Eppendorf Femtojet Manual - Duke University Light Microscopy ...

FemtoJet ®Operating ManualEppendorf AG22331 Hamburg · GermanyPhone: +49 40-5 38 01-0Fax: +49 40-5 38 01-556e-mail: eppendorf@eppendorf.comInternet: www.eppendorf.comApplication Hotline:Phone: +49 180-3 66 67 89e-mail:application-hotline@eppendorf.deBrinkmann Instruments, Inc.One Cantiague Road,P.O. Box 1019Westbury, New York 11590-0207(USA)Phone: 800-645-3050Fax: 516-334-7506e-mail: info@brinkmann.comInternet: www.brinkmann.comPrinted in Germanyeppendorf ® is a registered trademark

1 Safety precautions and application●Observe operating instructions!●Before plugging in the device, please compare your voltagerequirements with the specifications on the identification plate!●The unit must be connected to an earthed socket!●Electrical connections may be effected only when the device isswitched off!●The unit may be opened only by authorized service personnel!Potentially lethal voltage inside the unit.●Do not use the unit if damaged. This applies in particular when themains cable is damaged.●Electrical connection with devices not expressly mentioned in thisoperating manual is permitted only after obtaining prior approval fromthe manufacturer!●If the unit is not used in accordance with purpose for which it isintended, its protective function may be impaired.●Caution!Take care when mounting glass capillaries or tools!Mounting must always take place without the useof pressure! Capillaries which have been mountedincorrectly may break loose under pressure. Neverpoint mounted capillaries at persons.●Caution!Take care when using glass capillaries! Duringmounting or when placed upon a hard surface, theglass capillaries may break and glass splinters maycause injury. It is therefore essential to wear eyeprotection!3

1 Safety precautions and application●In the case of independently-manufactured capillaries, ensure that theouter diameter of the capillary corresponds to the specifications of thecapillary grip head.●In the case of extended periods of non-use, the unit should bedepressurized using the key and switched off before beingstored!●Use the unit only for the described purpose! The unit and in particularthe high pressure may not be used for purposes other thanmicroinjection!●Use only original Eppendorf accessories. Only original accessoryparts may be used. The only exceptions are auxiliary pieces ofequipment recommended by the manufacturer. Otherwise all claimsunder guarantee and liability become void immediately. Use otheraccessories only if you have a certificate from Eppendorf AGconfirming technical safety in use.●The responsibility for the correct functioning of the device passesonto the owner or user if the device is maintained or servicedimproperly by persons not belonging to the Eppendorf service team orif used for a purpose other than that intended. Eppendorf AG is notliable for damage occurring from non-observance of these notes.Guarantee and conditions of liability of the terms of sale and deliveryof Eppendorf AG are not expanded by the above notes.●The Eppendorf AG reserves the right to make technical alterations tothis device!4

2 Device descriptionThe FemtoJet ® enables minute quantities of liquid to be injected into cells.The relevant parameters (injection pressure p i and injection time t i )are set using the variable regulators. The key or connected hand orfoot control or the INJECT key of the connected micromanipulator areused to trigger these two parameters. If no injections are being performed,compensation pressure p c is applied permanently.5

2 Device description2.1 Overview12341 Display– Displays relevant parameters during injection or settingof parameters.– Guides operator through functions.2 Keypad– To trigger actions.– To call up the function menu.– To switch to and from Standby mode.3 Variable regulators– To set parameters injection pressure p i , injection time t i andcompensation pressure p c .– To select functions and parameters in Function mode.4 Tube connection– To connect the pressure tube to the device via the bayonet joint.6

2 Device descriptionStandard accessories:1 Pressure tube, 2 m1 Universal capillary holder with grip head for capillary diametersof 1.0 mm and adapter for Femtotips ®1 Connecting cable (Interface Cable 5171) to the Micromanipulator 5171or InjectMan ®1 Hand control for triggering injection and the Clean function1 Femtotips ® , Femtotips ® II, Microloader and CELLocate demo pack1 Mains cable1 Operating manualOptional accessories:1 Foot control for triggering the Inject function7

