E²dish User Manual
Contents
1. 3 Open the System Properties window by right clicking the My Computer icon and choosing Properties Alternatively open My Computer and click View system information in the System Tasks panel 4 Select the Hardware tab and click Device Manager This opens up the Device Manager as shown below M TE Action View Help e mema A Aleria USB Amplifiers Computer E i Disk drives Display adapters og DYDICO ROM drives f IDE ATA ATAPI controllers fy IEEE 1394 Bus host controllers H S Keyboards Fy Mice and other pointing devices H Monitors re Network adapters Ed y Ports COM amp LPT 5 You should see a node Aleria E drive attached to a node Aleria USB amplifiers or Other devices If this is not the case go to Action gt Scan for hardware changes to detect the Aleria E drive Return to step 2 2 6 Right click the node Aleria E drive and select Properties The Aleria E drive Properties screen appears as shown below Aleria Ezdrive Properties q xj General Drives Details O Alena E2drive Device type Aleria USB Amplifiers Manufacturer Unknown Location Location O Alena E 2drive Device status Thiz device i not configured corectly Code 1 To reinstall the drivers for this device click Reinstall Driver gl Reinstall Driver Use this device enable Device usage 7 Select the Driver tab and click Update Driver to start the driver install2. Display recording button is enabled as shown below H 24 My Network Places E ee My Computer M 42 Inspection of properties Inspect the properties of the recording in the Recording properties panel The panel should look like the one dis played lf you have co rrectly followed the steps in the section Making a re cording the NumberOfChannels property should equal Episodes Show channel fi Display recording 4 3 Voltage UV Bob Hoe Bol g ES 5 HR A new approach to electrophysiology for your neuronal cultures E dish pq Recording properties x E Recording details Mame Description Date 2 10 2009 StartT ime 6 28 59 PM EndT ime 6 43 59 PM NumberO fChannels b 10000 SamplingFrequencyL rit Hz TotalNumberO FS amples So000000 MNumberOfE pisodes 1 DataFormat Binary DataType DataPrecision 16 Ll nit Mizrawolte Gain 0 0331 71 2806 Mame The name of the recording 3 and that the SamplingFrequency property should equal 20000 The recording properties also include the date of recording the start time and end time and the total num ber of samples saved per channel Tutorial recording This is a test recording SamplingFrequency Displaying data of a specific channel Click Display recording in the Load recording panel to dis play the data recorded and wait until the progress bar completes The recording is plotted as shown below OC CC lan any alyan i
3. below is 3 2 2 In the Browse For Folder dialog that appears se lect the Desktop node and click OK The Location text box should now contain a folder path similar to C Documents and Settings lt User gt Desktop Changing the name of the recording Next change the name of the recording into Tutorial re cording and add This is a test recording as a description Setting the duration of the recording Set the duration of the recording to one and a half minu tes To do so check the Predefined length radio button and then enter 1 in the text box designated for minutes and 30 in the text box designated for seconds The Re cord to disk panel should now look like the one shown below Localicr Descriptor Format ay Recond bo disk a q x C Decumerts end Selling z Mame ol the recordng Tutorial recording 13 a best recording lina Double memiskiri Dustan Continuous Predefined length Leng foo foi 30 hem 00 00 00 Progres Elapsed ime Stat recording 3 5 Starting the recording Start the recording by clicking Start recording The data from channels 1 3 and 5 is now being saved to disk Du ring the recording a flashing recording indicator displa yed in the status bar reminds you that a recording is in progress shown below Wait until de recording finishes RECORDING A Start 3 6 Ending of a recording A message will appear indicating that
4. contact with its membrane Because the current must flow partly along the extracel lular medium and this path possesses a non zero electrical resistance given by the intrinsic resistivity p of the medium an extracellular potential Ve arises and is sensed by the micro electrode The magnitude of the extracellular potential Ve can be estimated using Ohms Law i Ve R xl where Re is the resistance along the extracellular path of the current which can be estimated at several KOhms The re sulting Ve is then in the order of 10 100 uV i e several orders of magnitude below the intracellular action potencial V Yet in dividual action potentials can be seen above noise Whole cell A Patch clamp techniques For patch clamp recordings the micropipette actually contacts the membrane and electrically isolates a patch typically as small as 1 um Suction is commonly used to attach the micro pipette to the cell and achieve a good seal The resistance of the seal is at least 1 GOhm compared to several KOhms with extracellular measurements With such high seal resistances little current leaks through the micropipette membrane gap Loose patch techniques The loose patch method can be considered an intermediate configuration between extracellular and patch clamp The mi cropipette isolates a patch of membrane but the seal does not reach 1 GOhm but remains in the range 10 50 MOhms Loose patch is used for rapid screening of cel
5. is optimal to attach a 12 well E2dish and so it is important gt to adequately centre the E dish on the substrate Make sure that the corners of the E2dish are not touching the walls of the culture dish and are equidistant from the sides It is recommended that you use the included template see Annex C as an aid to aligning the E2dish The template has been designed for the twelve reser voir EZdish but the two channel reservoir EZdish can be placed on any of the six available positions 4 Fill wells with cell culture media There are two different protocols depending on which type of substrate you will be using for the attachment of the E 2dish Glass substrates f you are using glass substrates cell cul ture medium 100 uL can be directly pipetted into the wells The microchannel s will fill soontaneously with the culture media This can be easily tested using a standard multimeter and measuring the resistance approximatelly 10 20 MOhms of the channel or by a visual check using a microscope Plastic substrates For plastic substrates prior to filling with culture media the E2dish should be exposed to CO2 gas using the CO2 chamber in order to prevent blockage of the micro channels with air bubbles CO gt is highly soluble in aqueous media and will replace the less soluble air mostly Nz in the microchannels This will result in the spontaneous filling of the microchannel with cell culture media Aleria Biodevices A
