in on a single 2016 µ Ω m below the surface of ; input resistance, 66 µ b m below the surface | ( Ω natureprotocols d ; input resistance, ). Access resistance, Ω ; depth of cell, 2 . 26. 4 , with >60% of the trials yielding good recordings for attempts made within the first hour after in vivo. g e ) Adapted ) Biocytin

in vivo

patchclamppractitioners. Inouroriginal study 2 e a g 8 –60 mV 400 ms –68 mV , change pipettes (to enable serial autopatching without human intervention), 1 s

20 mV Ω

10 mV , which is the generally acceptable range for patch pipette resistances, in 50 µ m Fig. 9 1 f s e b c

20 mV ). As described previously, under optimized 2.5 s 100 pA –300 pA 1 2 s 4 , wecompared the autopatched 20 ms

10 mV Ω . Patching different neuron types h d Fig. 9a 200 ms – d 2 ), and 9 . -

- - -

- © 2016 Nature America, Inc. All rights reserved. of Mechanical Engineering. E.S.B. acknowledges NIH 1R01EY023173, the New York the Technology Fee Fund, Invention Studio, and the George W. Woodruff School through the Institute for Bioengineering and Biosciences Junior Faculty Award, Translational/Clinical Research Grant Program and support from Georgia Tech Innovation in Research and Education (GT-FIRE), the Wallace H. Coulter & Science (TRIBES) Seed Grant Awards Program, the Georgia Tech Fund for the Georgia Tech Translational Research Institute for Biomedical Engineering an NIH Computational Neuroscience Training grant (no. 5T90DA032466), 0965945 and Computer and Information Science and Engineering (CISE) 1110947), National Science Foundation (NSF) (Education and Human Resources (Her) an (NIMH) 1-U01-MH106027-01), NIH Single Cell Grant 1 R01 EY023173, the Initiative (National Eye Institute (NEI) and National Institute of Mental Health manuscript. C.R.F. acknowledges the National Institutes of Health (NIH) BRAIN A online of version the pape Note: Any and Information Source Data Supplementary files are available in the spherical virtual reality treadmill recordings inawake head-fixed animals ( has beentried and testedinanesthetized mice. We havealsosuccessfully usedthe autopatcher toobtainwhole-cell patch ing, pharmacological infusion and other neuroscience strategies of importance. As afinal note, the protocol described above to the autopatcher setup spikes from neurons expressing channelrhodopsin-2 inaThy1-ChR2 transgenic mouse hyperpolarization of motor cortex neurons expressing the red-shifted optogenetic silencer Jaws lowered into the brain using the autopatcher toobtainwhole-cell patchrecordings and subsequently measured photo-evoked such thatthe tipof the fiberwas600µmlateral from and 500±50µmabove the tip of the electrode tip.Thisassemblywas optogenetic stimulation and autopatching. Inaseries of experiments, a200-µmoptical fiberwasattached tothe pipette brain regions. The algorithm isrobust enough toallowusage of optic fiberswiththe patchelectrode for simultaneous also usefulfor obtaining recordings inthe hippocampus—exhibiting adegree of generalizability of the algorithm across The algorithm usedfor autopatching wasderived inexperiments conducted inthe cortex of anesthetized mice, butitwas Generalizability of theautopatchingalgorithm structures ( were morphologically identified, 70% of neurons autopatched inthe cortex were pyramidal cells exhibiting apical dendritic staining of fine dendritic arbors, which are absent inthe ghost-stained neurons thatare found nearby. Ofthe cellsthat we were abletorecover the morphologies of ~75%of the recorded cellsbasedonthe intensity of staining, aswell in the brain slice containing the recorded neuron, and shortening the duration of recording (10–15min).Inourexperiments, of ‘ghost-stained’ (e.g., stained withbackground levelsof biocytin ejectedduring the approach of the patchpipette)cells following: configuring the autopatcher pipetteactuator atanangle (45 optimization strategies toobtainhigh-quality morphological reconstructions of autopatched neurons. These include the characterization of the recorded cellsvia standard immunohistochemical staining protocols. We developed several that avariety of celltypescanbepatch-clamped characteristics, weconcluded thatthese were glial cells. Although they are not comprehensive, these data do illustrate depolarized to−30mVvia steadilyincreasing current injections. Along withthisobservation and their lowcellcapacitance fired, typical of fast adapting neurons. 9% of the recorded cellslacked spiking activitywhen the membrane potential was of the neurons, steady (2sinduration) current injections overthe firing threshold resulted insingle action potentials suggestive of backpropagation of action potentials indendritic recordings and 2%hadspike firing thataccelerated. In9% burst firing patterns, 13% exhibited irregular spike characteristics, 4% exhibited spikes followed by smaller spikelet events hippocampus inamouse (91% of allcellspatched), typically 68%exhibited regular spiking characteristics, 4%exhibited In ouroriginal study C ard In allthese comparisons, no significant difference wasobservedbetweenautopatched neurons and those obtained via stand holding current required tovoltage-clamp neurons at−65mV, the resting membrane potential and the recording time. recordings withthose obtained via fullymanual patchclamping using commonly usedmetrics: the accessresistance, the 1DP1NS087724, an 1DP1NS087724, NIH Director’s Transformative Award (NIH 1R01MH103910) Stem Cell Foundation-Robertson Award, a NIH Director’s Pioneer Award ckno ell typesandmorphologiesof autopatchedneurons Including 0.4%(wt/vol)biocytin inthe internal pipettesolution whileautopatching enabled morphological in vivo w le dgm patchclamping protocols. These metrics may serveaspositivecontrols for success of the autopatching procedure. Fig. ents

9 We thank B.D. Allen and H.-J. Suk for feedback on the e r ), similartoobservations made inother studies . 2 4 , weanalyzed the celltypesobtained bythe autopatcher. Ofthe neurons recorded inthe cortex and 3 1 may enable the integration of the autopatcher into experiments involving extracellular record 14,3 3 . Fig. 9 f ), bothinafullyimmobilized setting in vivo

using the autopatcher.

2. 1. com/reprints/index.htm at online available is information permissions and Reprints are in available the details interests: CO G.L.H., A.C.S., G.T.F., C.R.F. and E.S.B. wrote the manuscript. M.L.M. developed the software included with the manuscript. S.B.K., I.R.W., and tested the autopatcher system. A.C.S. and G.T.F. assisted with experiments. AUT Friends of the McGovern Institute Fellowship. and an NIH BRAIN initiative grant (NIH G.T.F.1R24MH106075). acknowledges a

6 M . H PET 977–980 (2008). 977–980 representationsensory to synapses single of synapses. thalamocortical active Arenz, A., Silver, R.A., Schaefer, A.T. & Margrie,T.W.contribution The& A.T.Schaefer, R.A., Silver, A., Arenz, synchronously but weak by driven is Cortex B. Sakmann, & R.M. Bruno, OR I

N CONTR ° G G FI tothe vertical axis)soastominimizethe number NANC IB UT I I AL ONS l I . NTERESTS

S.B.K., I.R.W., G.L.H., C.R.F. and E.S.B. designed, built 11,3 3 0 natureprotocols ( 2 Fig. 9 and inhead-fixed animals ona

online online version of pape the The authors declare competing financial declare financial competing The authors Science h 2 ). Coupling of other devices 6 ( Fig. 9

312 , 1622–1627 (2006). 1622–1627 , in vivo in

| VOL.11 NO.4VOL.11 g ) and photo-evoked . Science protocol r http://www.nature.

321 | 2016 ,

- |

653

targeted patching (TPTP) patching targeted neurons unlabeled of electroporation single-cell and recordings patch-clamp system. olfactory cortex.unanesthetizedauditory the in 461 navigation. virtual during cells place hippocampal of dynamics GABAergicneurons mousein barrel cortex. mice. behaving of cortex barrel the in GABAergicneurons of dynamicspotential Membrane touch. active of coding sparse underlying (2006). 608–610 mice. awake of cortex barrel in potentialmembrane processing. sensory during cells granule 29 cortex. auditory in location source sound ofrepresentation brain. mammalian awake and anaesthetized the in neurons from recordings (2011). 527–532 connectomics. andphysiology, geneticssingle-cell USA Sci. Acad. Natl. Proc. (2004). cerebellargranule cellsduring sensory processing. 427 cortex. motor rat in cells pyramidal single of stimulation by evoked Komai, S., Denk, W., Osten, P., Brecht, M. & Margrie,T.W.Two-photon& M. P.,Brecht, W.,Osten, Denk, S., Komai, TargetedHausser,M. W.& Denk, M., Kano, B., Judkewitz, K., Kitamura, the Margrie,T.W.representationsin Spatiotemporal& A.T. Schaefer, soundsrepresentation ofSparse Zador, A.M. & Hromadka,T.,DeWeese, M.R. D.W.IntracellularTank, & D.A. Dombeck, F.,Collman, Harvey,C.D., Gentet,L.J. C.C. Petersen, Staiger,& F.,J.F.Matyas, M., Avermann, L.J., Gentet, mechanisms Synaptic C.C. Petersen, Kremer,& J.F.,Y.Poulet, S., Crochet, with behavior whisker Correlating C.C.H. Petersen, & S. Crochet, Chadderton,P.,Margrie, T.W., cerebellar Hausser,Integrationquantain ofM Margrie,T.W.synaptic The& D. J.P.,McAlpine,P.,Agapiou, Chadderton, B. Sakmann, & Margrie,T.W.,M. Brecht, E.A. Rancz, J. Eberwine, Chadderton,P., Margrie, T.W. Hausser,& IntegrationM. quantaof in Margrie,T.W.movements Whisker& B. Sakmann, Schneider,M., M., Brecht, | VOL.11 NO.4VOL.11 , 14127–14135 (2009). 14127–14135 , , 941–946 (2009). 941–946 , (2004). 704–710 , Pflugers Arch. Pflugers in vivo in et al. et et al. et et al. et . Neuron | Nat. Methods Nat. 2016 Unique functional properties ofsomatostatin-expressing Transfection via whole-cell recording whole-cell Transfectionvia Trends Neurosci. Trends Analysis of gene expression in single live neurons. live single in expressiongene of Analysis

444

65 |

, 491–498 (2002). 491–498 , natureprotocols

in vivo in , 422–435 (2010). 422–435 , 89 , 3010–3014 (1992). 3010–3014 ,

5 , 61–67 (2008). 61–67 , .