2 Device description2.2 Short descriptionDisplayDisplay of all relevant parameters and information.Function keysCleanTo apply the maximum pressure for cleaning the capillaryto the tube outlet.Auto / ManuTo switch between manual and automatic time-controlledinjection.CountTo reset to zero the counter for injections which have beenactuated.InjectTo execute automatic or manual injection.MenuTo switch the device to menu control; selected functionsmay be executed.StandbyWhen the key is pressed briefly:To switch the device between operating mode and standbymode.When the key is held down:As above, but also deairs the pressure accumulator.8

2 Device descriptionVariable regulatorIn the normal operating mode, the parameters for injection are set by thethree variable regulators. The arrangement of the variable regulatorscorresponds to the arrangement of the parameters in the two bottom linesof the display:From left to right: injection pressure pi, injection time ti and compensationpressure p c .In other operating modes, the variable regulators have either thesignificance indicated in the display or no significance at all.Tube connectionThe pressure tube is connected to the device via a bayonet joint.9

2 Device description2.3 Startup3321Diagram of rear of device– Electrical connection is effected via the mains socket with integratedmains switch (1).– Connection to an Eppendorf manipulator designed for injection iseffected via the 15-pin interface (2).– Optional switches (e.g. foot control, hand control) or a computer canbe connected to the 9-pin interfaces.Switching on the deviceSet the mains switch on the rear of the device to "I". The unit is switchedon, the display lights up, the unit runs through a warm-up routine and isthen ready for operation. The injection mode is shown in the display.After being switched on, the unit runs through an initialization phase andsupply pressure is built up. This takes approximately three minutes.10

2 Device descriptionSwitching off the deviceIf it is not going to be used for an extended period, the device shouldbe depressurized via the switch-off routine using the key. Thisguarantees a long service life of the device (see Section 3.1.3).The switch-off routine ensures that the system is dewatered and deairedcorrectly. Do not disconnect the device from the mains supply untildewatering and deairing have taken place. If only the mains switch isactuated, the existing pressure remains in the system.11

3 Mode of operation3.1 Display, variable regulators and keypadBefore operating the device for the first time, users should read thissection carefully. This section describes operation of the device only.Handling of the injection capillaries is described in Section 3.3.AUTO n= 0pi=150 hPapi[hPa] ti[s] pc[hPa]150 0.2 50AUTO n= 0pi=215 PSIpi[PSI] ti[s] pc[PSI]2.15 0.2 0.72DisplayThe display shows the following:1st line:injection mode AUTO or MANU,counter n for number of injectionsperformed2nd line (bold):The most-recently set parameter orrelevant parameterBottom lines:Parameters which affect injectionCompensation pressure p c which isapplied permanently in the normalmode is displayed as an actualmeasured value.The arrangement of the threeparameters shown, p i , t i , p c ,corresponds to the arrangement ofthe variable regulators.3.1.1 Setting the injection parametersThe injection parameters have to be adapted to the injection capillariesused and the cell type and liquid which are to be injected.The following injection parameters can be set using the variableregulators:Injection pressure p i 0 – 6,000 hPa (0.00 – 87.0 psi)Injection time t i 0.1 – 99.9 sCompensation pressure p c 0 – 6,000 hPa (0,00 – 87.0 psi)The settings are in increments of 1 hPa/0.1 s. The display can show hPaor psi, as required. The setting and internal pressure conversion arealways performed in hPa. Rounding for the display in psi can result inincrements of 0.01 to 0.02 psi.The display of pressure in psi varies according to the size of the value(e.g.: 9.99 may also appear in the display as 10.0).12

3 Mode of operationSlight deviations between the nominal value (specified value) anddisplayed compensation pressure p c (actual value) may occur fortechnical reasons. The relevant value is the actual value measured.The following settings are used as abasis for initial injection experimentsusing Femtotip ® / Femtotip ® II:Display:AUTO n= 0pc= 50 hPapi[hPa] ti[s] pc[hPa]150 0.5 50p i left-hand variable regulator: 150 hPat i central variable regulator: 0.5 sp c right-hand variable regulator: 50 hPaVariable regulators:3.1.2 Variable regulatorsThe variable regulators are set so that when they are turned slowly, thevalues are changed by 1 hPa / 0.1 second per notch (0.01 – 0.02 psi).If the regulators are turned faster, the values are altered in largerincrements.13