6. pressure regulator three way valve T VE cu Um source CO2 chamber B pressure valve n regulator three way valve SS le source CO2 chamber FIGURE 5 To fill the microchannels in the E2dish with culture medium f you are using Aleria Biodevices CO2 minichamber proceed as indicated in the next section f you have other CO2 vacu um systems proceed similarly Contact support aleriabio com for further support on this issue 4 1 Connections of the CO2 chamber Assemble the CO minichamber Ref 060 0004 and 060 0005 Place the black O ring supplied in the groove of the base and connect the three way valve to the cham ber cover The 2 m 5 mm ID tubing Ref 061 0002 should be connected to the CO gas cylinder pressure regulator and to the IN port of the 0 2 um filter Ref O60 0006 Next the 0 1 m 8 mm ID tubing Ref 061 0004 should be connected to the second port of the filter and to the top port of the three way valve on the chamber cover Use the tube clamp Ref 060 0007 to secure the 0 1 tubing to the three way valve Finally the 2 m 8 mm ID tubing Ref 061 0003 should be connected between the side port of the three way valve on the chamber cover and the vacuum source In your laboratory 4 2 Expel the air from the chamber Once the cover has been removed place the 60 mm cul ture dish with the attached E dish inside the CO2 cham ber on the chamber plate Turn on the vacuum pump for 30
7. seconds in order to expel air from the chamber Once this has been done check that the chamber cover cannot be removed as vacuum is holding it down 4 3 Fill the chamber with CO2 To turn on the CO2 gas turn the blue knob on the gas bottle to the open position and then turn the black lever to the up position Fill the chamber for 40 seconds When the chamber has completely filled you will hear a small pop sound as the chamber cover is released Turn the black lever to close the gas cylinder Repeat the cycle ex pel air fill with CO2 three times to ensure the complete exposure of the E dish to CO2 stages 4 2 and 4 3 4 4 Fill the wells with medium Open the chamber and fill the wells with cell culture me first apply vacuum A and then fill the chamber with CO B dium The microchannel will fill spontaneously without bubbles blocking the channel Tip After opening the chamber the microchannels re main filled with COz for just a few seconds Make sure the wells are filled swiftly 20 seconds maximum 5 Plate neurons in the wells Once the wells have been filled with medium you can seed the cells Good results are obtained with 50x105 to 105 cells per well when plating with Aleria Biodevices cryopreserved E16 hippocampus neurons on poly L lysine coated polystyre ne substrates Seeding densities for other neuron types and substrates should be adjusted to obtain channel guided axons after 1 2 weeks With wells pre fil
8. the insta llation process to begin LE MN 51 x Installing E2soft Aleria reee a Biodevices E2soft is being installed Please wait es Cancel 3 5 The Installation Complete screen appears You are remin ded to use Windows Update to check for any critical up dates to the NET Framework Please do so as Windows Update may include important security and performance updates Click Close to exit the Installation Wizard 5 x Installation Complete Aleria ines Biodevices E2soft has been successfully installed Click Close to exit Please use Windows Update to check for any critical updates to the NET Framework Cancel 11 Aleria Biodevices Driver installation Note Now you can connect the E drive to the USB port Make sure the power supply is also connected When the E2drive is connected to the PC for the first time it is necessary to select the appropriate driver software During installation this driver software has already been copied to your machine This makes installation straightforward To se lect the correct device driver follow the steps below 1 Connect the E drive to the PC If you already connected the device prior to or during installa tion of the software you must disconnect and reconnect the device A notification balloon appears followed by the Found New Hardware Wizard screen as shown below Found New Hardware Wizard Welcome to th
9. the recording has finished as shown below Click OK Information A The recording was succesfully saved 3 7 Verifying the file was correctly saved To verify that the recording exists in the folder you spe cified open the folder in Windows Explorer Inside the folder you should see two files a data file Episode_1 adf and a header file RecordingDescription ahf The data file contains the raw data of the recording the header contains the recording parameters such as the sampling frequency and the number of activated channels ri Fie Edit View Favorites Tools Help amp Geek O Fp search Folders 7 Address C Documents and Settings Administrator Desktop Tutorial recording gt Go Name Sze Type Episode 1 42 188KB Aleria Data File RecordingDescription 2KB Aleria Header File File and Folder Tasks y Other Places y Details y 4 Loading a recording The next steps demonstrate how to load a recording and dis play the data Note This tutorial assumes you have followed the previous steps of the E soft tutorial 4 1 Load a file Switch to the analysis window by clicking the Analysis tab shown below In the Load recording panel select the node lutorial recording located under the Desktop node E E2soft File Options Window Help Real time visualization Analysis F Load recording a y You will notice that an entry is added to the Episodes box and the
