3Brain BioCAM X User manual

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Welcome to

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Last updated 25.09.2019
User Guide
Essentials

Contents
5Introduction
6MEA technology
6CMOS-MEA technology
8At a glance
9Overview
10 BioChip
11 BioCAM X
13 Host PC
13 Desktop PC
13 Laptop PC
14 BrainWave X
15 Precautions
16 BioCAM X handling and use
17 BioChip handling and use
18 Cleaning procedure for BioChips
19 Sterilization procedure for BioChips
20 Get started
21 Install the BioCAM X
21 Desktop host PC
21 Laptop host PC
22 Plug the BioChip
23 Setupanonpre-conguredhostPC
23 Verify proper functioning
24 Quickguidetoyourrstuse
25 Basic concepts
25 Visualization of data
26 MEA Viewer control
27 Data formats
28 Online mode (Record mode)
28 Important notes
28 Open recorder
29 Visualize live data
30 BioChip Calibration
31 Record live data
32 Oinemode(Playbackmode)
32 Open recorded data
33 Quick analysis and save of results
34 Visualize recorded data
35 Understandingmaincontrolsaectinggraphs

36 Create a group of relevant channels
37 Visualize Instantaneous MFR
38 Visualize Raster
39 Troubleshooting
40 Electrodes with no-signal (saturation)
42 BioChip not properly initialized
44 Not working columns
45 BioChip with dirty surface

Introduction
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Welcome to the BioCAM X platform and thank you for your purchase. The BioCAM X platform is among
the most advanced systems for managing your experiments on the emerging generation of high-den-
sity CMOS-MEAs. This manual has been written to help you to take advantage of all functionalities
provided by your BioCAM X. Be sure to read this manual thoroughly, and to keep it handy when using
the BioCAM X platform.
Before any use of your BioCAM X platform, please read the “Precautions” on page 15, which contains
relevant information to preserver your BioChips and BioCAM X from possible damages.
MEA technology
Amongthedierentmethodologiesusedforelectrophysiologicalmeasures,metalmicroelectrodesin-
tegrated on-chip can provide multisite measures of extracellular signals with a high signal-to-noise ratio.
In addition, by applying voltage or current stimuli to the same microelectrodes it is possible to depolar-
ize cells or tissues, thus establishing bi-directional interfaces. As established over several decades of
research, both sensing and actuating performances of microelectrodes can be applied to study a wide
range of electrogenic cells and tissues, including neuronal and cardiac preparations.
Conventionalmicroelectrodearrays(MEAs)arebio-sensingchipsrealizedbymeansofthin-lmtech-
nologies and do not integrate on-chip any microelectronic circuit. Therefore, conventional MEAs are
passive devices made on silicon, glass or polymeric substrates. Each microelectrode can be made by
dierentmaterials(e.g.Pt,IrOx,TiN)anditisindividuallywiredon-chipandconnectedtoanexternal
amplieranddataacquisition(DAQ)instrument.DuetotheneedofthisMEAtechnologyofindividually
routing on-chip each electrode, space constraints and wiring encumbrance impede the realization of
dense and large electrode arrays. Thus, for conventional MEAs the typical electrode pitch is in the range
of 100 µm and the array includes from 60 up to 256 microelectrodes. In addition, given the distance
betweentheelectrodesandtheo-chipampliersandtheuseofinterconnectingwires,conventional
MEAs are subjected to inductive coupling noise.
CMOS-MEA technology
The electrode density and array sizes can be increased by changing the technology used to realize
microelectrode arrays (MEAs). High-density MEAs are realized with complementary metal–oxide–semi-
conductor technology (CMOS), as it is done for microelectronic devices (e.g., computer microproces-
sors) and light-imaging devices (e.g., camera sensor), and with post-processing methods to optimize the
electrode performances.
Briey,theCMOStechnologyallowstorealizeactiveelectrode-pixelsthatintegrateinsmallareasofa
fewsquaremicrometerselectrodes,ampliersandsignalconditioningcircuitsin-pixel,justunderneath
eachelectrodesite.On-chip,additionalamplicationstages,multiplexingandhigh-speedaddressing
circuits are provided.
In particular, the circuit architecture of 3Brain’s BioChips is based on the Active Pixel Sensor concepts,
as commonly used for light imaging CMOS cameras, and was designed to allow full array recordings at