3 Mode of operation3.1.3 KeypadInjectPerforms automatic or manual injection.Automatic injection is controlled via theinjection time t i , and the injectionpressure p i is used. In the second line,injection time t i is counted down from thenominal value.AUTO - INJECT n= 00.4 spi[hPa] ti[s] pc[hPa]150 0.6 50Manual injection is performed for as long as the key is held down.Injection pressure pi is used. In the second line, injection time ti is countedupwards from 0.0.If the FemtoJet ® is connected to the Eppendorf Micromanipulator 5171 orInject Man ® , the injection movement of the micromanipulator is alsostarted via the key. Both units then combine as a system to performsemi-automatic microinjection.Alternatively, the INJECT key of the connected micromanipulator or thehand/foot control can be used.If synchronization is set to "limit" (SYNC = LIMIT) in the INJECT functionof the micromanipulator, semi-automatic injection can only be triggeredvia the micromanipulator.CleanThe maximum available pressure forcleaning is applied at the tube outlet foras long as the key is held down. Once thekey is released, the pressure drops tocompensation pressure p c again. Thepressure curve is shown in the form ofa bar.CLEAN n= 0≡≡≡≡≡≡≡≡≡≡≡≡≡≡≡_____pi[hPa] ti[s] pc[hPa]150 0.2 5014

3 Mode of operationStandbyin the case of a brief interruptionWhen key is pressed briefly:The device switches to the Standbymode without deairing. The illuminationof the display switches off. "STANDBY"appears in the display with the suffix"Supply pressure = rxyz" as a code forsystem pressure p s being maintained.Application:For temporary interruptions or use ofas a Reset function.STANDBYSupply pressure 6789When key is pressed again:The device is reactivated and runsthrough a short warm-up routine.This lasts approximately one minute.Standbywith deairing of pressure accumulatorHold down the key (approx. 2 seconds;the display moves to "STANDBY"):The device switches to the Standbymode. The pressure accumulator iscompletely deaired, the illumination ofthe display switches off. "STANDBY"appears in the display with the note"Supply pressure = x" (x ~ 0).A noise can be heard at this stage.Application:For correct shutdown, with deairing of theentire pressure system in the event oflonger periods of non-use or standstill(weekend or storage) with subsequentswitch-off using the mains switch.Press the key again:The device is reactivated and runsthrough the warm-up routine.This takes approximately three minutes.Hold standbyto exhaustSTANDBYSupply pressure 016

3 Mode of operationResetTo reset the device to the initial mode bypressing the key briefly or via themains switch.The pressure accumulator is not deaired.3.2 Hand control / foot controlThe functions of the hand/foot controlcorrespond to the functions of theand keys on theFemtoJet ® .The centre key of the hand control hasno function.The function of the foot controlcorresponds to the key.17

3 Mode of operation3.3 Connecting the capillary holderUniversal capillary holderThe universal capillary holder is used to locate the injection capillaries.Prefabricated Eppendorf Femtotips ® / Femtotips ® II (6) are screwed intothe universal capillary holder using the adapter (5). To do so, the adapterfor Femtotips ® (5) is first screwed into the front rotatable knurled screw(4) and then the Femtotip ® / Femtotip ® II is attached. If self-pulledcapillaries are to be attached, the grip head 0 (7) needs to be screwedloosely into the front rotatable knurled screw (4). The self-pulled capillaryis pushed into the front opening of the grip head through the two O-ringsand fixed in position by tightening the grip head.1 2 3 4 5 67Universal capillary holder1 Tube connection2 Rear knurled screw3 Tension piece4 Front knurled screw, rotatable5 Adapter for Femtotips ® (microcapillaries for microinjection)6 Femtotip ® / Femtotip ® II7 Grip head 0, plastic, with two O-rings and sealing washerConnecting the universal capillary holderThe two-meter-long pressure tube is connected to FemtoJet ® (1) via thebayonet joint (2). The pressure tube (3) is screwed to the rear knurledscrew (4) of the universal capillary holder. Femtotips ® / Femtotips ® II arefitted at the front end of the universal capillary holder (5) or independentlymanufacturedcapillaries are clamped with the aid of the appropriate griphead.1 2 3 4 518