10. 8 um compared to 1 um In general the more membrane surface is confined the larger the expected signals To a first approxi mation the signal size can be expected to increase with the product of the confined surface and seal resistance following Ohms law Voltage signal size S x J x Rseal where S is the surface of the membrane patch in the micro channel J the current density per unit of surface and Rseal the resistance of the seal Yet as axons grow Into the microchan nel a gap remains between the membrane and the walls of the microchannel so that a gigaseal is rarely achieved The resistance of the microchannel measured end to end is typi cally in the range 10 20 MOhms and the voltage signal size is in the order of hundreds of uV see screen capture below from E16 hippocampus cultures Action potentials can clearly be seen above noise Voltage pV 8 8 8 00 00 03 00 00 06 00 00 09 00 00 12 000015 00 0018 00 00 21 000024 Do gt ACTION POTENTIALS FROM E16 aa HIPPOCAMPUS 14 DIV 4 100 3 0 w pl une i i A ebe i h Mule 00 00 17 800 00 oi m 00 00 18 0 Ly Ansys a x Anaya Fitering Select an analysis Select a hiter a non Apply Ready Annex B Amplifier Datasheet Component description Device Geometry Circular Dimensions diameter x high 165 mm x 30 mm 39 mm with plastic lockers Weight 660 g without plastic cover 710 g with plasti
11. Aleria tses ez Biodevices E2disn User Manual A NEW APPROACH TO ELECTROPHYSIOLOGY FOR YOUR NEURONAL CULTURES E2dish User Manual For technical support contact support aleriabio com For new orders order status enquires sales aleriabio com Aleria Biodevices March 2009 Contents 1 Materials supplied 2 Introduction to E2dish technology 5 Conventional micropipette electrophysiology is cumbersome 5 ER technology automates electrophysiology by integrating the micropipette in the substrate 5 Use your proven protocols No need to change to new substrates 6 Advantages of the E2dish 3 Quick start guide to using the E2dish 7 Procedure overview 7 On the day of seeding 9 On the day of recording 4 E2soft and driver installation manual 10 System requirements 10 E2soft installation 12 Driver installation 5 E soft Tutorial 6 Troubleshooting 7 Frequently Asked Questions Annex A Theory of operation of E2dish technology Annex B Ezdrive amplifier datasheet Annex C Ezdish substrate alignment template 10 13 17 18 19 21 22 1 Materials supplied E2drive benchtop amplifier CO gt Chamber kit Units Component description Reference Units Component description Reference 1 E2drive 600A amplifier 020 0001 1 CO2 minichamber 060 0004 1 Plastic cover with electrodes 020 0003 cl A AC DC adapter 060 0001 l Enamber plate li USB cable 060 0002 1 Sili
12. aph will be updated to show the lowpass filtered signal on which the threshold detection is performed as well as the threshold that has been automatically compu ted on the basis of the noise in each channel Mor Debrction riu Parameters have bes odia compubed Ori thee bas dl the rose level in paih dhara Fou Gat adiit Ehe v ghue of be pr ambler indirin Pp herp Deer raya Hiii Mit ers bora De ik met fio gt ai ceed gt le H or fe So fo Hi deeds fit ii ou imi chews fit Soe fo i Cal i Charmed 1 157 bursis Charred dari 5 4 Change the threshold values In the screen that pops up you are able to change the value for each threshold and the graph is automatically updated to show the new position of the threshold While the popup screen is shown you can continue to use the zooming functionality of the graph You can also specify for each channel the minimum amount of time the signal has to remain above the threshold in order for a burst to be detected Change the values of the thresholds and click Recompute to perform the burst detection based on the new parameters Note how the number of detected bursts changes 6 Troubleshooting The Aleria E drive is not recognized or is not installed correctly The Aleria E2drive needs a custom driver to be installed in your system in order to work properly Normally this driver is copied to your system during installation of the E soft soft ware and i
13. arge O displaced into the cell to produce the rising phase of an action potential depends on multiple parameters but a first estimate can be calculated as Q CxV where C is the capacitance of the cell and V de magnitude of the action potential AP Although the values for C and V vary from cell to cell we can use 100 pF and 100 mV as physiologi cally plausible values for capacitance and AP size resulting In O 10 picoCoulombs This AP generating charge O flows into the cell during a short period of time T during which the membrane potential is driven from resting potential to the peak AP value Assum ing T 500 us we can estimate that the generation of an AP in a neuron with a capacitance of 100 pF requires a pulse of inward Na current of a magnitude of I 0Q T fN or l 20 nA The ionic current flows along closed loops partly inside and partly outside the cell and with a gen erator locus believed to reside at the Axon Hillock axon nillOck Electrophysiological recordings typically involve either ac cessing the intracellular space to measure V or confining to measure its magnitude The biophysical basis of EZdish measurements can be easily related to more conventional techniques Extracellular electrophysiology Usually a glass micropipette with an internal metal wire often Ag AgCl is used as the sensing electrode The tip of the micropipette is positioned in the proximity of a cell not necessarily in physical
14. ass Bandstop notch Frequency Hz Parameters Filter order Specify order le Minimum order Frequency specifications Fs Hz fro000 Low cutoff Hz 100 High cutoff Hz 3000 Magnitude specifications Passband ripple fo peak to peak dB Realize model Filter equation yin 0 00926728558408825 x n O x n 1 0 074138284672706 x n 2 O x n 3 0 25948399635447 1 x n 4 O x 5 0 518967392708942 x n 6 O x r 7 0 6487099908861 78 vin 8 7 Save Cancel In the Filter design screen set the Design method to IIR Butterworth the Response type to Bandpass the Filter order to 8 and the Low and High cutoff frequency to 100 and 3000 respectively Click the Realize model button to view the frequency response of the filter and then click Save In the dialog that appears type Bandpass noise 16 removal and click OK In the Analysis panel your newly created filter is now selected Click the Apply button to apply the filter to the data The plot is automatically upda ted Depending on the size of your data and the number of active channels in the graph the filtering may take up to several seconds 5 3 Performing a burst detection Perform a burst detection by selecting the Burst detec tion option in the Analysis panel and click Run to filter and analyze your data Depending on the size of your recor ding this operation may take up to a minute The gr