Nat. Protoc. Nat. 30 , 92–100 (2007). 92–100 , PLoS Biol. PLoS Nature Nat.Neurosci. In vivo In Neuron

, low-resistance, whole-celllow-resistance, , 428 , 647–652 (2006). 647–652 ,

Nature

6 69 , 124–137 (2008). 124–137 , Nat. Neurosci. Nat. , 856–860 (2004). 856–860 , Nat. Neurosci. Nat. , 1160–1175 (2011). 1160–1175 ,

in vivo in 428 , 607–612, (2012). , 856–860, J. Neurosci. J. : bridging : Nature

9 , , Nature

30. 29. 28. 27. 26. 25. 24. 23. 22. 21. 20. 19. 34. 33. 32. 31.

Ann. NY Acad. Sci. Acad. NY Ann. fields. biological intact-system other andneuroscience of automation the Neurophysiol. J. (2014). e97310 cells. glial and neurons cortical of responses visual and for method zap and rhodopsin.microbial (2007). 6.22.1–6.22.15 Methods Nat. neurons of electrophysiology patch-clamp whole-cell Automated generation.sequence neuronal (2012). 849–853 behavior.during excitability of manipulation single-cell rats. moving freely rats. moving freely in vivo in (Humana Press, 1995). Press,(Humana W.)233–258 Walz, & Baker,G. A., Boulton, (eds. 26 Vol. Protocols. (2013). 1331–1339 cortex. visual in modulation gain state-dependent states. cortical of control (2014). doi:10.1152/jn.00629.2014 recording. patch-clamp whole-cell automated (2007). channelrhodopsin-2.expressing micetransgenic in Arenkiel, B.R. Arenkiel, C.R. Forest, & E.S. Boyden,Kodandaramaiah, S.B., N. Pak, touch The L.J. Graham, & Gener,T. D., Marinazzo, A.E., Schramm, A.S. Chuong, recording Whole-cell M.R. DeWeese, Kodandaramaiah,S.B.,Franzesi,G.T., Chow, Forest,C.R.B.Y., & Boyden,E.S. of model chain synaptic a for Support M.S. Fee, & D.Z. Jin, M.A., Long, upon emerge fields place Hippocampal A.K. Lee, & B.-J. Lin, D., Lee, in recordings Whole-cell M. Brecht, & B. Sakmann, I.D., Manns, A.K., Lee, recordings whole-cellHead-anchored M. Brecht, & J. Epsztein, A.K., Lee, Margrie,T.W. Plant, T.D., Eilers, J. & Konnerth, A. in Patch-Clamp Applications and ApplicationsPatch-Clamp in A. Konnerth, & J. Eilers,T.D., Plant, brain of mechanisms P.Cellular Golshani, & P.O.,J. Polack,Friedman, Thalamic C.C.H. Petersen, & S. Crochet, L.M.J., Fernandez,J.F.A., Poulet, R.R. Harrison, . et al. et Neuron

Closed-loop, ultraprecise, automated craniotomies.automated ultraprecise, Closed-loop, et al. et et al. et 9 et al. et et al. et

, 585–587 (2012). 585–587 ,

39 113

, 911–918 (2003). 911–918 , Targeted whole-cell recordings in the mammalian brain mammalian the in recordings Targetedwhole-cell Noninvasive optical inhibition with a red-shifted a with inhibition optical Noninvasive 1305 Neuron

Microchip amplifier for amplifier Microchip , 3943–3953 (2015). 3943–3953 , in vivo in In vivo In Nat. Neurosci. Nat. Nat. Protoc. Nat. Box 2 Box , 63–71 (2013). 63–71 , Nat. Neurosci. Nat.

whole-cell patch recording of intrinsic of recording patch whole-cell 51 light-induced activation of neural circuitry neural of light-inducedactivation | , 399–407 (2006). 399–407 , Autopatching Nature

4 17 in vivo in , 385–392 (2009). 385–392 ,

, 1123–1129 (2014). 1123–1129 , 468

15 , 394–399 (2010). 394–399 , . , 370–372 (2012). 370–372 , in vitro in Curr. Protoc. Neurosci. Protoc.Curr. Nat. Neurosci. Nat. In vivo In , Neuron in vivo in Science PLoS ONE PLoS robotics:

54 , and , , 205–218 ,

337

in vivo in

9 38

, , ,

SUPPLEMENTARY METHODS

Saline bath testing prior to in vivo experiments

Once the autopatcher equipment has been set up (I. HARDWARE SETUP, Steps 1-6 in the main protocol), the steps below can be performed using a saline bath as a proxy for the intact brain to ensure the autopatcher hardware and software are functioning properly before attempting in vivo experiments.

Saline bath test step 1: Follow Steps 21-24 in the main protocol and initialize the software programs for autopatching. The computer screen appears as shown at the beginning off Supplementary Video 1 (0 – 60 seconds).

Saline bath test step 2: Place a beaker containing 0.9% saline solution underneath the amplifier headstage in place of the head fixation apparatus.

Saline bath test step 3: Using a “helping hand” or other holder, place the Ag-AgCl pellet of the ground electrode inside the saline solution. Plug the gold coated pin at the other end of the ground electrode to the back of the amplifier headstage (Step 25).

Saline bath test step 4: Follow Step 27 in the protocol and fill a recently pulled patch pipette with freshly thawed internal pipette solution and install it in the pipette holder. The Ag-AgCl electrode in the pipette holder should be immersed in solution contained inside the pipette.

Saline bath test step 5: Follow Step 28 in the main protocol and set the pressure states in the autopatcher control box’s front panel.

Saline bath test step 6: Lower the pipette using the micromanipulator until the pipette tip is immersed in the saline bath.

Saline bath test step 7: Use the ‘Check resistance’ function in the amplifier control software to measure the pipette resistance.

TROUBLESHOOTING: Optimal pipette resistance for autopatching should be 5-7 MΩ. If the pipettes do not have the suitable pipette resistance, follow instructions in Box 1 to program the pipette puller to pull pipettes suitable for autopatching. If needed, repeat Saline bath test steps 1-7 until a program for pulling suitable patch pipettes has been derived before proceeding to the next step.

Saline bath test step 8: Follow instructions in Step 30 of the main protocol and de-noise the setup.

TROUBLESHOOTING: Refer to the troubleshooting section accompanying Step 30 in the main protocol.

PAUSE POINT: Ensure that the peak to peak noise amplitude in voltage clamp is less than 40 pA before proceeding to the next step.

Nature Protocols: doi:10.1038/nprot.2016.007

Saline bath test step 9: Install a manometer inline between the autopatcher control box and the pipette holder to monitor the pressure applied to the pipette during autopatcher operation.

Saline bath test step 10: Check the pressure delivered to the pipette.

TROUBLESHOOTING: The inline manometer installed in Saline bath test step 9 as well as the manometer on the front panel of the autopatcher control box should indicate high positive pressure (800 mBar). If you are unable to set the high positive pressure, it is indicative of a leak. Check the tubing between the autopatcher control box and the pipette holder, as well as the pipette holder for leaks.

Saline bath test step 10: Following Step 31 in the main protocol, Click ‘OK’ in the autopatcher software GUI. The autopatcher will commence the steps performed during automated patch clamping. It will perform an ‘Initial pipette assessment’ (Step 32 in the main protocol) and update the ‘Initial pipette resistance assessment’ indicators in the software GUI.

TROUBLESHOOTING: The variation in pipette resistance should not be greater than 100 KΩ. If the variation is higher than this value, it is indicative of internal clogging of the pipette. Refer to troubleshooting section accompanying Step 32 in the main protocol.

Saline bath test step 11: After 20 seconds, the autopatcher will perform the ‘Regional pipette localization’ (Step 33 in the main protocol) and lowers the pipette in the saline bath by the depth specified during the initial autopatcher setup (Saline bath test step 1).

TROUBLESHOOTING: If the motor does not move, the motor is not communicating with the software. Refer to the configuration manual in Supplementary Data 1 - ‘Autopatcher software.zip’ for instructions to correctly configure the autopatcher software.

Saline bath test step 12: After lowering the pipette, the autopatcher will switch the pressure applied to the pipette to low positive pressure (25 mBar) and this should be indicated by the inline manometer.

Saline bath test step 13: The autopatcher will now commence ‘Neuron Hunting’ (Step 34 in the main protocol). As this is being performed in a saline bath, no ‘neuron detection’ will occur and the autopatcher will lower the pipette in increments of 2 µm until it has reached the depth specified in the ‘Pipette depth to stop neuron hunting (micrometers)’ entry box in Saline bath test step 1, and then retract the pipette back to the point where the saline bath test was initiated.

TROUBLESHOOTING: As the pipette is moving through a saline solution, very little (<100 KΩ) variation in pipette resistances should be seen in the ‘NEURON HUNT RESISTANCE MONITOR’ graph. If the observed variation is higher than 100 KΩ, it is indicative of internal clogging of the pipette. Refer to suggestion 3 in the troubleshooting section accompanying Step 34 of the main protocol.

Nature Protocols: doi:10.1038/nprot.2016.007 Saline bath test step 14: As no ‘neuron detection’ occurs in the saline bath testing, the experimenter can force the autopatcher to execute the gigasealing protocol by pressing the ‘SKIP TO GIGASEALING’ button in the autopatcher software GUI before the pipette reaches the depth specified in the ‘Pipette depth to stop neuron hunting (micrometers)’ entry box in Saline bath test step 1. The autopatcher will then commence gigasealing (Step 34 of the main protocol). 10 seconds after gigasealing commences, the autopatcher will release positive pressure and the inline manometer installed in Saline bath test step 9 and the pressure gauge in the software should indicate 0 mBar of pressure. Between 15-25 seconds later, the autopatcher applies low negative suction pressure and the manometer should indicate -15 mBar for several seconds.

Saline bath test step 15: Press the ‘START OVER’ button. The autopatcher will stop performing the gigasealing protocol and pull back the pipette to the position at which it commenced the saline bath test in Saline bath test step 10.