3 Mode of operationOther accessoriesClamping piecefor fastening the universal capillary holder.Not required for TransferMan ® NK or for Eppendorfmicromanipulators, which have an adjustablecannula holder.The universal capillary holder is mounted to themicromanipulator by placing the clamping piece intothe tool holder of the Eppendorf Micromanipulatorand then tightening it with the fastening screw.The universal capillary holder is positioned withthe correct length into the tool holder and then fixedinto position. The position of the capillary can bechanged by loosening the fastening screw.Grip head 0,incl. 2 sets of O-rings and sealing washer.Using grip head 0 supplied, capillaries with an outerdiameter of 1.0 mm to 1.1 mm can be used. In thecase of outer diameters which differ from thesemeasurements, the relevant grip heads can beordered as non-standard accessories for theuniversal capillary holder (see Section 7, Orderinginformation). The transparent grip head allows theuser to visually monitor the position of the capillary.19

3 Mode of operation3.4 Information on working practicesThe following pressures can be used:Name Pressure range CommentsCompensationpressure p c0 – 6,000 hPa0.00 – 87.0 psiInjection pressure p i0 – 6,000 hPa0.00 – 87.0 psiClean pressuremaximumup to 7,000 hPamaximum up to 102 psiConstant workingpressure p w0 – 6,000 hPa0.00 – 87.0 psiSelect Function 4 viathe key.System pressure p s[supply pressure]maximumup to 7,000 hPamaximum up to 102 psiThis is used directlyfor .In certain phases ofinitialization, thesystem pressure maytemporarily increaseto a maximum of8,000 hPa (117 psi).20

3 Mode of operation3.4.1 Compensation pressure p cThe compensation pressure p c ensures that no medium flows into thecapillary. Capillary forces would make liquid flow out of the cell culturedish into the injection capillary and thus dilute the injection material. Toprevent this, a permanent compensation pressure p c is set. This shouldbe selected so that there is a permanent slight flow-out of liquid from theinjection capillary. The individual pressure level can be determined in apreliminary test. For example, a fluorescent dye can be added to thecapillary to enable outflow to be monitored directly. The pressure requiredis heavily dependent on the surface tension and viscosity of the injectionmaterial. Experience has shown that compensation pressures of between30 hPa and 300 hPa (0.44 – 4.4 psi) are appropriate values for ready-touseEppendorf Femtotips ® injection capillaries.Capillary suctionCompensation pressure p cHydrostatic pressure P hydPressure in the capillaries3.4.2 Injection pressure p iInjection pressure p i is applied during the injection process. This pressureis generally higher than the compensation pressure. In many cases, apressure between 50 hPa and 500 hPa (0.73 – 7.2 psi) is appropriate forFemtotips ® . The volume injected is dependent on the injection pressureand the injection time. The injection pressure must be greater thanthe internal pressure of the cell into which injection should take place.However, if the cell or the cell nucleus swell up during injection, theinjection pressure is too high.If the compressor starts up occasionally during experiments, this does notimpair the function of the FemtoJet ® . Pressure is always available up tothe maximum value of 6,000 hPa (approx. 87.0 psi), even when thecompressor is topping up the supply pressure.21

3 Mode of operation3.4.3 Injection time t iInjection time t i gives the period for which injection pressure p i ismaintained. t i is usually selected from a range between approx. 0.3 s and1.5 s. The relevant factor for the injected volume is the product of theinjection pressure and the injection time.Depending on the setting on the Eppendorf Micromanipulator which isconnected, the injection time t i is measured from the point at which thekey is pressed. The injection pressure is built up immediately, so itis already present during the injection movement and during penetrationof the cell. Once the end of injection time has been reached, the deviceswitches to compensation pressure p c . At the same time, the micromanipulatoris moved back into the working level. When "Injection at Z limit" isset, measurement of the injection time t i does not begin until the Z limithas been reached. Once t i has elapsed, the FemtoJet ® switches back top c and the micromanipulator moves back into its starting position in theworking level.3.4.4 Clean functionThe Clean function is used to flush out blocked capillaries. The maximumavailable pressure is applied to the outlet with the key. It is appliedfor as long as the key is pressed. The maximum pressure of theFemtoJet ® is in the range of 7,000 hPa (102 psi). The value cannot bechanged.The pressure curve up to maximum pressure appears in the display in theform of a bar chart. By pressing the key briefly, a lower pressurecan also be obtained.22