15. ation procedure Then follow the steps described in the E2soft and driver installation manual 17 7 Frequently asked questions Q Can reuse the E2dish A Generally speaking no Bubble free attachment of the E2dish to the substrate and clean non blocked integrated micropipettes are important to achieve successful record ing Once used for culture the E2dish often retains debris within the microchannel Moreover repeated handling leads to attachment of particles on the bottom side precluding ad equate seal against the substrate Cleaning procedures with solvents often result in toxicity due to leaching during culture of the solvent used for cleaning o Can record with my amplifier inside the incubator A It is not recommended High humidity will shorten the life of your amplifier o s it important that the E2dish is centrally placed in the culture dishes A Yes this is very important otherwise the reservoir holes will not line up with the electrodes which will either result in unstable recordings due to the electrodes touching the walls of the reservoir or damaged electrodes Use the template supplied in annex C o have trouble aligning the E dish using the alignment template A The alignment template has been designed to use with a 60 mm culture dish and small size deviations may cause prob lems If you downloaded the alignment template from Aleria Biodevices web make sure you select the No scaling opt
16. c cover Electrical characteristics Operating Temperature 10 C to 60 Supply Voltage from external AC DC adaptor 12V DC 2 Supply Current from external AC DC adaptor 400 mA 500 mA Number of channels 6 Maximun Input Signal peak to peak 2 2 mV Maximum Output Signal perADC 5 0V peak to peak Bandwidth 3dB of maximun gain 0 8 Hz 3 9 kHz Gain 2300 67 dB Input Noise Voltage grounded channel lt 1 uVRms Noise density grounded channel 18 nV VHz Spring Loaded Pin Max Number of 1 000 000 cycles Electrodes Ag Electrodes spring loaded pin interconnection material Stainless Steel Spring loaded pin material shell plating 20 um Gold over Nickel Inverted Microscope compatibility Yes Notes 1 Depending on microscope stage dimensions Data acquisition Number of bits 16 Sampling frequency all channels acquired Max sampling frequency only one channel acquired Power supply External AC DC Adaptor 10 ksps channel 60 ksps MASCOT 8613 regulated 12V MASCOT 9793 regulated 12V Plug in EU mains UK mains Output voltage 12V DC Max output current 400 mA 500 mA Output jack ext int length 5 5mm 2 1 mm 9mm Positive pin Center USB connection USB cable standard Male Male A Mini B Length Z TM ICSP In Circuit Serial Programming PCB connection Header r
17. cated in lt Installation folder gt My Screenshots Jl wo Prinkscreen ioj x File Edt View Favorites Tools Help N X a Qee Q gt Fp seach i gt Folders FF Address lo C Program Files Aleria Blodevices E2soft My Screenshots 3 11 2008 Go Name Size T Date Modified 155KB JPEG Image 11 3 2008 5 53 PM screenshot_2 155KB JPEG Image 11 3 2008 5 53 PM Details a screenshot JPEG Image Dimensions 1272 x 680 Size 154 KB Date Modified Monday November 03 2008 5 53 PM 3 Making a recording The next steps demonstrate how to make a recording 3 1 14 Choosing the channels from which to record By default all channels are activated for data acquisition To record from channels 1 3 and 5 only deactivate chan nels 2 4 and 6 by clicking the corresponding buttons in the Select channels panel Your panel should now look like the one displayed below gf e Select channels Acquire from channels DEME EM SER All Note that by deactivating channels 2 4 and 6 the sam pling frequency for channels 1 3 and 5 is doubled The E2drive automatically redistributes its sampling power over the number of active channels 3 2 Saving the recordings 3 3 3 4 By default recordings are saved to the folder lt Installa tion folder gt My Recordings In this case we will Save our recording onto the Desktop 3 2 1 In the Record to disk panel click the browse button shown
18. cone tube 2 m 5 mm ID 061 0002 1 Polyurethane antivibration pad 061 0001 A A eee 170x170x30 mm 1 Silicone tube 0 10 m 8 mm ID 061 0004 1 Electric cable with crocodile clips 060 0003 1 0 2 um gas filter 060 0006 1 E2soft Installation CD 1 Tube clamp 060 0007 1 E dish User Manual CO gas cylinder optional Units Component description Reference 1 CO gas cylinder with pressure 020 0002 regulator 2 L 2 Introduction to E2dish Technology A NEW APPROACH TO ELECTROPHYSIOLOGY FOR YOUR NEURONAL CULTURES The E2dish aims at making electrophysiology on neuronal cultures simple cost effective and high throughput for the research and drug screening oriented neuroscience community How it all works Conventional micropipette electrophysiology is cumbersome In conventional electrophysiology a micropipette is painstak ingly manipulated to approach the membrane of a cell A short distance lt 50um between micropipette and membrane suf fices for extracellular recordings whereas physical contact and seal are needed for loose patch whole cell or cell attached single channel patch clamp measurements Common to all configurations is the need for the flow of transmembrane cur rent to be constrained by a seal or alternatively by the intrinsic resistivity of the extracellular medium in order to generate measureable signals micropipete electrode a culture substrate neuron FIGURE 1 Conventional electrophysiology re
19. e Found New Hardware Wizard This wizard helps you install software for Alena E2drive If your hardware came with an installation CD 42 or floppy disk insert it now What do you want the wizard to do Install from a list or specific location Advanced Click Next to continue lt Back Cancel 2 Installing driver software Since the driver software is already present on your system Windows can install the software automatically Select nstal the software automatically Recommended and click Next Windows will now search for the device driver 2 1 f the driver is located automatically proceed to step 3 If the driver software is not found click Back and proceed to step 2 2 2 2 Check Install from a list or specific location Advanced and click Next 2 3 In the following screen click Browse and select the fol der nstallation folder Driver Click OK and then click Next 2 4 Depending on your Windows version and security set tings you may be presented with a Security Alert similar to the one shown below Click Yes to proceed with the installation 12 Security Alert Driver Installation x A The driver software you are installing for Alenia EZ dive has not been properly signed with Authenticode TM technology Therefore Windows cannot tell if the software has been modified since it was published The publisher s identity cannot be verified because of a problem The third part