Nature Protocols: doi:10.1038/nprot.2016.007 Assembling the Control Box

This chapter contains step-by-step instructions to assemble the autopatcher control box. After all the parts are sourced, the box can be assembled in about 3 days. Alternatively, the box can be purchased fully assembled from Neuromatic Devices Inc.

Assembly

Tools Needed

1. Antistatic mat, grounded (e.g., Digikey ‪SCP225-ND) ‪‪‬‬‬‬‬‬‬‬‬‬‬‬‬‬ 2. Antistatic wrist strap, grounded (e.g., Digikey ‪SCP172-ND)‬ ‪‪‬‬‬‬‬‬‬‬‬‬‬‬‬‬ 3. Soldering iron & solder (e.g., Mouser 578-WES51) 4. “Helping hands” for clamping components to be soldered (e.g., Sparkfun TOL-11784) 5. Wire cutters (“snips”) (for cutting wire and cable ties) (e.g., Digikey 170M-ND) 6. Hex key (a.k.a. Allen wrench), for ¼”-20 hex-head screws (e.g., McMaster Carr 5709A31) 7. Torx screwdrivers, sizes T25 and T30 (e.g., McMaster Carr 5756A45) 8. Flathead screwdrivers, assorted sizes (e.g., McMaster Carr 7020A51) 9. Wire strippers (e.g., Digikey JIC-2030-ND) 10. Adjustable wrenches, small (2) (e.g., McMaster 5385A11 and 5385A12) 11. Multimeter (e.g., Digikey 705-1051-ND) ‬‬‬‬‬‬‬‬‬ 12. Handheld manometer (‪Dwyer 475-5-FM),‬ for testing finished box ‪‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬ 13. Scissors (for cutting Teflon tape and 1/8” o.d. tubing) 14. Razor knife (for cutting ¼” and larger o.d. tubing) 15. Tape measure

Preassembly Preparation

1. Order parts in the parts list file “Autopatcher parts list.xlsx”. More detail about ordering the circuit boards and laser cutting the mounting plates can be found below.

2. Have printed circuit boards made and assembled.

Figure S3.1 – The pressure regulator board is on the left and two valve switching boards are on the right.

Figure S3.1 shows the assembled printed circuit boards. The Gerber files included with this supplementary material can be sent to a PCB fabrication house such as pcbfabexpress.com for production of the boards themselves. These boards and the required components can then be soldered together by hand or sent to an assembly house such as pcacorporation.com for assembly. Two lists of the components used to populate the boards are included with the Gerber files and are called “pressure_board_2012-10-11_04_bom.xlsx” and “valve-relay_board_2012-11-16__04_bom.xlsx”. You will need one pressure board and two valve boards. Whenever handling the boards or their components, observe antistatic precautions by working on a grounded antistatic mat and wearing an antistatic wrist strap that is grounded to the antistatic mat. Failure to do so may result in destruction of some of the components.

3. Laser cut front and rear panels and internal mounting plates.

Figure S3.2 shows the acrylic plates used to mount the internal components of the autopatcher.

Figure S3.2 – The acrylic plates used to mount the autopatcher components inside the metal box.

Use the “.ai” files included with this supplementary material called “Autopatcher panels front & back.ai” and “Autopatcher structural base, platform & supports.ai”. The blue lines in these files should be etched and the black lines should be cut. The “.ai” files are shown in Figure S3.3. They can be sent to customlasercutting.com or pololu.com to be fabricated. The front and back panels are 1/8” black acrylic and the structural plates should be made from transparent 1/4" acrylic, customlasercutting.com keeps both materials in stock so you don’t have to order the acrylic in the parts list which will reduce the lead time.

Figure S3.3 – Renderings of the design files used to make the mounting base plate and mounting platform.

4. Solder wires to BNC relay.

Place the BNC relay (Tohtsu CX-230) flat on the bench with the label down as shown in Figure S3.4 Strip the end of an 8” length of red 24 gauge wire and solder it to the tab that is on top. Strip the end of an 8” length of black wire and solder it to the tab that is on the bottom as shown in the figure.

Figure S3.4 – BNC Relay with wires soldered to coil terminals.

5. Solder wires to unmarked switches.

Strip the ends of two 6.5” lengths of orange 24 gauge wire and two 16” lengths of yellow 24 gauge wire. Solder one end of one of the orange wires to the tab that is nearest the end of one of the unmarked rocker switches (CH758-ND), do not use the power switch (CH755-ND). Solder one end of one of the yellow wires to the other tab of the same switch as shown in Figure S3.5. Repeat for the second unmarked switch.

Figure S3.5 – Photograph of where to solder the yellow and orange wires to the unmarked rocker switch.

6. Solder wires to power switch and barrel connector.

Cut an 8” length and a 22” length of red 18 gauge wire and a 26” length of black 18 gauge wire. Strip one end of the black wire and solder it to the middle tab of the barrel connector (SC1049-ND) as shown in Figure S3.6. Strip one end of the 22” red wire and solder it to the tab of the barrel connector that is above the middle tab when the middle tab is oriented to the left (see Figure S3.7). Strip the other end of the 22” red wire and solder it to the tab on the power switch (CH755-ND) that is closest to the edge. Strip the end of the 8” red wire and solder it to the other tab as shown in Figure S3.7.

Figure S3.6 – Photograph showing where to solder the 18 gauge, red and black wires on the barrel connector.

Figure S3.7 – Photograph showing where to solder the orange wires onto the marked power switch.

7. Test the potentiometers.

Using the multimeter, test the potentiometers before soldering them. The resistance between tabs 1 and 3 (see Figure S3.8 and the data sheet for the potentiometers) should measure approximately 10 KOhms. Turning the potentiometer shaft counterclockwise to the maximum extent should cause the resistance between tabs 1 and 2 to drop to approximately zero (~50 Ohms) and the resistance between tabs 2 and 3 to increase to approximately 10 KOhms. Turning the shaft all the way clockwise to the maximum extent should have the opposite effect (resistance between tabs 1 and 2 should increase to approximately 10 KOhms, resistance between tabs 2 and 3 should increase to approximately 50 Ohms).

Figure S3.8 – Photograph showing where to solder the yellow, orange, and black wires onto the potentiometers. It also shows the numbered tabs which are used to test it.

8. Solder wires to potentiometers.

Cut three 8” lengths each of orange, yellow, and black 24 gauge wire. Because overheating can damage the potentiometers, it is important to apply only as much heat to the potentiometer tabs as is necessary to melt the solder onto them. To this end, strip a generous length (~1”) from one end of each of the wires. See Figure S3.8 for the arrangement of wires on each potentiometer; a yellow wire is to be connected to tab 2 (which is the tab that is offset with respect to the other two), a black one to tab 1, and an orange one to tab 3. Insert each stripped end halfway through the appropriate potentiometer tab and twist back onto the rest of the stripped end, so that ~½” of twisted wire is available. This allows the soldering to begin well up on each wire, far from the potentiometer tab, with heat applied to the tab only once the wire is thoroughly heated and “tinned” with solder, so that the tab is heated only for a second or two to allow a bridge of solder to form between the tab and wire. After soldering each potentiometer, retest its function as was done in the previous step to ensure that it was not damaged by the soldering. If any potentiometer is found not to function properly, procure a new one and try again more carefully.

Base plate Assembly

1. Attach feet to the bottom panel.

Wipe the bottom face of the bottom panel with 70% ethanol to remove oil and wipe dry. Attach four rubber feet to the bottom panel, with their centers roughly 2” from the left or right side edge and 2.5” from the front or back edge. These locations correspond to the locations of the base plate support legs that will stand on the back panel. Note that the top and bottom panels of the chassis are identical. Refer to Figure S3.9 for the proper locations.

Figure S3.9 - Photograph showing the location of the rubber feet that support the control box. They are applied to the bottom panel of the rack mount enclosure.

2. Initial chassis assembly.

Attach the side panels of the chassis to the bottom panel of the chassis, using four of the screws included with the chassis kit. If using the laser-cut acrylic front and rear panels (as is recommended), the metal front and back panels that come with the chassis kit can be discarded. Do not attach the front, back, or top panels at this stage as shown in Figure S3.10.

Figure S3.10 – Photograph showing the rack mount enclosure partially assembled. The front, back, and top of the box will be assembled in a later step.

3. Add the base plate support legs.

Attach the four ¼” hex (1” length) standoffs to the bottom of the base plate, using ¼”-20 (¾” length) screws through the four corner holes as shown in Figure S3.11. All of the other components to be attached to the base plate will be attached to its top face.

Figure S3.11 – Photograph of the base plate with the vertical standoffs mounted underneath.

4. Add all vertical standoffs to the tops of base plate and platform.

Attach the four 3”-long hex standoffs to the top of the base plate, using 10-32 (5/8” length) screws through the four holes flanking the outline of the USB-6211 DAQ board on the base plate. Attach eight of the twelve ¼“ o.d., 5/8”-long standoffs to the top of the base plate, using 4-40 (7/16” length) screws through the holes at the corners of the PC board outlines (marked “Pipette Valve Board” and “Manometer Valve Board”) on the base plate as shown in Figure S3.12. Similarly, attach the remaining four ¼“ o.d., 5/8”-long standoffs to the top of the platform plate using 4-40 5/16”-long screws through the holes at the corners of the PC board outline (marked “Pressure board”) as shown in Figure S3.13. Set the platform aside for now.

Figure S3.12 – Photograph of the base plate with the vertical standoffs mounted to the top.

Figure S3.13 – Photograph of the vertical standoffs mounted to the top of the mounting platform.

5. Add the valve support standoffs. Strap the two ¼” hex (1.5” length) standoffs to the top of the base plate, lying down on their sides, using 8” black cable ties running through the length of each one and through the two holes flanking each of the rectangular outlines at the ends of the outlines of the groups of valves as shown in Figure S3.14. Once cable ties are tightened, cut off the excess length using wire cutters.

Figure S3.14 – Photograph of the valve support standoff on the base plate. There are two places where these standoffs need to be added. One standoff should go by each valve control circuit board.

6. Assemble the DAQ board.

Plug in two connector blocks (included with the board) into the USB-6211 and affix the included pinout stickers to them. For consistency with this assembly manual, use the “Single-Ended Signal Name Labels”, which denote pin 1 “PFI0 in” and so on as shown in Figure S3.15.