3 Mode of operation3.4.5 Manual or automatic injectionIn the Manu operating mode, injection is performed for as long as thekey or hand / foot control is held down. The duration (t i ) ofinjection is measured and shown in the display. In the case ofcombination with an Eppendorf Micromanipulator, the injection movementis triggered.In the Auto operating mode, the built-in timer facilitates a reproducibleinjection time. The injection runs automatically for the duration of set timet i . It is triggered by the key or the hand / foot control being pressedbriefly. During injection, the Inject key is inactive, i.e. pressing it again hasno effect. In combination with an Eppendorf Micromanipulator 5171 /InjectMan ® , the automatic injection procedure can also be triggered fromthe operating panel of the micromanipulator.3.4.6 Constant pressure/constant working pressureUsing the Constant Pressure function, it is possible to select a higherpressure permanently if required. This function is helpful when specialinjection techniques require either a high pressure up to a maximum of6,000 hPa (87 psi) or a permanent flow of injection solution(see Section 3.7.5). Control accuracy drops in the pressure range from2,000 – 6,000 hPa (29 – 87 psi).3.4.7 General noteFemtoJet ® is fitted with an integrated compressor. For this reason, thecompressor may start up or excess pressure may be let off briefly before,during and after injections or during periods when the FemtoJet ® is notbeing used. This is not a malfunction of the device and does not impairinjection.In the case of injections under high pressure at very short intervalsor in the case of the Clean function being carried out several timesin succession, the pressure in the system accumulator may dropsignificantly. This is indicated by a warning or an error message. In thiscase, wait a short time until the supply pressure has stabilized again.It is then possible to continue working immediately.23

3 Mode of operation3.5 The first injectionThe following are available:● Adherent cells in a Petri dish (possibly on CELLocates to observespecific cells after injection),● Injection liquid (purified, e.g. by centrifugation),● Femtotips ® / Femtotips ® II; other capillaries as an alternative● Microloader,● 0.5 – 10 µl pipette to take the Microloader,●A universal capillary holder on the micromanipulator which has beenpreadjusted with a Femtotip ® .Filling the Femtotips ® with the Microloader:– Select function 0 "Capillary exchange" by pressing . Connectthe pressure tube.– Aspirate approximately 0.5 – 5.0 µl of centrifuged injection fluid withthe Microloader without touching the base of the vessel. Using theMicroloader, fill the Femtotip ® through the rear opening, inserting thetip approximately up to the centre of the Femtotips ® and releasing thefluid there.– Hold the Femtotip ® vertically, loosen the cap by turning it and allow itto drop vertically.– Screw the Femtotip ® into the capillary holder.– Complete Function 0 by pressing . Compensation pressure isapplied.– Move the capillary into the cell medium (Petri dish) with the micromanipulator.– Focus the cells with the microscope, position the capillary slightlyabove the cells but do not allow it to come into contact with the baseof the Petri dish. Focus the capillary.– Using the key, check that the capillary is not blocked. Eitherthe fluid can be seen flowing out or it is rendered visible by thefluorescent substance it entrains. As a rule, any air bubbles presentare blown out.– Perform a sample injection using the preset parameters(e.g. p i ~ 150 hPa, t i ~ 0.5 s, p c ~ 50 hPa, corresponding top i ~ 2.18 psi, t i ~ 0.5 s, p c ~ 0.73 psi). Injection can be monitoredvisually when the cell size changes by approx. 5 % to 10 % or viafluorescence. If required, adapt parameters p i and/or t i .24

3 Mode of operation3.6 Connecting an Eppendorf MicromanipulatorConnection to an Eppendorf Micromanipulator designed for automatedinjection is effected with the 15-pin interface.13 4 58276FemtoJet ® (rear) with all connection options1 Foot control (optional) with Inject function2 Hand control with Inject and Clean functions3 FemtoJet ®4 Micromanipulator 5171 or InjectMan ®5 Connection socket of micromanipulator forTransmission cable 5246 ("Y cable")6 Transmission cable 5246(accessory for Micromanipulator 5171 and InjectMan ® )7 Interface cable 5171(Connection from FemtoJet ® to Transmission cable 5246 "Y cable")8 Interface of FemtoJet ® to micromanipulator25