20. el just above the first graph Notice how all graphs exhibit Change the voltage range of channel 1 by clicking the the same voltage range which facilitates comparison of Auto button located below the graph The software au activity across channels tomatically computes an appropriate range based on the last 10 seconds of activity The graph should now look E E2soft E l xj Fie Options Window Help Real time visualization Analysis HE Autoscale J Freeze i Printscreen Voltage UV Voltage UV Range 455 to 578 1 uv gt Auto Range 455 to 578 1 uv Auto al Gala p 8 Select channels oF x si Record to disk ii Mais yem Acquire from channels Location C Archivos de programa leri Duration Continuous Predefined length ea Detected ME PEPR BEE Name of the recording My recording ngh Bo JOO JOO hh mm ss Listen tochannet 1 y Description T Progr olume apsed time ni Vv Wis yi Start recording j Format Ready FIGURE 10 E2soft main window 13 Aleria Biodevices 2 3 2 4 Freezing image Freeze the graphs by clicking the Freeze button shown below Real time visualization is stopped until you click the Freeze button again Creating screenshots Create a screenshot of the graphs by clicking the Prints creen button shown below The screenshot is automati cally saved to the My Screenshots folder in JPEG format By default this folder is lo
21. established avoiding the use of micromanipulators Then AgCl macroelectrodes are lowered into the wells and recordings can be easily per formed with the E drive amplifier Use your proven protocols No need to change to new substrates Aleria Biodevices provides the integrated micropipettes of the E technology in the form of multiwell silicone units the E2dish The wells are 6 mm in diameter and 7 mm deep Each pair of wells is connected by integrated micropipettes implemented as 3 5 um high x 25 um wide x 1mm long mi crochannels on the bottom side of the polymer unit Fig 3 These E wells are placed on the cell culture substrate be It glass coverslips polystyrene dishes or other types of plastic substrates Fig 3 The cells are seeded in the two wells to E2dish well Il FIGURE 2 The E2dish employs substrate integrated microchan nels that function as electrophy siological micropipettes The cell spontaneously sprouts axons into the microchannel to attain a loose patch configuration Polymer Aleria Biodevices microchannel microchannel microchannel FIGURE 3 The integrated micropipette connects the wells of the E2dish A The E2dish can be attached on your preferred cell culture substra te with the microchannel facing down B FIGURE 4 Growth inside the microchannel E16 mouse hipoccam pus 14DIV adhere to the substrate and grow along the microchannel As sprouting axo
22. ion of the printer settings o What can do if bend the electrodes A If an electrode is slightly bent It can be easily straightened with tweezers However after straightening it is likely that the silver chloride coating will be damaged and so the electrode will have to be recoated If the electrodes are damaged at high er levels please contact support aleriabio com o My recordings are unstable noisy A First check that the fan and lights have been switched off inside the cabinet before starting recordings Make sure the electrodes are not touching the walls of the reservoirs and are not damaged If you still record a noisy signal you should make sure that the E drive is connected to the main building earth Check with the building maintenance personal if you are unsure about this 18 Q don t remember on which side of the E2dish is the microchannel A Place the E2dish in a clean and sterile dish and check under the microscope o There are bubbles in the microchannel what can do A When placing the E2dish on the substrate of your culture make sure you press gently the device from the centre to the outer part in order to remove air bubbles If you still have bub bles make sure that you remove the cover of the culture dish before you place the E2dish in the CO2 minichamber as this would result in an inadequate CO2 exposure o Which should be the final volume of culture medium for each well A The ma
23. ivity will be visible on the screen t can be saved and later analysed using the data analysis functions of the E2soft pack age see software guide for details FIGURE 9 The amplifier must sit on the antivibration pad provided during recording Tip If you are recording data with a drifting or unsta ble baseline this could be due to a silver chloride elec trode that requires re coating electrodes should be rechlorided every few weeks depending on frequency of use or any subsequent damage Tip If you have a noisy baseline check that the electrodes are not touching the reservoir walls of the E disli 4 E soft and driver installation manual This section provides system requirements and instructions for installing the Aleria E2soft software and the device driver on a Windows computer System requirements Operating Windows XP 32 bit Service Pack 2 and higher system Windows XP 64 bit Service Pack 2 and higher Minimum Pentium 4 2 4 GHz processor Minimum RAM 512 MB Minimum 20 MB disk space An additional disk space of 186 MB is required if the Microsoft NET 2 0 Framework is not present on the target machine In most cases the Microsoft NET 2 0 Framework is already installed Recommended A minimum screen resolution of 1280 x 1024 pixels with a 32 bit color depth is recommen ded display settings Note When using a laptop make sure it has a power supply with a 3 pin AC adapter wi
24. led with 50 uL of medium you should add up to 50 70 uL of cell suspension to reach the desired seeding density A B 089 VO 08 66 089 VO FIGURE 6 Schemes of an E2dish filled with culture media A and of an amplifier cover with electrodes B Note the two arrows showing the locating dowels to align the cover of the amplifier to the device 6 Cell growth Wait until the axons have sprouted into the channel usually 10 14 days drilled holes A new approach to electrophysiology for your neuronal cultures E2dish holding keys L FIGURE 7 The device has six drilled holes on its base A The E2dish should be placed so that the reservoirs are on either side of the drilled holes B As a result the microchannel will sit over them so that you will be able to be observed using an inverted microscope C Finally place the electrode cover on top of the amplifier making sure that the two locating dowels align with each other see Fig 6B Secure the cover with the white holding keys D On the day of recording 1 Connect the E2drive amplifier Connect the E2drive amplifier to your computer using the USB cable and connect the provided power supply FIGURE 8 USB and power connections 2 Start E2soft Start E2soft on your PC You should see 6 signal traces on your screen picking up electrical noise as the device has not been connected to the amplifier yet 3 Place the E dish in the am