Figure S3.15 – Photograph of the assembled connectors on the DAQ board.

7. Add the DAQ board.

Strap the USB-6211 to the top of the base plate using two 11” black cable ties daisychained together. Once cable ties are tightened, cut off the excess length using wire cutters. Affix the tab at the end of the DAQ board to the base plate using one of the 10-32 (5/8” length) screws through the hole in the tab and through the hole in the base plate, with one of the 10-32 nuts underneath the base plate to secure the screw. Figure S3.16 shows how the DAQ should be assembled to the base plate.

Figure S3.16 – Photograph of the DAQ attached to the base plate.

8. Assemble the venturi vacuum generators.

Add two adapters to each vacuum generator (Air-Vac “pump”): one 1/8" Tube OD X NPT Female adapter (McMaster 5779K123) to the end and one 1/8" Tube OD X 1/8" NPT Male adapter (McMaster 5779K102) to the top port. Wrap the threads using Teflon tape to prevent leaks. Tighten with adjustable wrench until snug as shown in Figure S3.17.

Figure S3.17 – Photograph showing how to assemble the vacuum generators. The one on the left is assembled, the one on the right shows how to apply the Teflon to the adapters before tightening them on.

9. Add the vacuum generators.

Strap the vacuum generators to the base plate, with the top port (with the NPT Male adapter) pointing upward, and the NPT Female adapter pointing toward the edge of the base plate, away from the DAQ board. For each vacuum generator, use two 1/8” black cable ties, each one running through one of the two holes in the body of each vacuum generator and through one pair of the holes flanking the outline on the base plate. Tighten cable ties well and cut off excess length with wire cutters as shown in Figure S3.18.

Figure S3.18 – Photograph showing how to attach the vacuum generators to the base plate.

10. Assemble the manual pressure regulators.

Attach two 1/8" Tube OD X 1/8" NPT Male adapters to each of the four manual regulators, again first wrapping the threads with Teflon tape to prevent leaks (if the adapters did not come with threads already wrapped in tape) and tightening with wrench until snug as shown in Figure S3.19.

Figure S3.19 – Photograph showing how to attach the adapters to the manual pressure regulators.

11. Add the manual regulators.

Strap three of the manual regulators to the base plate, taking care to use the correct one in each position. In the position marked “V_lo” on the base plate, use one of the two 0-60 psi range regulators (McMaster 41795K34). In the position marked “P_lo”, use the 0-5 psi range regulator (McMaster 41795K31). In the position marked “P_hi”, use the 0-30 psi range regulator (McMaster 41795K33). Use two 8” black cable ties per regulator to strap each one securely to the base plate using the four holes flanking the outline of each regulator, with the regulators oriented so that the arrows on their sides are pointed up. Cut off excess cable tie length with wire cutters as shown in Figure S3.20.

Figure S3.20 – Photograph showing how to attach the manual pressure regulators to the base plate.

12. Assemble the electronic vacuum and pressure controllers.

Add the eight 1/8" Tube OD X 10-32 UNF Male adapters (McMaster 5779K241) to the Parker controllers, two per controller, tightening with wrench until snug. Teflon tape is not necessary on these threads. Attach the cable that comes with each controller to the jack on the top of the controller as shown in Figure S3.21.

Figure S3.21 – Photograph showing how to attach the tubing adapters and cables to the vacuum and pressure regulators.

13. Attach the electronic vacuum and pressure controllers to the platform.

Attach the four controllers to the platform using 8-32 (7/16" length) screws for the vacuum controllers (“VSO…”) and 4-40 (3/8" length) screws for the pressure controllers (“OEM…”). Take care to place each controller in its correct location: in the position marked “V_hi”, use part # 990-005203-005; in the position marked “V_lo”, use part # 990-005201-002; in the position marked “P_lo”, use part # 990- 005101-002; in the position marked “P_hi”, use part # 990-005101-015. Orient the controllers according to the markings on the platform, with the sides labeled “SOURCE” (for the vacuum controllers) or “SUP” (for the pressure controllers) facing the right hand side of the platform. This step is shown in Figure S3.22.

Figure S3.22 – Photograph showing how to mount the electronic pressure regulators to the mounting platform acrylic plate.

14. Mount the platform to the 3” standoffs.

Mount the platform above the DAQ board, oriented so that the Parker vacuum controllers are in the rear and the small standoffs for the pressure board are in the front. Fasten the platform to the tops of the 3” standoffs that were previously attached to the base plate, using the remaining four 10-32 (5/8” length) screws. This step is shown in Figure S3.23.

Figure S3.23 – Photograph showing how to mount the platform to the base plate.

15. Add the PC boards to the base plate and platform.

From this stage onward (or at least whenever coming into contact with the PC boards), work with the components on a grounded antistatic mat and wear an antistatic wrist strap grounded to the mat. Mount the three PC boards to the three sets of four ¼“ o.d. (5/8” length) standoffs that were previously mounted on the base plate and platform, using the twelve 4-40 (5/16” length) screws through the corner holes on the boards to secure them to the standoffs. The two valve boards go on the base plate, the pressure board goes on the platform. Make sure that the boards are oriented correctly, as indicated on their outlines on the base plate and platform. The valve boards should be oriented so that the terminal blocks marked “VALVES4-1” face the valve support standoffs previously strapped to the base plate, and the pressure board should be oriented so that the phone-jack-style connectors face toward the Parker regulators (and the back panel of the box). See Figure S3.24.

Figure S3.24 – Photograph of circuit boards, manual regualtors, vacuum generators, DAQ, and mounting platform all mounted to the base plate.

16. Install the valves.

Loosen all eight screws on the “VALVES4-1” terminal block of the pipette valve board, and the middle four screws on the “VALVES4-1” block of the manometer valve board. Mount four valves (Lee Company, LHDA0533115H)‬ on the pipette valve board, and two on the manometer valve board, with their nozzles pointing up; the valves’ pins should be inserted into the holes of the terminal blocks, and the valves should be lined up neatly with their “heads” resting on the valve support standoffs previously strapped to the base plate. The pins of the four valves on the pipette valve board will fill the available eight terminals of their terminal block. The pins of the two valves on the manometer valve board should be inserted into the middle four terminals (i.e., pins 3-6) of their terminal block. Tighten the screws of the terminals, making sure that the valves remain aligned with each other neatly. Once the screws are tightened, strap each set of valves down tightly using 8” cable ties through the holes flanking the outlines of the valves on the base plate. After tightening and trimming the loose ends of the cable ties, retighten the screws on the valve terminal blocks to ensure that the connections are secure. See Figures S3.25-26.

Figure S3.25 – Photograph of solenoid valves installed on the circuit board.

Figure S3.26 – Photograph of solenoid valves installed on the second circuit board.

17. Assemble the BNC switch assembly.

Attach one of the two 2-foot BNC cables to the connector on the end of the Tohtsu BNC relay. Attach the second 2-foot BNC cable to the connector on the relay that is on the same side as the large blue solenoidal coil. Attach the male BNC-binding post adapter to the third BNC connector on the switch. See Figure S3.27.

Figure S3.27 – Photograph of BNC relay assembly.

18. Attach the BNC switch assembly to the base plate.

Align the BNC switch and attached BNC-binding post adapter with the outlines of these components on the base plate (the outline of the adapter is marked “DAQ AO0”), with the BNC switch standing up on its end so that the long axis of the blue solenoid is vertical. Use an 8” cable tie through the holes of the binding post adapter and the matching holes in the base plate to securely strap the adapter to the base plate. See Figure S3.28.

Figure S3.28 – Photograph showing where to mount the BNC relay on the base plate.

19. Attach the female BNC-binding post adapter to the base plate.

Align this adapter with its outline, marked “DAQ AI0”, and strap it to the base plate with another 8” cable tie. See Figure S3.29.

Figure S3.29 – Photograph showing where to attach the BNC adapter to the base plate.

20. Attach the securing cable ties and cable tie holders to the base plate.

Locate the four holes near the left and right edges of the base plate. Thread four large white cable ties each through one large (not small) adhesive-backed cable tie holder (do not remove the paper squares covering the adhesive backing at this stage), and continue threading the ends of the cable ties through the holes in the base plate edges. Insert the cable ties’ ends in their respective heads to complete a loop with each one, but do not pull these loops tight yet. See Figure S3.30.

Figure S3.30 – Photograph of base plate attached to the adhesive cable tie holders.

21. Attach the base plate to the bottom panel of the chassis.

Wipe the inside of the bottom panel with 70% ethanol and wipe dry. The tabbed edges of the side panels indicate the front of the box; stand the base plate on the bottom panel, with the edge marked “BASE PLATE” oriented toward the front. Center it between the left and right side panels, so that the side edges are ~1.5” from the side panels. Position the base plate so that it is ~2.5” from the back panel and ~2.25” from the front panel. Remove the backing from one of the attached cable tie holders at a time and adhere it to the bottom panel, aligned with the base plate hole and offset slightly so that it is roughly centered with the edge of the plate; do not tighten the cable ties yet. Repeat these steps for the other three cable ties and holders. When all four holders are affixed, tighten all four cable ties. Cut off the loose ends of the cable ties with wire cutters. See Figure S3.31.

Figure S3.31 – Photograph of base plate attached to the bottom panel of the enclosure.

3.3.3 - Back Panel Assembly

Figure S3.32 shows the laser cutter image of the back panel. Black lines depict cuts, blue lines depict etches. Red lines depict the outlines of components and are not used for cutting or etching.

Figure S3.32 – Laser cutter design for the back panel.

1. Attach the panel-mount USB B cable.

The two screws supplied with the cable go through the back panel’s holes flanking the rectangular hole marked “USB” to affix the large rectangular block on one end of the cable to the interior face of the back panel. See Figure S3.33.

Figure S3.33 – Photograph showing where to attach the USB cable to the back panel.

2. Attach the through-wall coupling for ¼” o.d. tubing to the hole marked “Pressurized air <60 psi”. See Figure S3.34.

Figure S3.34 – Photograph of the through wall coupling for ¼” tubing attached to the back panel.