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sucientlyhighsamplingfrequencyforeachelectrode.
InBioChipsthelocalin-pixelbuersunderneatheachelectrodeallowtolocallyadaptsignalsfromhigh
impedance (electrode-side) to low impedance (wiring-side) to avoid the induction of coupling noise
fromelectricalwiringbeforeamplication.Additionally,theon-chipmultiplexingandaddressingcircuits
allow to minimize the number of wiring outputs even though the large number of electrodes that have
tobesimultaneouslymeasured.Indeed,eachelectrodesignalisnotindividuallywiredo-chip.Rather,
signalsofmultipleelectrodesaremultiplexedathighfrequencyoverafewnumberofwires.Inthisway,
several thousands of electrodes can be recorded on only a few tens of output wires.
The in-pixel circuit implemented in BioChips allows recordings from a large signal bandwidth that
includeseldpotentialsandspikes.InsteadofimplementinganAC-couplingsolutionthatwouldrequire
largeareasfortheintegrationofadequatecapacitors,theBioChipsin-pixelcircuitintegratesanau-
to-zeroing circuit that is regularly calibrated to the electrode DC voltage and that subtracts this cali-
bratedDCvoltagefromtheelectrodesignalbeforetherstamplicationstage.Thisprocessiscalled
calibrationanditalsoallowstoadaptthecircuitperformancetodierentexperimentalconditions.In
particular,allCMOSdevicesarephotosensitiveandphoto-generatedchargesgiverisetodierentDC
driftingtimesthatcanbringin-pixelamplierstosaturate.BioChipsmitigatetheseeectsandhence
allowexperimentsunderlightstimulationconditions(asrequiredforinstanceforretinastimulation)by
usinghighercalibrationfrequenciestokeeptheDCinputsignaltotheworkingpointoftheamplierand
toavoidthesaturationoftheamplier.

At a glance
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Overview
The BioCAM X platform is a high resolution electrophysiology system capable to perform in-vitro elec-
trophysiological measures on electroactive cells and tissues. Even though, to some extents, the plat-
form has some similarities with more conventional Microelectrode Array (MEA) systems, it is a radically
newtechnologytoperformextracellularmeasures.DierentlythanconventionalMEAsystems,the
BioCAM X platform relies on active CMOS-MEA chips, which integrate thousands of miniaturized active
electrodes over large areas. Combined to sub-millisecond temporal resolution, the dense integration of
electrodes results in a spatial resolution that enables novel analysis, can increase the statistical signif-
icance of your experiments and opens new imaging approaches to investigate electroactive prepara-
tions.
The 3Brain’s BioCAM X platforms was tested so far for in-vitro electrophysiological experiments on
neuronal cell cultures, brain slices, retina preparations and cardiac cultures. Electrophysiological signals
rangingfromsloweldpotentialstofastsinglecellspikingactivitycanberecordedsimultaneously.
ExamplesofrecordingsprovidedbydierentlaboratoriesusingtheBioCamplatformsareavailableon
the 3Brain website and uploads of novel examples based on your research are warmly welcome.
Overall, the platform consists of three basic components: A) a multiuse BioChip cartridge available in
dierentmodelsandincorporatingtheCMOS-MEAsiliconchip;B)theacquisitionBioCAMXhardware
thatreadsoutelectrophysiologicalsignalsfromtheBioChip;C)thehostPCequippedwiththeacqui-
sitionboardandtheBrainWaveXsoftwaretoacquire,visualize,storeandanalyseexperimentaldata
recorded with the BioCAM X platform.

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BioChip
The BioChip cartridge integrates on an electronic substrate the CMOS-MEA chip and a reservoir cham-
ber to maintain cultures or tissues contacting the electrode array either under cell culture media or per-
fused media, respectively. The cartridge is 54 mm x 54 mm (2-1/8 in x 2-1/8 in) in size and is conceived
to be placed in an incubator for cells or tissue cultures.
• Contact pads : gold-plated pads for connection between the BioChip and the BioCAM X. Properly
cleaned and non-oxidized pads are needed for a stable connection.
• PCB :theprintedcircuitboardsubstrateoftheBioChip.Dierentcolorsareavailableandaccordingto
the layout of the CMOS-MEA.
• Glass ring :borosilicateglassringdeningthereservoir.Externaldiameter28mm(1-7/64in),internal
diameter 25mm (0-63/64 in), height 5mm (0-13/64 in).
• Reservoir : chamber for the bath solution used for the biological preparation. The volume of solution
that can be contained is of about 2mL.
• CMOS-MEA : chip integrating thousands of active electrodes for high-resolution electrophysiological
recordingsfromcellsortissuesplacedonitsactivearea.Dierentapplication-specicMEAlayouts
according to the BioChip model are available.
Contact pads
Glass ring
PCB
Reservoir
CMOS-MEA