25. ls in a culture The moderate seal resistance precludes measurements of in tracellular potential but allows detection of action potentials 19 Aleria Biodevices E2dish as an automated loose patch configuration The measurement techniques above require the confinement of a patch of membrane and the presence of the tip of the pipette acting as a sensor The electrical resistance of the confinement varies amongst techniques from a few KOhms in extracellular recordings continement effected by the ex tracellular medium itself to 10 50 MOhms in loose patches confinement by the pipette loosely attached to the cell and up to several GOhms in gigaseal patch clamps confinement by tight physical contact between pipette and membrane Confined Common to all the techniques A membrane path above is the need to manually ma nipulate the micropipette in close proximity to the cell E2dish follows an alternative strategy shown in figures A B and C A micropipette A can be integrated in a substrate using microfabrication technology B and neurons cultured in the vi cinity of the tip will sprout randomly into the integrated micropipette mi crochannel C The patch of mem brane grown in the microchannel is usually larger than the confined patch in conventional patchclamp For example for a L 200 um long axon inside the microchannel with a radius of R 0 5 um the total confined membrane is OL O x Surface 2 xm x Rx L 62
26. m or any portion of it may result in severe civil or criminal penalties and will be prosecuted to the maximum extent possible under the law Cancel Back 3 1 Click Next The License Agreement appears 3 2 Select Agree after reading the license agreement and click Nextto proceed The Select Installation Folder screen appears All xi Select Installation Folder Alerla merses Biodevices The installer will install E2soft to the following folder To install in this folder click Next To install to a different folder enter it below or click Browse Folder C Program FilestAleria Biodevices E 2soft Browse Disk Cost Install E2soft for yourself or for anyone who uses this computer Everyone Just me Cancel lt Back 3 3 Select the folder to which you want to install Aleria E soft To view the available drives that you can install to along with each drive s available disk space click Disk Cost Installing Aleria E2soft for Everyone ensures that a Start menu folder and a Desktop shortcut is created for each A new approach to electrophysiology for your neuronal cultures E2dish user on the system If you do not want this to happen then click Just me loj xi Confirm Installation Aleria moros Biodevices The installer is ready to install E2soft on your computer Click Next to start the installation 3 4 Click Next and the nstalling screen appears for
27. move the E2dish from the packaging Before removing the E2dish from the packaging note the side that is labeled microchannel Cut open the packaging and remove the E2dish carefully holding it with flat ended tweez ers Do not touch the microchannel side of the E dish to keep it clean 2 Sterilize the E2dish Once the E2dish has been removed it can be placed on a cul ture dish with the microchannel side facing up Sterilize it un der UV for 15 minutes 3 Mount the E2dish on the culture substrate The E2dish can now be attached to a clean and dry substrate on which the culture will be performed t is important that the microchannel side is down forming an enclosed channel when attached to the substrate see Fig 3 To remove any air bubbles in the microchannel the E2dish should be gently pressed using tweezers from the central to the outer part of the device Once the E2dish is attached to your substrate do not move it or re attach it to the same substrate as this might disturb and unbind your cell adhesion promoter e g poly lysine In addition if you re attach the E dish to a new clean substrate you may experience adhesion problems due to the residues of the cell adhesion promoter from the previous treated substrate Tip If you don t remember which side contains the microchannel place the E2dish in a clean sterile dish watch out for dust and check under the microscope Tip The geometry of a 60 mm cell culture dish
28. ns enter the embedded micropipette a loose patch recording configuration is spontaneously achieved after 1 2 weeks Fig 4 The culture should be checked regularly un der the microscope for growth inside the micropipette Single cell and population activity can be easily recorded over a long time period by placing the culture inside the E2drive amplifier Advantages of the E2dish 1 Simple to use Undergraduate students can seed the cells and record neuronal activity without supervision and with very little training 2 Single unit and population activity can be recorded 3 Your cultures grow on your preferred substrates plastic or glass using your standard protocol 4 With our multiwell arrays high throughput electrophysi ological screening is possible and cost effective E2dishes are available in two well and twelve well versions 3 Quick start guide to using the E2dish This section will provide you with important information on how to handle the E2dish Procedure Overview VAL AV ADIN FROM PACKAGING On the day of seeding Note Stages 1 5 must be carried out in a laminar flow hood and nitrile or latex gloves should be worn as it is very important that the E2dish is not exposed to dust and moisture from your fingers This would have a negative effect on the adhesion to the substrate Note 60 mm culture dishes must be used with the E dish in order to fit the E drive amplifier 1 Re