3. Attach the remaining 0-60 psi manual regulator to the hole below the large hole in the back panel.

Turn the knob counterclockwise until the screw is completely out of the body of the regulator. Remove the hex nut on the black shaft (not the smaller hex nut on the shaft of the knob), put the shaft through the hole in the back panel (with the body of the regulator on the interior side of the panel), then replace the nut on the shaft and gently tighten. Rotate the body of the regulator so that the white arrow marked “flow” is pointing up, with the two connectors vertically aligned. With regulator properly oriented, tighten the hex nut securely with a small adjustable wrench (take care not to scratch the face of the back panel). Reinsert the shaft with attached knob and screw clockwise a few turns so that it will not fall out. See Figure S3.35.

Figure S3.35 – Photograph of manual regulator mounted to the back panel.

4. Attach the panel-mount pressure gauge to the back panel.

Wrap threads on the fitting at the back of the pressure gauge with Teflon tape to prevent leaks, and attach the remaining 1/8" Tube OD X NPT Female adapter (McMaster 5779K123). Tighten with adjustable wrench until snug. Use the hardware supplied with the gauge to affix it in the large hole in the panel, ensuring that it is correctly oriented before tightening the nuts. Do not over tighten the nuts, as this can bend the back panel. See Figure S3.36.

Figure S3.36 – Photograph showing the pressure gauge mounted to the back panel and the 1/8” NPT to 1/8” tubing adapter also attached to the pressure gauge.

5. Leave the hole marked “24V 3A” empty for now.

6. Attach small standoffs to the rear edges of the chassis.

Use six ½” long 4-40 screws to attach six ¾” long, ¼” o.d. round nylon standoffs to the two holes at the rear edges of each of the side panels and the bottom panel. The standoffs should stick into the interior of the box and will support the rear panel, which will be pulled against them by cable ties. See Figure S3.37.

Figure S3.37 – Photograph showing where to attach the standoffs to the enclosure which will support the back panel.

7. Attach the back panel to the chassis.

Place the back panel in position between the side panels, resting on the back edge of the bottom panel and pushed against the six standoffs. First wiping the surfaces lightly with 70% ethanol and wiping dry (do not overdo it with ethanol on acrylic, as it can crack it), attach a pair of opposing large cable tie holders to each of the four corners (see Figure S3.38). Take care that the two holders of each pair are well aligned with each other so that the back panel will not be pulled either up or down by the cable ties. Thread a cable tie through each pair and loosely secure. When all cable ties are in place, gently tighten each one. Only tighten enough that the back panel does not wiggle unacceptably; over tightening will result in unsightly (but harmless) bending of the back panel.

Figure S3.38 – Photograph showing how to attach the back panel to the enclosure using the adhesive cable tie holders.

3.3.4 - Assembling front panel

Figure S3.39 shows the laser cutter image of the front panel. The black lines are to be cut, and the blue lines are to be etched. The red lines are only to indicate the outlines of the components in their final positions and are not to be used for cutting or etching.

Figure S3.39 – Laser cutter design of the front panel. Blue lines are etched, the black lines are cut, and the red lines are just for reference.

1. Add the 1/8” o.d. through-wall connector.

Add the (McMaster 5779K675) to the hole immediately under the “PIPETTE” label. In this and the subsequent steps, be careful not to scratch the front surface of the panel; acrylic is easily scratched. See Figure S3.40.

Figure S3.40 – Photograph showing where to attach the 1/8” through wall connector.

2. Add one of the larger (¼-20) torx-head screws (McMaster 96710A733) to the hole underneath the ¼” o.d. through-wall connector.

Also, attach a ¼-20 nut (McMaster 90499A029) to the screw on the reverse side of the front panel and tighten to hold the screw in place. This screw is only ornamental. See Figure S3.41.

Figure S3.41 – Photograph showing where to insert the ornamental torx-head screw

3. Attach the front panel to the chassis.

Using the four smaller (10-32) torx-head screws (McMaster 96710A578) inserted through the corner holes of the panel, attach the front panel to the enclosure. Secure the screws in position using four nuts (McMaster 90675A195). See Figure S3.42.

Figure S3.42 – Photograph showing where to insert the torx-head screws to attach the front panel to the enclosure.

4. Attach standoffs to manometer support plate.

Attach the 14 7/8” long hex standoffs (McMaster 92319A770) to the etched side of the manometer support plate using 14 5/8” long ¼-20 screws (McMaster 92196A539). Make sure that the standoffs are mounted on the etched side of the plate and are all oriented so that their cross-sections are well aligned with the etched hexagons. Inadequate orientation of the standoffs to match their etched outlines will result in inability to mount the manometers properly. See Figures S3.43-44.

Figure S3.43 – Laser cut design of the manometer support plate. The blue lines are etched, the black lines are cut. See the included files for the actual design file that can be used to make this plate.

Figure S3.44 – Photograph showing where to attach the ¼”-20 standoffs to the manometer support plate. 5. Prepare the manometers for mounting.

For each of the two manometers, use a flat-head screwdriver to remove the bottom end cap that holds in the 9V battery. Remove the batteries, caps, and screws. These will not be used. Gently extract the connectors for the 9V batteries out of the bottom of the manometers.

Figure S3.45 – Photograph showing how to prepare the manometers for mounting in the control box.

6. Add manometers to the manometer support plate.

Gently slide the manometers into their respective positions between the hexagonal standoffs on the manometer support plate. If necessary, loosen the screws attaching the standoffs to the plate to allow the standoffs to rotate into alignment with the sides and bottom of the manometers. The manometers are correctly positioned when they are pinned between the standoffs against their sides and with their bottom edges snug against the standoffs at the bottom edge of the plate. The 9V battery connectors should be pulled out of the open bottoms of the manometers so that they are accessible in a later step. Once the manometers are in place, tighten the screws as necessary so that the standoffs are secure. See Figure S3.46.

Figure S3.46 – Photograph showing where to insert the manometers into the manometer support plate.

7. Attach the manometer support plate to the front panel.

Being careful not to allow the manometers to fall out of their positions between the standoffs attached to the manometer support plate (although they should be snugly pinned between these standoffs), position the support plate behind the front panel so that the manometer buttons and display panels are visible through the cutouts in the front panel. The threaded holes of the six standoffs flanking and underneath the manometers should be aligned with the corresponding holes in the front panel. Make sure that the 9V battery connectors are pulled out of the open bottoms of the manometers, with their wires running under the bottom edge of the manometer support plate so that the connectors will be accessible after the plate is secured in place. Affix the manometer support plate to the front panel using six of the larger (¼-20) torx-head screws (McMaster 96710A733) through the holes on either side of each manometer cutout and through the holes at the bottom edge. After making sure that the manometer buttons and display panels are aligned with the cutouts, tighten the screws securely. See Figure S3.47.

Figure S3.47 – Photograph showing where to insert the other torx-head screws to attach the manometer support plate to the front panel.

8. Add the BNC bulkhead connectors.

(Digikey A33802-ND) to the two pairs of holes under “AMPLIFIER” and “DIGITIZER”. They should be oriented so that the nuts are on the inside of the box. For best appearance, orient the hexagonal surrounds of the connectors uniformly. Tighten the nuts using adjustable wrenches, taking care not to scratch the front of the panel. See Figure 3.48.

Figure S3.48 – Photograph showing where to attach the BNC bulkead connectors.

9. Add the panel-mount LED.

(Digikey 679-2976-ND) to the round hole under “POWER”, tightening the nut on the reverse side. Leave the leads unattached for the time being. See Figure S3.49-50.

Figure S3.49 – Photograph showing where to attach the panel mount LED indicator. This is the view from outside the box.

Figure S3.50 – Photograph showing how to attach the panel mount LED. This view is from inside the box.

10. Add the switches above the manometer 11. s.

Orienting the blank switches (with wires previously soldered on) so that tabs with the orange wires are on the left sides of the tabs with the yellow ones (or, more generally, so that the tabs marked “C1” are to the left of the ones marked “5E4”, as viewed from the front of the box), feed the wires through the rectangular cutouts above the manometers and snap the switches into place in the cutouts. See Figure S3.51.

Figure S3.51 – Photograph showing where to attach the unmarked switches on the front panel. This view is from the inside of the box.

12. Add the power switch.

The front-panel power switch was wired to the rear-panel 24V barrel connector in a previous step. Feed the barrel connector and wires through the rectangular cutout beneath the “POWER” label on the front panel. Orient the switch so that the “0” is underneath the “1” and snap into place in the cutout. See Figure S3.52.

Figure S3.52 – Photograph showing where to connect the power switch on the front panel.

13. Attach the 24V barrel connector to the back panel.

Remove the nut and washer from the connector and feed it through the hole marked “24V 3A” on the rear panel. Put the washer and nut back on and tighten to secure the connector, making sure not to scratch the panel. See Figure S3.53.

Figure S3.53 – Photograph showing where to attach the barrel connector to the back panel.

14. Add the potentiometers.

Add the 4 potentiometers to the four holes flanking the unmarked switches. The wired tabs of the potentiometers should be pointing up. Tighten the nuts with a wrench until snug. See Figure S3.54.

Figure S3.54 – Photograph showing where to mount the potentiometers to the front panel.

15. Add the knobs to the potentiometers.

Attach the knobs to the potentiometers, tightening their built-in set screws until snug. See Figure S3.55.

Figure S3.55 – Photograph showing the knobs attached to the shafts of the potentiometers.

16. Add three of the larger (¼-20) torx-head screws (McMaster 96710A733) to the holes along the top edge of the front panel, and secure in place with ¼-20 nuts (McMaster 90499A029). These screws are also only ornamental. See Figure S3.56.

Figure S3.56 – Photograph showing the additional torx-head screws attached to the front panel. These are purely ornamental.

17. Add and connect the internal BNC cables.

Connect the 2’ BNC cable that is connected to the end of the BNC relay (i.e., the one that is sticking straight up from the vertically-mounted relay) to the interior side of the “AMPLIFIER – CH1 COMMAND INPUT” BNC connector on the front panel. Connect the other 2’ cable, which is connected to the back side of the BNC relay (i.e., the connector that is underneath the blue solenoid) to the back side of the “DIGITIZER – ANALOG OUTPUT 0” front-panel connector. Attach the BNC tee (Mouser 523-31-208-RFX) to the back side of the “DIGITIZER – ANALOG INPUT 0” connector. Use a 1’ BNC cable (Mouser 523- 115101-19-12.00) to connect one arm of this tee to the back side of the “AMPLIFIER – CH1 PRIMARY OUTPUT” connector. Use the second 1’ BNC cable to connect the other arm of the tee to the female BNC-binding post adaptor that is strapped to the base plate. See Figure S3.57.