3 Mode of operation3.7.3 Switching on/off the loudspeakerFUNC 2Push Inject to selectBeeperON OFFTurn pi toselect functionFUNC 2Use the key to switch theloudspeaker on or off. The currentlyselected setting (in this case: ON) isunderlined. Acoustic error messages arenot emitted if the settingis OFF.3.7.4 Switching on/off the background illumination of the displayFUNC 3Push Inject to selectIllumination ON OFFTurn pi toselect functionFUNC 3Use the key to switch theillumination on or off. The currentlyselected setting (in this case: ON) isunderlined.In the normal operating mode,illumination switches off after threeminutes if no operating control has beenactuated. When an operating control isactuated, the illumination lights up again.3.7.5 Constant pressureFUNC 4Continuous flowINJECT = startTurn pi toselect functionFUNC 4Continuous flowpw= 50 hPaTurn pi topw[hPa]select function 50FUNC 4This function can be used to apply aconstant working pressure pw in therange from approx. 0 to 6,000 hPa(87.0 psi) at the pressure outlet. Thepressure unit psi can also be used.The working pressure p w is set withthe right-hand variable regulator (p c ),the specified value is shown in largecharacters. The actual value is shownin the bottom right of the display.The Clean function can be activated. Thepressure of the Clean function is shownin the bottom right of the display in theform of p w .27

3 Mode of operation3.7.6 Dewatering the pressure accumulatorFUNC 5Drain pressure supplyINJECT = startTurn pi toselect functionFUNC 5– Briefly opens the dewatering valveof the pressure accumulator; anycondensed humidity present is blownaway.– When this function is performed, thenoise of air being blown away can beheard.This function is automatically performedevery time the unit is switched on. It canbe performed manually as described ifcondensation is suspected to be present.28

4 Care and maintenance4.1 CleaningBefore cleaning the FemtoJet ® , disconnect the plug.Ensure that no fluids enter into the inside of the injector in order to avoidshort-circuits in the electrical installation as well as corrosion.Wipe painted parts and aluminum surfaces using a cloth and milddetergent and then with a dry cloth.Warning: Do not use any corrosive, solvent or abrasive detergentsor polishes.4.2 DisinfectionClean the FemtoJet ® before disinfecting.Wipe every part of the FemtoJet ® , including accessories and connectorcables, with disinfectant.Spray disinfection is not advisable, as disinfectants can enter into themicroinjector.The disinfection method used must comply with current disinfectionregulations and guidelines.4.3 MaintenanceRegular maintenance is not required.The dealer is responsible for warranty and service.29

5 Troubleshooting5.1 Potential errors– Pressure loss due to capillary not fitted with capillary holderconnected.Solution: Connect the capillary or select Function 0, Change capillary,via the key and variable regulator p i .– Capillary is blocked.Solution: Press the key for long enough.Change capillary.Purify injection fluid (e.g. by means of centrifugation).– Compressor runs for a long time.Solution: Take a short break.Do not use the key too often within a short period.If possible, set a lower compensation pressure p c.Perform self-test (Section 5.1.1).– Valve rattlesSolution: Connect tube to filled capillary. Pressure control is designedfor the tube connected.– No communication with the micromanipulator.Solution: Check cables and connections.5.1.1 Self-testFUNC 6Self-TestINJECT = CompleteCLEAN = Leak onlyConnect closed tube !Turn pi toselect functionFUNC 6The FemtoJet ® is checked for leaks inthe self-test.This self-test can also be used if a leak issuspected.The test runs automatically.The pressure tube must be connected toa closed capillary (e.g. melted closed orfilled) for the pressure control to workproperly.30

5 TroubleshootingFUNC 6Self-TestCompressor _____ sPs leak _____ _____ hPaPw leak _____ _____ hPaInjection _____ _____ hPaps=7000 pw= 10 hPakey: Complete self-test– Checks compressor run time up tothe point when a specific pressureis reached and thus for leaks in thearea of the compressor.– In the case of two differentpressures, checks for leaks in thepressure accumulator (p s ) and forthe internal tightness of the valves.– Checks for leaks in the area ofworking pressure (p w ) in the caseof two different pressures.– Checks behavior during injectionat different pressures (shortcontinuous test).If certain values are outside the specifiedrange, an error message is emitted. If thisrecurs, inform Service.FUNC 6Self-TestPs leak _____ _____ hPaPw leak _____ _____ hPaps=7000 pw= 10 hPakey: Self-test to search for leaks– Checks for leaks in the pressureaccumulator (p s ) in the case of twodifferent pressures, as well as forinner tightness of the valves.– Checks for leaks in the area of workingpressure (p w ) in the case of twodifferent pressures.If certain values are outside the specifiedrange, an error message is emitted. If thisrecurs, inform Service.31