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BioCAM X
The BioCAM X is the station where BioChips are plugged into for electrophysiological measures. This
instrument is compatible with all BioChip types including also those with electrical stimulation capabili-
ties. It contains all the electronics, real-time hardware and logic to control the CMOS-MEAs chips on the
BioChipcartridgesandtoacquireandpre-processlarge-volumeofelectrophysiologicaldatabefore
sending it to the host PC.
Magnetic plate
Lock button
Peltier-Element
BioChip bay
• Lock button : two-position button for locking (end position) or unlocking (mid position) the BioChips.
• BioChip bay : bay for inserting the BioChip and connecting it to the BioCAM X.
• Magnetic plate : ferromagnetic stainless steel to attach magnetic holders (e.g., for perfusion).
• Peltier-Element : active element to control the temperature of your preparation on BioChips

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Power button Camera Link USB BCExt connector Power port
• Camera-link :SDRminiCameraLinkconnectortoconnecttheBioCAMXtotheacquisitionboard
mounted in the host PC. Electrophysiological data and control signals are managed through this
interface.
• USB :thisportisusedonlyforrmwareupdatesoftheBioCAMX.Fornormaluseitisnotneededto
connect this interface.
• BCExt connector : plug the BCExtAdapter cable to access extended functionalities such us triggering
and stimulation.
• Power port : plug for the power supply unit.
BioChip slot Bay Barrier & BioChip connector Reference connector LED1 LED2
• BioChip slot : the slot through which the BioChip (contact pads side) is inserted into the BioCAM X for
electrical interfacing.
• Bay Barrier & BioChip connector : immediately behind the BioChip slot, a protective movable Bay Bar-
rierblocksaccidentaloverowsofliquidsthatmightgetintouchanddamagetheinternalelectronics.
Behind the Bay Barrier, the BioChip connector allows to connect the BioChip’s contact pads to the
internal circuitry. Electrical contacts are established only once the locking system is activated.
• Reference connector : connector for the pseudo-reference electrode that need to be placed in the
bath solution of the BioChip cartridges.
• LED1 : a solid blue LED indicates whether the BioCAM X is powered (LED ON) or not (LED OFF). A sol-
idvioletLEDindicatesafailureinBioCAMrmware.Inaddition,theLED1blinksviolettoindicatethat
the temperature control is ON.
• LED2 :asolidgreenLEDindicateswhethertheBioCAMXisacquiringsignal(LEDON)orisinstand-by
mode (LED OFF).
• Power button :push-buttontopowerONorOFFotheBioCAMX(theinstrumentneedtobecon-
nected to a power line using the provided adaptor).

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Host PC
Depending on your order the host PC can be a desktop PC or a laptop. The way the BioCAM X instru-
ment is connected to the host PC changes according to these two cases.
Desktop PC
When connected to a desktop workstation, the connection is direct between the BioCAM X and the
hostPCthatisequippedwithaCameraLinkacquisitionboard.Thetypicalcongurationisasfollow
(tower desktop not shown).
BioCAM X with
BioChip
Desktop host PC
with BrainWave X
Laptop PC
When connected to a laptop PC, you need an adapter to connect the BioCAM X to the host PC. Such
adapterisaThunderbolt-CameraLinkadapterboxthatenclosestheCameraLinkacquisitionboard.
ThetypicalcongurationforalaptophostingPCisillustratedherebelow.
BioCAM X with
BioChip
Laptop host PC
with BrainWave X
Thunderbolt-
Camera Link adapter

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BrainWave X
BrainWave X is a complete software application developed to exploit all the functionalities of your
BioCAMXplatform.Itisequippedwithadvancedtoolsadaptedtomanagehigh-resolutionelectro-
physiologicaldataacquiredfromBioChips.Whenlauncheditappearsasfollows(withoutexperiments
ongoing).
• Main Menu : access main functions by clicking on the main menu.
• Tool Bar : displays tools provided by the data controls currently shown in the Workspace. Tools are
refreshed according to the currently focused controls and for some controls can be missing.
• Panels : dockable panels that are automatically updated according to the currently selected experi-
ment loaded into the Workspace. Panels allow you to modify the experiment’s options and parame-
ters and can be shown or hidden by choosing the Window menu on the Main Menu.
• Left Status Bar :notiesyouaboutongoingoperations.
• Workspace :arenaforvisualizingthroughtabsonlineandoineexperiments.
• Right Status Bar :notiesyouaboutthestatusofthecontrolleddevices.ItcomprisesalsoLEDicons
for BioCAM and BioChip devices. If the LED icon is on (blue) the corresponding device is properly
connected and recognized.
Main Menu
Tool Bar
Panels
Workspace
Left Status Bar Right Status Bar