Figure S3.57 – Photograph showing where the BNC cables from the BNC relay and BNC adapters are connected.

3.3.5 - Wiring

Observe antistatic precautions (use antistatic mat and wrist strap) when touching or wiring to boards. Do not touch the boards unnecessarily.

1. Wire the power switch and connector to the pressure board.

Refer to Figure S3.58 when wiring the pressure board. Connect the free red lead from the power switch on the front panel, and the black lead from the 24V barrel connector on the back panel, to the terminal block labeled “PWR_IN_GND_24V” on the pressure board. All terminal blocks on the PCBs are labeled with the names of the inputs in the same spatial order in which they are to be wired to the terminals of the block. For the “PWR_IN_GND_24V” block, for example, connect the red 24V wire from the power switch to the left hand terminal, and the black ground wire from the barrel connector to the right hand terminal. For this and all subsequent steps of wiring to the terminal blocks on the boards, use wire strippers to strip a ~2mm stretch from the end of each wire. Use a small flat-head screwdriver to loosen the screw of each terminal, then insert the striped end of the wire and tighten the screw to secure the wire. See Figure S3.58.

Figure S3.58 – Drawing showing the wiring connections on the pressure control board.

2. Wire the front panel LED to power outputs of the pressure board.

Connect the red (power) and black (ground) leads of the LED to the terminal block labeled “PWR_OUT_24V_GND”. The odd terminals of this 8-terminal block are +24V, and the even ones are ground, so any two adjacent terminals can be used. For example, the ground wire can be connected to the rightmost terminal, and the red wire can be connected to the one immediately to its left. See Figure S3.58.

3. Plug the 24V power supply (Mouser 418-TRG70A24-12-V) into the wall using the power cord (Digikey 993-1039-ND, or any similar cord), and plug it into the barrel connector under “24V 3A” at the back of the box.

4. Turn on the power switch to test.

The green LED on the front panel should come on. If it doesn’t, check all connections and try again. Troubleshoot using the multimeter if necessary.

5. Turn off the power switch AND UNPLUG THE POWER SUPPLY FROM THE BOX before proceeding with further wiring.

Wiring powered electrical circuits is not advised.

6. Wire the Parker pressure controllers to the pressure board.

Plug the phone-jack-style connector on the cable attached the P_LO Parker pressure controller into the phone jack labeled “PRESSURE_LO”. Plug the connector on the cable attached to the P_HI controller into the jack labeled “PRESSURE_HI”. (not shown)

7. Wire the Parker vacuum controllers to the pressure board.

The vacuum controllers come with short Ethernet-type cables that are cut off on one end. For each of these two cables, strip ~1” of the black outer insulation from the cut end, and strip ~1/8” of the insulation from the ends of the white/orange, orange, white/green, and blue wires (the other four wires in each of the cables are not used and can be cut off or simply bent out of the way). Plug the “Ethernet” connectors into the jacks on the tops of the controllers. Connect the four stripped wires of the cable attached to the V_HI controller to the “V_HI_WO_O_WG_B” terminal block in the following order (and as indicated in the name of the block), from left to right as looking at the hole side of the block: white/orange-orange-white/green-blue. Similarly, connect the stripped wires of the cable attached to the V_LO controller to the “V_LO_WO_O_WG_B” terminal block in the same order. See Figure S3.59.

Figure S3.59 – Photograph showing where and how to connect the two vacuum pressure regulator cables.

8. Wire the manometers to the pressure board.

To each of the two 9V battery connectors, which should be accessible behind the manometer support plate, attach another 9V battery connector (“Shielded 9V Battery Strap”, ‪Mouser 534-81-8TB)‬ to mate with it and provide free wires that will be connected to the pressure board (although a logical approach would be to simply cut the connectors off of the wires running out of the manometers, these wires are not long enough to reach the pressure board by themselves). The four wires are to be wired to the terminal block labeled “PWR_OUT_GND_9V”. Wire both black wires to the terminal on the left side, when viewing the block from the front (i.e., the side with the holes that the wires enter). Wire both red wires to the terminal on the right side. See Figure S3.60. Note that this appears to violate the naming convention we have adopted for all the terminal blocks on the boards, because the black wires are wired to the terminal that is labeled “9V”, and the red wires to the terminal labeled “GND”, according to the label. However, there is no inconsistency: the use of the second set of 9V battery connectors to mate with the first reverses the color convention of the wires, so that in this case (and only in this case), the red wires are to be connected to ground and the black wires to +9V. Secure the battery connectors to a small cable tie holder affixed to the back of the manometer support plate. See Figure S3.61. ‪‪‪‪‪

Figure S3.60 – Photograph showing where to connect the 9V battery connector wires to the circuit board.

Figure S3.61 – Photograph showing how the 9V battery connectors from the manometers connect to the adapter wires from the circuit board.

9. Plug in the power supply and turn on the power switch and test the manometer power.

Pushing the “on/off” button on each manometer should cause it to turn on. If not, check the wiring and troubleshoot with the multimeter.

10. Turn off the power and unplug the power supply before proceeding.

11. Wire both valve board power input terminals to the pressure board +24V power output terminal.

Cut an 8” length each of red and black 24 gauge wire, for the pipette valve board, and a 15” length each of red and black 24 gauge wire for the manometer valve board. Wire the power input terminal block (PWR_IN_GND_24V) of each to the PWR_OUT_24V_GND block of the pressure board, to which the front-panel LED is already wired. Following the naming convention, the red (+24V) wires should be connected to the terminals on the left sides (as seen from the side of the blocks containing the holes) of the two-terminal input blocks on the valve boards, and the black (ground) wires should be connected to the terminals on the right sides. On the PWR_OUT_24V_GND block of the pressure board, the terminals are alternately connected to ground and +24V, beginning with a ground terminal on the right side (to which the black wire of the LED should be connected). Any of the +24V terminals can be used for the red wires, and any of the ground terminals can be used for the black ones. See Figure S3.62.

Figure S3.62 – Photograph showing where to connect the red and black wires on the pressure control board.

12. Wire the potentiometers to the pressure board.

Connect the triplets of orange, yellow, and black wires from the potentiometers to the POTS/AO_PH_PL_VL_VH_GND_SIGNAL_5V block of the pressure board, in the following way: Connect the wires of the leftmost potentiometer (“high pressure”) to the rightmost three terminals (pins 10-12) in the order orange-yellow-black (i.e., the third terminal from right (pin 10) gets the orange wire, the second from right gets the yellow wire, and the rightmost gets the black wire). Connect the wires of the second potentiometer (“high suction”) to the leftmost set of three terminals (pins 1-3) in the same order (orange-yellow-black). Connect the wires of the third potentiometer (“low pressure”) to the set of terminals second from right (pins 7-9), and the wires of the fourth potentiometer (“low suction”) to the set second from left (pins 4-6), again in the order orange-yellow-black in each case. See Figure S3.63.

Figure S3.63 – Photograph showing how to connect the potentiometers to the pressure control board.

13. Wire the “PRESSURE/SUCTION” front panel switches to the pressure board.

Connect the orange wires from the two blank front-panel switches above the manometers to the same terminals of any of the four orange wires just connected (all are connected the +5V supply). That is, loosen the screw of any of terminals 1, 4, 7, and 10 of the POTS/AO_PH_PL_VL_VH_GND_SIGNAL_5V block on the pressure board, and connect each of the two orange switch wires alongside the orange potentiometer wire already there.

14. Wire the “PRESSURE/SUCTION” front panel switches to the manometer valve board.

Referring to Figure S3.64, connect the yellow wire from the left hand switch “HIGH PRESSURE/SUCTION” to terminal 2 (second from left, when looking at the holes) of the TTL_IN_VALVES4-1_BNC_GND block of the manometer valve board. Connect the yellow wire from the right hand switch (“LOW PRESSURE/SUCTION”) to terminal 3 (third from left) of the same block.

Figure S3.64 – Drawing of the manometer valve control board. It also shows where to connect the wires from the front panel pressure selector switches (unmarked switches) to the manometer valve board.

15. Wire the BNC-binding post adapters to the DAQ board.

Cut two 2.5” lengths each of yellow and black 24 gauge wire and strip the ends. Wire one end of a yellow wire to each binding post’s red terminal. Wire one end of a black wire to each binding post’s black terminal. Twist each pair of wires together along most of their length to shield from electrical noise (see Figure S3.65). Connect the yellow wire from the BNC-binding post adapter labeled “DAQ AO0” (i.e., the one attached to the BNC relay) to the “AO0” terminal (pin 12) on the DAQ board. Connect the black wire from the same adapter to the “AO GND” terminal (pin 14). Connect the yellow wire from the BNC-binding post adapter labeled “DAQ AI0” to the “AI0” terminal (pin 15) on the DAQ board. Connect the black wire from the same adapter to the “AI8” terminal (pin 16). Tighten the binding post adapter knobs very securely.

Figure S3.65 – Photograph showing where to connect the wires from the BNC adapters to the DAQ.

16. Connect the DAQ board’s DGND and AO GND terminals.

Cut a 1” length of black wire, strip both ends, and bend it into a U to form a short jumper. Connect the ends to the “AO GND” terminal (pin 14, into which the black wire from the nearest binding post adapter was just inserted; both wires will be screwed into the terminal together) and to the “DGND” terminal that is nearest it (pin 11).