29. ou O00 000 oe 00 i 00 00 00 800 Time hhmm ss Remove a channel from the graph by clicking on one of the numbered buttons located above the graph Using these buttons you can toggle the appearence of a chan nel in the dataset Add the channel again to the plot by clicking on the same button 4 4 Zooming Zoom into a portion of the dataset by left clicking in the main graph and while holding the mouse button drag ging out a rectangle Release the mouse button to zoom into the region you selected 15 Aleria Biodevices 4 5 Un zooming Right click within the main graph to return the previous zoon level 5 Analyzing a data The Analysis panel contains functionality to filter your data and apply a burst detection algorithm dl Analysis of x Analysis Filtering Select a filter High frequency noise removal Edit New Select an analysis Burst detection Aun 5 1 Reducing noise Reduce noise in your recording by applying one of the already provided filters to your data or create a new filter of your own 5 2 Creating a Filter Apply a 100 3000 Hz bandpass filter to the data To do so click the New button to display the Filter design screen to create a new digital filter Filter design xj Design method IIR Butterworth y C FIR JE quiripple Magnitude response dB gt Response type 3 3 Lowpass 3 D C bi E Highpass E Bandp
30. ow 5 sguare pins Pitch 2 54 mm 0 1 inches The physical distribution of the channels to be used with the E2soft Software is shown in the figure below The ground pins are connected internally then all channels have a common reference ground Fig 11 ORIENTATION SIGNAL ORIENTATION SIGNAL FIGURE 11 Plastic cover spring loaded pin distribution The number of channels used from 1 to 6 depends on the user application It is possible to ground any channels by hard ware using the jumpers installed on the printed circuit board PCB CH cH J2 GB NOT USED T O o 7 GND J1 O LL Ww D 7 Poe O O Z solo 0 EO S OW O N 9 2 2 9 J3 O cua FIGURE 12 Jumper settings for channel connection inside PCB 21 Annex C E dish substrate alignment template SOC SOC Download the template at www aleriabio com 22 Notes 4 Aleria ww 47 Biodevices Parc Cientific de Barcelona c Baldiri Reixac 15 21 08028 Barcelona Spain www aleriabio com info aleriabio com Kg Hg Rg Hg Mg hig Py Hg Mg high Eee ee ee S
31. plifier Take out the dish from the incubator and place it in the Edrive amplifier There are 6 holes in the amplifier dish holder Fig 7A Position the dish so that each hole in the base is aligned between the two reservoir holes in the E2dish Fig 7C This way the holes will be underneath each of the 6 microchannels so that they can be observed using an inverted microscope Tip If you are using the E2drive for the first time remember to chloride the electrodes of the device A simple chloriding procedure is to submerge the elec trodes in a bleach solution for about 20 minutes The wires should turn from a bright metal to a unifor mally blackened colour once coated with the solution 4 Position the amplifier s cover The cover with the integrated AgCI electrodes can now be positioned over the top of the amplifier dish holder and into the respective reservoirs of the E2dish Fig 7D Check that the electrodes are not touching the reservoir walls of the E2dish as this will affect the quality of the recordings Lightly push down the electrode cover and make sure It seats cor rectly on the face of the amplifier While maintaining light fin ger pressure on the cover the three white holding keys can be turned to keep it in place 5 Recording The amplifier must sit on the polyurethane pad provided Fig 9 for vibration isolation Turn off the hood lights and fan to avoid electrical and mechanical interference The electrical ac t
32. quires manipulation of micropipettes with micrometer resolution Approaching the cell with a micropipette and obtaining elec trophysiological measurements is a low throughput process requiring trained personnel and expensive micromanipula tion and electronics instrumentation E2dish well Integrated micropipette Polymer neuron MICROCHANNEL culture substrate E technology automates electrophysiology by integrating the micropipette in the substrate The E electrophysiology enabling product family of Aleria Bi odevices offers a simpler method for all neuroscience labs to gather electrophysiological data from their cultures The core concept can be summarized as et the cells in your culture do the hard work and approach the micropipette rather than the other way around The E technology involves cell culture wells with integrated microchannels acting as embedded micropipettes patent pending Effectively the micropipette of conventional elec trophysiology Fig 1 which usually approaches the cell at approximately 45 degrees is laid horizontally and integrated with the cell culture substrate in the E dish Fig 2 by using microfabrication technology The integrated micropipette is lo cated at the level of the culture so that a subset of nearby cul tured neurons will sorout axons that will soontaneously enter the microchannel At this point a loose patch electrophysi ology configuration is spontaneously
33. ra mework x For the following components HET Framework 2 0 x86 Please read the following license agreement Press the page down key to see the rest of the agreement MICROSOFT SOFTWARE SUPPLEMENTAL LICENSE TERMS MICROSOFT NET FRAMEWORK 2 0 MICROSOFT WINDOW S INSTALLER 2 0 MICROSOFT WINDOWS INSTALLER 3 1 Microsoft Corporation or based on where you lve one of ite affiliates licenses this supplement to you f you are licensed to use Microsoft Windows operating system software the software You may use this supplement ou may not use iti you do not have a license for the software ou may use a copy of this supplement with each valid licensed copy of the software View EULA for printing Do you accept the terms of the pending License Agreement IF You choose Dont Accept install will close To install you must accept this agreement Accept Don t Accept 3 E2soft Setup Wizard After all pre requisites have been installed the Welcome to the Setup Wizard screen appears Follow the instructions given to proceed successfully with the installations of the software nixi Welcome to the E2soft Setup Wizard T Aler se ait My tee Sa 2 Bilodevices The installer will guide you through the steps required to install E2soft on pour computer WARNING This computer program i protected by copyright law and international treaties Unauthorized duplication or distribution of this progra