Precautions
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BioCAM X handling and use
The BioCAM X instrument is made with a solid metal case and with optimized electronics that make
the system robust to mechanical and vibrational noise. Even if it is recommended, the installation on an
anti-vibration table is not mandatory.
Moreover, the instrument has been design to be shielded from external electrical environmental noise.
However, to guarantee optimal recording performances simple precautions as listed here below should
be followed:
• Position the BioCAM X far from potential strong noise sources, such as fridges, incubators etc… In-
deed, if not well shielded these instruments might generate electromagnetic noise.
• ConnecttheBioCAMXtoadequatepowerlineswithlownoiselevels.
• Take care to avoid that electrical cables connected to the BioCAM X (in particular, the one for the
power supply and for the Camera Link) do not pass near other power supplies.
• Check that the cable connectors are well inserted into the BioCAM X and the Host PC before operat-
ing the platform.
The system allows for the integration of a perfusion system as it is needed for maintaining tissues
during electrophysiological measures. Even if precautions have been taken in designing the system to
preventliquidstoenterincontactwiththeinternalelectronics,spilloutofelectrophysiologicalsolution
that might get into the BioChip slot should be strictly avoided. However, should this happen please
follow these steps:
1. Immediately switch OFF the BioCAM and remove the BioChip.
2. Clean accurately the BioChip Bay of the BioCAM X with ethanol 96% and let it dry. Execute the same
procedure for all the BioChip parts but for the BioChip reservoir and in particular for the BioChip con-
nector pads.
3. In case after the cleaning with ethanol, there are still doubts that some liquid could have been
penetrated into the BioCAM X, do not switch on the system for at least one day, allowing the liquid
to be completed dried and preserving internal circuitry from potential oxidative and short-circuit
eects.
Some precautions have to be also taken when manipulating the pseudo-reference, which from one
side (connector) is connected to the BioCAM X and whose other end (platinum wire) needs to be placed
into the bath solution inside the BioChips:
• Handle the reference with care by wearing gloves and avoid touching the platinum wire (i.e. the part
of the reference that is in contact with the electrophysiological solution).
• Clean regularly the reference to guarantee optimal recording performances. The platinum wire
of the reference should be rinsed with deionized water before starting and after an experimental
session. Alternatively, for a heavier cleaning, the platinum wire only can be immersed in HCl 0.1M for
about 1 minute and then abundantly rinsed with deionized water.
• When the BioChip is inserted and the reference is positioned in the electrophysiological solu-
tion, the operation of switching ON or OFF the BioCAM X might cause electrical glitches that can
potentially damage the circuitry of the CMOS-MEA chip. To avoid this, it is a good practice to follow
theoperationalsequenceofpluggingtheBioChip,switchingONtheBioCAMXandtheninsertthe
referenceintotheliquid.Inananalogousway,removethereferencefromtheelectrophysiological
solution before switching OFF the BioCAM X and unplug the BioChip.