17. Wire the pipette valve board TTL inputs to the DAQ board.

Cut one 5” length of black 24 gauge wire and 5, 5” lengths of yellow 24 gauge wire. Use the black wire to connect the TTL_IN_VALVES4-1_BNC_GND terminal block’s rightmost (ground) terminal to the DGND terminal (pin 5) of the DAQ board. Use the yellow wires for the remaining connections. Connect the first (leftmost, when looking at the holes) terminal of the TTL_IN_VALVES4-1_BNC_GND block to the “PFI7 OUT” terminal (pin 9) of the DAQ. Connect the second terminal of the block to the “PFI6 OUT” terminal (pin 8) of the DAQ. Connect the third terminal to the “PFI5 OUT” terminal (pin 7) of the DAQ. Connect the fourth terminal to the “PFI4 OUT” terminal (pin 6) of the DAQ. Connect the fifth terminal (second-to-rightmost, “BNC”) pin of the terminal to the AO1 terminal (pin 13) of the DAQ.

18. Wire the BNC switch to the pipette valve board.

Connect the red and black wires soldered to the BNC switch to the terminal block labeled “BNC_OUT_HI_LO”, positioning the wires so that they cannot touch the mechanical flap on top of the solenoid and potentially interfere with its free movement. The red wire should connect to the terminal on the left side (pin 1), the black wire to the terminal on the right. See Figure S3.66.

Figure S3.66 – Drawing of the valve control board (not the manometer valve control board). It also shows where to connect the digital pins from the DAQ as well as the wires from the BNC relay.

3.3.6 - Internal Plumbing

1. Add the reducing coupling to the ¼” through-wall bulkhead connector.

Cut a 1” length of ¼” o.d. black tubing (‪McMaster 5648K251)‬ and ‪insert it into the interior side of the ¼” through-wall connector (McMaster 5779K677)‬ on the rear panel. Attach the ¼”-1/8” reducing coupling (‪McMaster 5779K352)‬ to the other end of the black tubing so that it is sticking into the interior of the box.‪‪‪‪‪‪‪‪‪‪‪‪‪‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

2. Connect the reducing coupling to the rear-panel manual regulator.

Cut a 6” length of 1/8” o.d. blue tubing (‪McMaster 5648K224).‬ This is the type of tubing that will be used in all the subsequent steps in this section unless otherwise indicated. Insert one end into the open orifice of the reducing coupling, insert the other into the lower connector of the manual regulator (‪McMaster 41795K34)‬ mounted in the rear panel. Ensure that this tubing is connected to the input of the regulator, as indicated by the arrow on the regulator’s side showing the direction of airflow.‪‪‪‪‪‪‪‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

3. Insert a 1.5” length of tubing into the other connector of the rear-panel manual regulator.

Ensure that this is the output connector of the regulator, as indicated by the arrow on the regulator’s side. 4. Attach a tee adapter (McMaster‪ 5779K31) to the other end of this 1.5” length. The‬ “base” of the tee should be connected to the tubing, with the two arms oriented up and down. ‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬ 5. Connect the rear-panel pressure gauge (McMaster‪ 3846K4)‬ to the top arm of the tee using a 2.5” length of tubing. ‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬ 6. Attach a 4” length of tubing to the bottom arm of the tee. 7. Attach a second tee to the other end of the 4” length. One arm of the tee should be connected to the tubing, so that the base of the tee is pointing perpendicular to it.

8. Connect the base of this second tee to the input connector of Venturi_hi using a 1.5” length of tubing. 9. Connect the other arm of the second tee to an arm of a third tee using a 2” length of tubing. 10. Connect the base of the third tee to the input connector of the V_lo manual regulator using a 1.5” length of tubing. 11. Connect the output connector of the V_lo manual regulator to the input of Venturi_lo using a 5” length of tubing. 12. Connect the other arm of the third tee to an arm of a fourth tee using a 1” length of tubing. 13. Connect the base of the fourth tee to the input of the P_lo manual regulator using a 1.5” length of tubing. 14. Connect the other arm of the fourth tee to the input of the P_hi manual regulator using a 2.5” length of tubing. 15. Connect the output of Venturi_hi to the input connector of the V_hi electronic vacuum controller using a 6” length of tubing.

The input connectors of all of the Parker controllers are labeled “SOURCE” (vacuum controllers) or “SUP” (pressure controllers) and should all be on their right-hand sides, closest to the Venturis and manual regulators.

16. Connect the output of Venturi_lo to the input connector of the V_lo electronic vacuum controller using a 6” length of tubing. 17. Connect the output of the P_lo manual regulator to the input connector of the P_lo electronic pressure controller using an 11” length of tubing. 18. Connect the output of the P_hi manual regulator to the input connector of the P_hi electronic pressure controller using an 11” length of tubing. 19. Attach a tee to the output of each of the four electronic pressure controllers.

Use approximately a 1” length of tubing between each controller and one arm of the tee. The base of each tee should be perpendicular to the output connector of the controller to which it is attached.

20. Attach a reducing elbow adapter (McMaster‪ 5779K661)‬ to the left hand port on the top of each manometer. ‪‪‪‪‪‪

Use a 1” length of black ¼” o.d. tubing (‪McMaster 5648K251)‬ to connect each port to its adapter. ‪‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

For the following steps, refer to Figures S3.67 for the manometer valve board tubing connection diagram and Figure S3.68 for the pipette valve control board tubing diagram when connecting the tubing in the following steps. To attach the 1/8” o.d. tubing (McMaster‪ 5648K224)‬ to the nozzles of the valves, it is necessary to first stretch the openings of the tubing lengths with a small pointed object, such as the tip of a multimeter probe (such as one from the DT830B‪ LCD Digital Voltmeter Ammeter Ohm Multimeter,‬ Amazon B005KGCI0Y‪ ),‬ to enlarge them enough that they can fit over the nozzles. ‬‬‬‪‪‪‪‪‪‪‪‪‪‪‪‪‪‪‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

Figure S3.67 – Schematic showing where to connect the tubing from the pressure regulators and manometers to the valves on the manometer valve control board.

Figure S3.68 – Schematic showing how to connect the valves to the pressure regulators and pipette connection on the valve control board.

21. Connect the manometers to the manometer valves.

Connect the elbow adapter on the high-range (left hand) manometer to the middle nozzle of the valve connected to pins 5 and 6 of the manometer valve board, using a 14” length of tubing (see Figure S3.67). Connect the elbow adapter on the low-range (right hand) manometer to the middle nozzle of the valve connected to pins 3 and 4 of the manometer valve board, using a 12” length of tubing.

22. Connect the Parker controller outputs to the manometer valves.

Refer to Figure S3.67 for this step. Connect the base of the tee on the output of the V_HI Parker vacuum controller (the one that is nearest to the back panel) to the distal (i.e., farthest from the board) nozzle of the valve connected to terminals 3 and 4 of the manometer valve board, using a 14” length of tubing. Connect the base of the tee on the output of the V_LO Parker vacuum controller to the distal nozzle of the valve connected to terminals 5 and 6 of the manometer valve board, using a 15” length of tubing. Connect the base of the tee on the output of the P_LO Parker pressure controller (the one that is nearest to the back panel) to the proximal (i.e., closest to the board) nozzle of the valve connected to pins 5 and 6 of the manometer valve board, using a 17” length of tubing. Connect the base of the tee on the output of the P_HI pressure controller to the proximal nozzle of the valve connected to pins 3 and 4 of the manometer valve board.

23. Connect the Parker controller outputs to the pipette valves.

Refer to Figure S3.68 for this step. Connect the open arm of the tee on the output of the V_HI Parker vacuum controller (nearest the back panel) to the distal nozzle of the valve connected to pins 7 and 8 of the pipette valve board, using an 8” length of tubing. Connect the open arm of the tee on the output of the V_LO Parker vacuum controller to the proximal nozzle of the same valve, using a 7” length of tubing. Connect the open arm of the tee on the output of the P_LO Parker pressure controller (nearest the back) to the distal nozzle of the valve connected to pins 3 and 4, using a 5” length of tubing. Connect the open arm of the tee on the output of the P_HI Parker pressure controller to the proximal nozzle of the same valve, using a 4” length of tubing.

24. Connect the pipette valves to each other.

Again refer to Figure S3.68 for this step. Using 4” lengths of tubing, connect the proximal nozzle of the valve connected to pins 1 and 2 to the middle nozzle of the valve connected to pins 3 and 4, connect the distal nozzle of the valve connected to pins 1 and 2 to the middle nozzle of the valve connected to pins 5 and 6, and connect the distal nozzle of the valve connected to pins 5 and 6 to the middle nozzle of the valve connected to pins 7 and 8. The proximal nozzle of the valve connected to pins 5 and 6 is left unconnected.

25. Connect the pipette valves to the front panel through-wall coupling.

Refer to Figure S3.68 for this step. Connect the middle nozzle of the valve connected to pins 1 and 2 to the (inside opening of the) through-wall “PIPETTE” coupling on the front panel. The internal wiring and plumbing of the control box is now complete.

The following steps explain how to finish the assembly and adjust the manual pressure regulators to be within the range required by the electronic pressure regulators.

26. Prepare air filter assembly components.

Cut two 3” lengths of ¼” ID tubing using a razor knife. Cut two lengths of ¼” OD black tubing: one 3”, the other 18”. Screw a ¼” ID fitting into each end of the shutoff valve using an adjustable wrench.

27. Assemble air filter assembly.

Using 70% ethanol to lubricate the inside walls of the 3” lengths of ¼” ID tubing, jam one length all the way onto each of the two barbs of an inline filter. Jam one end of the 3” length of black tubing all the way into the open end of one of the 3” lengths of ¼” ID tubing, again lubricating with 70% ethanol as necessary. Similarly, jam one end of the 18” length of black tubing into the open end of the other 3” length of 5/8” tubing. Affix a hose clamp onto each length of ¼” ID tubing: thread the hose clamps over the black tubing and onto the ¼” ID tubing roughly ½” from the end into which the black tubing is inserted. Tighten the clamps securely using a screwdriver so that the ¼” tubing is visibly compressed. Attach the 3” length of black tubing to one fitting of the shutoff valve.

28. Install air filter assembly.

Insert the free end of the 18” length of black tubing into the ¼” fitting at the back of the control box. After confirming that the house air supply pressure does not exceed 60 p.s.i., connect the pressurized air supply to the open fitting of the shutoff valve (this may require replacement of the nozzle on the wall spigot with a 1/4" Tube OD X 1/4" NPTF Male Pipe fitting, the same as are screwed into the ends of the shutoff valve). Do NOT turn on pressurized air yet. See Figure S3.69.