34. s installed right after you connect the E2drive to your PC for the first time The driver may not have been cor rectly installed or may have disappeared from your system after installation In that case the E2drive might not be recog nized by Windows or might not work properly Follow the procedure below to check the status of the E2drive and its driver 1 Ensure that the E drive is disconnected to the PC Check that the USB port works properly by attaching a USB memo ry stick or some other USB device like a mouse or keyboard 2 Attach the E drive You may receive one of the following message boxes 2 1 USB Device Not Recognized In case a message box appears that informs you that the USB device is not recognized like the one shown below the cause of the problem is hardware related In that case contact the technical support of Aleria Bio devices A USB Device Not Recognized x One of the USB devices attached to this computer has malfunctioned and Windows does not recognize it For assistance in solving this problem click this message 2 2 Found New Hardware In case a message box appears that informs you that new hardware is found like the one shown below wait until the New Hardware Found wizard is shown Then follow the procedure described in the E2soft and driver installa tion manual If the New Hardware Found wizard does not appear automatically proceed to step 3 4 Found New Hardware EJ Aleria Ezdrive
35. t tutorial demonstrates how you can similar to the one shown below You can also manually use the Aleria E2soft software to view realtime specify the voltage range by typing directly into the text data make a recording and load the recording for boxes next to Range After changing the value in a text box press Enter to apply the change further analysis Please follow the steps below Channel 1 Note This tutorial assumes that the Aleria E2soft software and the E2drive device driver have been successfully installed If this is not the case plea se follow the installation procedure of the previous section prior to starting this tutorial TE iKi l hi Voltage pV 1 Loading the software Connect the E drive and Load Aleria s E2soft by double clicking on the desktop icon or by selecting on your Start menu Start gt Programs gt Aleria gt E2soft The main window appears Fig 10 0 30 40 50 60 FO G 30 100 Time ms Range 55 3 to 50 0 E Auto 2 Viewing real time data The six graphs visualize real time activity of channels 1 to 6 Each graph displays a hundred milliseconds of activity and is refreshed ten times per second By default the Y axis dis 2 2 Changing the voltage range of all channels plays a voltage range trom 200 to 200 microvolts Change the voltage range of all channels simultaneously by clicking the Autoscale button shown below located 2 1 Changing the voltage range of a chann
36. th earth connect Alternatively Aleria Biodevices provi des a cable with crocodile clips that should be connected to one of the three non painted metallic screws of the E2drive and to a metallic structure in your lab grounded to the main building earth E2soft Installation Note Do not connect the E2drive before finishing the installation of the software 1 Loading E soft Insert the E soft Installation CD into your computer s CD ROM drive If the installer does not load automatically go to My Computer and double click the CD ROM drive icon 10 2 Automatic check of files The installer will automatically check if the Visual C Runt ime Libraries and the NET 2 0 Framework are present on your system If they are not the installer will ask your permission to install them Please follow the procedure described below 2 1 Visual C Runtime Libraries If the Visual C Runtime Libraries are not present on your system the installer presents you with the screen shown below Click Install to install the Visual C Run time Libraries x The following components will be installed on your machine Visual C Runtime Libraries x86 Do you wish to install these components IF You choose Cancel setup will exit 2 2 Microsoft NET 2 0 Framework If the Microsoft NET 2 0 Framework is not installed on your system the installer presents you with the screen depicted below Click Accept to install the NET 2 0 F
37. ximum volume the wells of the E2dish admit is ap proximately 120 uL o While recording is my culture sterile A The cover of the amplifier should maintain the sterility of your culture However it is highly recommended that you al ways work under the flow hood o How long can record from the E2dish A The E technology does not limit the recording time This will only depend on your experimental conditions and on the remaining disk space left on your computer o have trouble aligning the E dish using the alignment template A The alignment template has been designed to use with a 60 mm culture dish and small size deviations may cause prob lems If you downloaded the alignment template from Aleria Biodevices web make sure you select the No scaling option of the printer settings Annex A Theory of Operation of Edish Technology Electrical activity in neurons is asso ciated with the transmembrane flow of ions mostly Nat K and Ca in the millisecond time scale resulting in a relatively fast change in mem brane voltage the action poten tial AP Most electrophysiological techniques attempt to measure this change in membrane potential current clamp modes or the actual ionic currents underlying it voltage clamp modes During an action potential the membrane can depolarize up to 100 mV above its resting value for approximately 1ms as a re sult of the inflow of positive charges Na The actual ch
38. y INF does not contain digital signature information Do you still want to install this driver software 3 Driver version If more than one device driver exists on your machine for instance an older version you may be presented with the screen shown below Select the latest version of the Aleria E2drive device driver and click Next This installation may take up to a minute on slower machines Found New Hardware Wizard i E Please select the best match for your hardware from the list below Phy a Alera Ef drive Description Manufacturer Aleria E e J i Alera EZ drive Alera Biodevices cc windowsinf oem2s ir gt This driver is not digitally signed Tell me why driver signing is important Back Cancel 4 Completing installation Once the device driver has been installed correctly you will be presented with the screen shown below If you receive an error notification click here Otherwise click Finish to exit the Found New Hardware Wizard Found New Hardware Wizard Completing the Found New Hardware Wizard The wizard has finished installing the software for Alenia EZ drive Click Finish to close the wizard lt Back Lancel Note Please be aware that this procedure must be repeated for each USB port the E2drive is connec ted to Once the device driver is installed you can use the E2drive unimpededly with that USB port 5 E2soft tutorial This quick star
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