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BioChip handling and use
BioChips are low-power active electronic circuits and it is highly recommended to avoid the following
operations that might result in a damaging of their electronics:
• Do not touch the contact pads of the BioChip. Electrostatic charges might damage the on-chip
circuits and might result in damaging the device. Always manipulate the BioChip by holding it on the
sides and ideally by using plastic gloves.
• Do not hold the BioChip by its glass ring to avoid detaching it.
• DonottouchtheactiveareaoftheBioChipswithanytoolsexceptthosespecicallyallowed(see
“Cleaning procedure for BioChips”). In particular, hard or semi-hard objects (e.g. metal or plastic tools)
can irreparably damage the electrodes and the on-chip circuits.
• For cleaning or for sterilization avoid immersing the entire BioChip in water or ethanol. Prolonged
immersion in water of the entire BioChip might cause oxidation of the contact pads. In ethanol, some
parts of the BioChip might be deteriorated. As a general rule only the chamber can be wet while the
rest of the chip should stay dry.
• Do not place the BioChips in autoclaves, ovens or under UV-light exposure for sterilization. These
methods might deteriorate the CMOS devices, resulting in malfunctioning or in reduced lifetime. For
the BioChip sterilization, please refer to the “Sterilization procedure for BioChips”.
• Maintain the pH of the electrophysiological solutions used for neuronal cultures or brain tissues pos-
sibly at physiological conditions (7-7.5). Important changes in the pH of the solution might damage the
electrodes.
During recordings (both cultures and brain tissues) users have to comply with the following precautions:
• Before contacting the BioChip into the BioCam, clean the metal pad contacts with a tissue paper
soaked with ethanol 96% and let it dry for few seconds.
• Avoidliquidstospilloutandstrictlyavoidliquidstoenterincontactwiththepads(theBioCAMsys-
temisequippedwithaBayBarriertoprotecttheconnectingsocket).Incaseusersexperienceliquid
spill out, follows instructions provided in the “BioCAM X handling and use” section.
Inordertoensureagoodqualityoftherecordings:
• Avoid using HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) in the solutions used for
electrophysiological recordings, since we observed that it can interfere with the electrodes making
the chip unstable and the recordings very noisy.
• BioChipsarelight-sensitivedevicesandtheirperformancesmightbeaectedbyatoohighintensity
direct exposure or by noisy light sources.

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Cleaning procedure for BioChips
Normal cleaning
It is a good practice to clean the BioChips immediately after the experimental recordings both for cell
cultures as well as brain tissues. First, rinse abundantly the BioChips with Double Deionized Water
(DDW),thenllthechamberswithadetergentasWPI-Enzol(WPI)orTerg-A-zyme(Alconox)andgently
pipette for few seconds. Leave the detergent for few minutes (typically 3-5 min) and then pipette again.
Aftertheuseofdetergent,rinsetheBioChipsabundantlywithDDW,thenleavethechamberlledwith
DDW for 1-2 minutes and repeat this operation 3-4 times in order to assure to wash out completely the
detergent.
Intense cleaning
In case the chips are very dirty, users can mechanically remove cellular debris with a soft brush. Be
aware that this operation can damage the sensing area of the chip, so it is suggested only if, after a
normal cleaning procedure, the chips still present dirtiness and in any case it has to be performed very
mildly and carefully.
Finishing and storage
BiochipscanbedriedbyusingagentleuxofNitrogenair(donotdirectlyexposetherecordingareato
theuxtoavoidpotentialelectrodedamages).AlternativelylettheBioChipsdryonabenchbycovering
them with a plastic petri dish to avoid dust deposition on the recording area.
The Biochips area out of the chamber can be cleaned with a tissue soaked in ethanol 96%. It’ a good
practice to manipulate chips always wearing gloves.
Once the BioChips are dried they should be stored in a closed box in order to protect them from dust
and dirtiness.
As a general rule, avoid sterilizing or using ethanol in the BioChip chamber without having preventively
cleanedthechipfromcellulardebrisofpreviousexperiments.Alcoholcanxbiologicalmaterialsonthe
recording area of the chips, resulting in a degeneration of the signal recording performances.

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Sterilization procedure for BioChips
As indicated in the previous section, BioChips cannot be put in autoclaves, ovens or under UV-light
exposure for sterilization purpose. These procedures risk to deteriorate the chips resulting in not optimal
recording performances and in a shorter lifetime.
If still not done, perform a cleaning procedure as indicated in the “Cleaning procedure for BioChips”
previous section and adopt the following procedure (that has to be performed under a biological hood)
to sterilize the chips:
1. Clean the area of the BioChips outside the glass ring (including the external part of the glass ring
itself) by using a tissue soaked with ethanol 96% and put inside sterile plastic petri dishes.
2. Fill the reservoir completely with ethanol 70%. Also dip with the same solution the border of the glass
ring (the tiny upward-oriented side of the ring). Hence, wait for 20 minutes.
3. Suck the ethanol with a pipette or with a vacuum pump always using sterile tips.
4. Fill3/4ofthereservoiroftheBioChipswithsterileDDW,waitfewsecondsandsucktheliquidalways
with sterile tips. Repeat these operations 4 times to ensure to wash out completely the ethanol. When
removing sterile DDW for the last time, be sure to have completely dried the chamber. Aspirate the
liquidontherecordingareaalwaysbykeepingthetipclosetotheglue/CMOSborderbutnottouch-
ing the CMOS to avoid any risk to damage the electrodes.

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