Figure S3.69 – Photograph showing the filter assembly attached to the back panel of the control box.

29. Terminate outputs of manual regulators and Venturi devices.

Disconnect the blue tubing from the outputs of the two Venturi devices and from the P_hi and P_lo manual regulators (not the V_hi manual regulator or the regulator mounted on the back panel). Leave the other ends of the four lengths of tubing connected to the inputs of the Parker regulators. Connect terminated (e.g. with closed stopcocks) short lengths of 1/8” OD tubing to the outputs of the two Venturi devices and two manual regulators (see Figure S3.70).

Figure S3.70 – Photograph showing some of the plumbing partially completed with the addition of some caps added to stop leaks during the pressure testing steps.

30. With the shutoff valve turned off, turn on the house air supply.

The shutoff valve is off when the handle is perpendicular to the valve’s long axis. Check for air leaks.

31. Turn the knobs on all four manual regulators counterclockwise until approximately 5/8” of threading is exposed on the knob shafts.

This closes off the flow through them. Refer to Figure S3.71. If this amount of threading is already exposed, skip this step. If you turn a knob too far counterclockwise (>3/4” of shaft exposed) so that the shaft falls out of the body of the regulator, simply screw it back in, trying not to wipe off the grease.

Figure S3.71 – Photograph showing a manual pressure regulator completely closed so no air can flow through it.

32. Turn on pressure to box using the shutoff valve. 33. Turn the input regulator knob clockwise to raise the input air pressure to ~40 p.s.i. 34. Plug the power supply into the jack at the back of the box & turn on the power switch on the front of the box. The green light next to the power switch should come on. 35. Connect the high-range manometer to the output of Venturi_hi.

Disconnect the blue tubing from the input to the manometer, and disconnect the terminated length of tubing from the output of Venturi_hi. Connect the two using a length of 1/8” o.d. tubing.

36. Increase the input air pressure until the manometer reads roughly -466 mbar.

Monitoring the reading on the high-range manometer, adjust the rear-panel regulator to increase (or possibly decrease) the input pressure until the manometer reads approximately -466 mbar. This is the midpoint of the acceptable range of (-)414-517.5 mbar. Once the pressure is set correctly, manually rotate the locking nut on the shaft of the rear panel manual regulator to advance it down the knob shaft until it is snug against the body of the regulator, and tighten the locking nut with your fingers (rather than a wrench, so as not to overtighten). The knob should now not move when turning is gently attempted.

37. Label the rear-panel gauge with the target input pressure.

Write the rear panel’s current reading (e.g., 49 p.s.i.) on a sticky label (e.g., Electron Microscopy Sciences, cat. no. 77560-Y, or any other convenient label, or simply paper tape) and affix it to the rear panel beneath the gauge under the words “Adjust to”. Record this target pressure in the documentation for that particular box. See Figure S3.72.

Figure S3.72 – Photograph showing where to display the required input pressure to the control box.

38. Determinate the output of Venturi_hi and connect the high-range manometer to the output of Venturi_lo. 39. Adjust the output of the V_lo manual regulator so that the output of Venturi_lo is ~-202 mbar.

This is the midpoint of the acceptable range of (-)180-225 mbar. Tighten the locking nut on the regulator once the pressure is set correctly.

40. Reterminate the output of the V_lo manual regulator, and connect the high-range manometer to the output of the P_hi manual regulator. 41. Adjust the output of the P_hi manual regulator to ~1396 mbar.

Turn the regulator knob clockwise to raise the reading on the manometer to approximately 1396 mbar, which is the midpoint of the acceptable range of 1241-1551 mbar. Tighten the locking nut with your fingers.

42. Reterminate the output of the P_hi manual regulator, and connect the high-range manometer to the output of the P_lo manual regulator. 43. Adjust the output of the P_lo manual regulator to ~186 mbar.

This is the midpoint of the acceptable range of 165.4-206.8 mbar. Once the pressure is correctly set, tighten this regulator’s locking nut too.

44. Turn off air supply to box, remove all terminated tubing lengths and reconnect the high-range manometer, Venturis, and manual regulators to their correct lengths of 1/8” OD tubing, and turn air supply back on.

Reconnect the manometer to the valve board, reconnect the P_hi manual regulator to the P_hi Parker controller, and so on.

45. Terminate the “PIPETTE” output port on the front panel of the control box.

See Figure S3.73. The Parker regulators will not be able to attain the intended pressure and vacuum levels if this port is left open.

Figure S3.73 – Photograph showing the pipette output from the box, terminated with a plug so there is no flow. This is necessary for the pressure testing step.

46. Test the range of the High Pressure setting.

With the switch above the high-range manometer set to “Pressure”, turn the knob on the left side of the switch clockwise to the maximum extent of its movement range. The manometer should read slightly more than 1000 mbar.

47. Adjust the High Pressure setting to approximately 800 mbar. 48. Test the range of the High Vacuum setting.

With the switch above the high-range manometer set to “Suction”, turn the knob on the right side of the switch clockwise to the maximum extent of its range. The manometer should read slightly less than - 300 mbar (e.g., -332 mbar).

49. Adjust the High Vacuum setting to approximately -150 mbar. 50. Test the range of the Low Pressure setting.

With the switch above the low-range manometer set to “Pressure”, and beginning with the knob to its left turned all the way counterclockwise, turn knob clockwise until the low-range manometer reads OFL, indicating that the attainable low pressure level exceeds the range of the manometer.

51. Adjust the Low Pressure setting to approximately 2.5 kPa. 52. Test the range of the Low Vacuum setting.

Note that the display on this manometer is limited to 10 kPa, and the low vacuum output can be set to higher than this, so that setting it to -11.5 kPa will result in an apparent reading of -1.5 kPa. With the switch above the low-range manometer set to “Vacuum”, and beginning with the knob to its right turned all the way counterclockwise, turn knob clockwise until the reading exceeds (absolute value) -10 kPa and continue turning until the knob reaches the maximum extent of its range. The reading on the manometer should be around -3 kPa, indicating an actual pressure of -13 kPa.

53. Adjust the Low Vacuum setting to -1.5 kPa.

Bear in mind that the display on this manometer is modulo 10 kPa, as noted above. Start at 0 kPa and increase vacuum to -1.5 kPa, making sure you haven’t gone past -10 kPa and are actually setting it to - 11.5 kPa.

54. Turn off the pressure to the box. This will make it possible to hear the clicking of the BNC switch in the next steps. 55. Connect the USB port on the back of the box to a USB port on the computer on which LabVIEW and the autopatcher software is installed. 56. Open the “BNC switch – continuous run” VI. “VI” = “virtual instrument”, what National Instruments calls individual LabVIEW programs. 57. Set the “Device name” control to the assigned name of the DAQ board.

Click on the down arrow on the right side of the “Device name” control on the VI front panel. A menu should drop down showing the name (e.g., “Dev9”) of the DAQ board in the box. Click on this name. If there are no other National Instruments devices connected to the computer, there will be only one choice. If there is any uncertainty as to which device name to select, start “NI MAX”, the National Instrument Measurement and Automation Explorer program that is installed with NIDAQmx (the National Instruments device drivers), and expand the “Devices and Interfaces” list in the left hand column to see what device name corresponds to the USB-6211 board.

58. Start the “BNC switch – continuous run” VI by clicking on the arrow in the control bar at the top of the VI. 59. Toggle the “Program in command” control between “Autopatcher” and “Clampex” and listen for a loud click coming from the BNC switch whenever this control is switched from one to the other.

If there is no loud click, check all connections with a multimeter. When “Program in command” is set to “Clampex”, the voltage across the pins of the BNC switch should be zero. When it is set to “Autopatcher”, the voltage across the pins should be 12V.

60. Set the multimeter to check resistance (by audible alarm to indicate shorts if it has this functionality). 61. With the “Program in command” control set to “Autopatcher”, test the resistance between the “Amplifier Ch1 Command Input” connector and the “Digitizer Analog Output 0” connector on the front panel. The resistance should be infinite (open circuit, no connection). 62. With the “Program in command” control set to “Clampex”, again test the resistance between the above two connectors on the front panel. The resistance should be zero (i.e., these connectors are connected to each other). 63. Stop the “BNC switch – continuous run” VI using the large “STOP” button and close the VI. 64. Turn on the pressure to the box again. 65. Open the “Pressure switch – continuous run” VI. 66. Set the “Device name” control to the assigned name of the DAQ board as was done for the previous VI. 67. Connect the “PIPETTE” output port to the input of another high-range manometer, using a length of 1/8” OD tubing. The high-range manometer of a second control box can conveniently be used for this purpose. Turn on the second control box to power this manometer. 68. Start the “Pressure switch – continuous run” VI. 69. Observing the reading on the external manometer, cycle through the “Pressure state” control settings.

The reading on the external manometer should correspond to the “Pressure State” setting. For example, if the state is set to “Pressure hi”, the manometer reading should be approximately 800 mbar. The reading may be discrepant by a few percent with respect to the readings on the manometers of the control box being assembled, reflecting the precision limits of the manometers (and perhaps pressure loss due to the resistance of the tubing).

70. Disconnect the external manometer from the “PIPETTE” port.

71. Organize cables and tubing using cable tie holders and cable ties.

Refer to Figure S3.74. Attach small cable tie holders to the rear panel at a location conducive to securing the tubing connecting the Parker controllers to the manometer valves and the BNC cables connected to the BNC switch. Secure cables and tubing with cable ties.

Figure S3.74 – Photograph showing how to organize the tubing and cables in the control box with the adhesive cable tie holders.

72. Attach the top panel to the control box. Secure using four of the black screws that come with the chassis. 73. Connect control box to amplifier, digitizer, and to pipette holder.

Use BNC cables to connect the four BNC connectors on the front panel to the indicated connectors on the MultiClamp 700B and Digidata. Connect the “PIPETTE” port on the control box to the pipette holder using the 1/8” diameter tubing (McMaster 5648K221) to the rig, with a barbed fitting (McMaster 5116K16) used to connect to a short length of 0.04” i.d. tubing (McMaster 1883T3) that connects to the port of the electrode holder. The tubing lengths, particularly the 0.04” i.d. tubing, should be as short as possible.

74. Begin autopatching. The assembly of the control box is now complete.