5 Troubleshooting5.2 WarningsAs a rule, warnings are emitted if certain operating conditions are not (yet)reached or if an action is not possible.Acknowledge the warning with the key and the unit will return tonormal status.WARNING 001Please wait forsupply pressureINJECT = ContinueWarning 1Supply pressure has not yet beenreached.The minimum supply pressure requiredfor an injection or the Clean function wasnot present when the function wastriggered.Possible cause:Multiple triggering of Clean or very rapidinjection at a high injection pressure.Solution:Wait until the supply pressure has built upand trigger again.WARNING 002Check capillarye.g. broken/missing ?INJECT = ContinueWarning 2Compensation pressure cannot bemaintained.Possible cause:Capillary or capillary holder not attachedor only connected loosely.Solution:Check all connections and tighten.Activate Function 0 to change thecapillary and when the capillary holder isopen.32

5 TroubleshootingWARNING 003Switch on manipulatoror disconnect cableINJECT = ContinueWarning 3Micromanipulator does not reactA micromanipulator is connected butdoes not react to a start pulse from theFemtoJet ® .Possible cause:Micromanipulator is switched off.INJECT function on manipulator is notreleased.No limit is set on the micromanipulator.In the INJECT function on the micromanipulator,synchronization is set tolimit (SYNC = LIMIT).Solution:Switch on the micromanipulator andmake it operational.Disconnect the cable to micromanipulator.With SYNC = LIMIT, trigger semi-automaticinjection via the micromanipulatoronly.5.3 Error messagesError messages are emitted if certain operating conditions are notreached or the device records a deviation from specified conditions.Acknowledge the error message with the key and the unit willreturn to normal status. The measures described under “Solution” shouldbe carried out. If the error message recurs after acknowledgement withthe key, inform Service.33

5 TroubleshootingERROR 010Supply pressurecannot be reachedINJECT = ContinueERROR 011Supply pressure istoo highINJECT = ContinueERROR 012Supply pressure isdoes not fallINJECT = ContinueError 10Supply pressure cannot be reachedThe compressor runs without themaximum supply pressure beingreached.Possible cause:High compensation pressure when thecapillary holder is not attached or ispoorly attached.Technical cause:Compressor does not start up. Leak incompressor or pressure accumulator.Solution:Check the capillary holder and tube forleaks or tighten up the tube connections.Service.Error 11Supply pressure is too highSupply pressure has exceeded themaximum permissible supply pressure.Possible cause:supply pressure is not relieved by therelevant standby function.Technical defect.Solution:Switch off using the key (holddown for approximately two seconds).Service.Error 12Supply pressure does not drop farenoughPossible cause: Technical defectSolution: Service34

5 TroubleshootingERROR 013Supply pressure isdoes not raiseINJECT = ContinueERROR 014Supply pressureleak is too highINJECT = ContinueERROR 015Supply pressureleak is too highINJECT = ContinueERROR 016Valve 1 pressureleak is too highINJECT = ContinueERROR 020Working pressurecannot be reachedINJECT = ContinueError 13Supply pressure does not risePossible cause: Technical defectSolution: ServiceError 14Massive leak in pressure accumulatorPossible cause: Technical defectSolution: ServiceError 15Leak in pressure accumulatorPossible cause: Technical defectSolution: ServiceError 16Leak through Valve V1 is too largePossible cause: Technical defectSolution: ServiceError 20Working pressure has not been reachedCompressor runs without supplypressure being reached within a specifictime.Possible cause:Capillary holder not attached or poorlyattached. (Broken) capillary with largeopening.Technical defect.Solution:Check the capillary. Check the capillaryholder and tube for leaks or tighten up thetube connections.Service.35

5 TroubleshootingERROR 021Inject pressurecannot be reachedINJECT = ContinueERROR 022Compensation pressurecannot be reachedINJECT = ContinueERROR 023Inject n pressureoverflow too largeINJECT = ContinueERROR 024Working pressuredoes not riseINJECT = ContinueError 21Injection pressure has not been reachedPossible cause:Capillary holder not attached or poorlyattached. (Broken) capillary with largeopening.Technical defect.Solution:Check capillary. Check capillary holderand tube for leaks or tighten up tubeconnections.Service.Error 22Compensation pressure has not beenreachedPossible cause:Capillary holder not attached or poorlyattached. (Broken) capillary with largeopening.Technical defect.Solution:Check capillary. Check capillary holderand tube for leaks or tighten up tubeconnections.Service.Error 23Increase in working pressure is smallerthan requiredPossible cause: Technical defectSolution: ServiceError 24No increase in working pressurePossible cause: Technical defectSolution: Service36

5 TroubleshootingERROR 025Working pressuredoes not fallINJECT = ContinueERROR 026Working pressureleak is too highINJECT = ContinueERROR 027Working pressureleak is too highINJECT = ContinueError 25No drop in working pressurePossible cause: Technical defectSolution: ServiceError 26Working pressure leak rate is too highPossible cause:Capillary holder not attached or poorlyattached. (Broken) capillary with largeopening.Technical defect.Solution:Check capillary. Check capillary holderand tube for leaks or tighten up tubeconnections.Service.Error 27Working pressure leak rate is too highPossible cause:Capillary holder not attached or poorlyattached. (Broken) capillary with largeopening.Technical defect.Solution:Check capillary. Check capillary holderand tube for leaks or tighten up tubeconnections.Service.37

5 TroubleshootingERROR 028Working pressureleak is too highINJECT = ContinueERROR 030Sensoroffset supplyis too lowINJECT = ContinueERROR 031Sensoroffset supplyis too highINJECT = ContinueERROR 032Sensoroffset workis too lowINJECT = ContinueERROR 033Sensoroffset workis too highINJECT = ContinueERROR 034Valve 1 offsetis too lowINJECT = ContinueError 28Working pressure leak rate is too highPossible cause:Capillary holder not attached or poorlyattached. (Broken) capillary with largeopening.Technical defect.Solution:Check capillary. Check capillary holderand tube for leaks or tighten up tubeconnections.Service.Error 30Sensor offset supply pressure is too lowPossible cause: Technical defectSolution: ServiceError 31Sensor offset supply pressure is too highPossible cause: Technical defectSolution: ServiceError 32Sensor offset working pressure is too lowPossible cause: Technical defectSolution: ServiceError 33Sensor offset working pressure is toohighPossible cause: Technical defectSolution: ServiceError 34Valve 1 offset is too lowPossible cause: Technical defectSolution: Service38

5 TroubleshootingERROR 035Valve 1 offsetis too highINJECT = ContinueERROR 036Valve 2 offsetis too lowINJECT = ContinueERROR 037Valve 2 offsetis too highINJECT = ContinueERROR 040Self-Calibrationis missingINJECT = ContinueERROR 041Fullscale Calibrationis missingINJECT = ContinueError 35Valve 1 offset is too highPossible cause: Technical defectSolution: ServiceError 36Valve 2 offset is too lowPossible cause: Technical defectSolution: ServiceError 37Valve 2 offset is too highPossible cause: Technical defectSolution: ServiceError 40Self-calibration is missingPossible cause: Technical defectSolution: ServiceError 41Full-scale calibration is missingPossible cause: Technical defectSolution: Service39

6 Technical dataVoltage/Frequency: 100 V – 240 V ± 10 %, 50 – 60 HzSet voltage:adapts automaticallyFuse for 100 – 240 V: T1.0 A, 250 V (2 pcs.)Power consumption: 50 WMax. current consumption: < 0.5 AProtection class:IOvervoltage category: II (IEC 61010-1)Pollution degree: 2 (IEC 664)Ambient temperature: 15 °C – 35 °CAmbient relative humidity: max. 70 %Weight:6.2 kgDimensions: Width: 220 mmDepth: 170 mmHeight: 280 mmInjection time t i :0.0 – 99.9 sin 0.1 s incrementsInjection pressure p i : 0 – 6,000 hPa (87 psi),in 1 hPa (0.01–0.02 psi) incrementsRegulating accuracy: 1 % of value set in each case,at least ± 1 hPaCompensation pressure p c : 0 – 6,000 hPa (87 psi),in 1 hPa (0.01–0.02 psi) incrementsRegulating accuracy: 1 % of value set in each case,at least ± 1 hPaClean rinsing pressure: max. approx. 7,000 hPa (102 psi)RS 232 computer Baud rate: 9,600interface: Start bit: 1Data bits: 8Parity: nonStop bit: 1The device is approved with thesign.Technical specifications subject to change!40

7 Ordering informationOrder no.5247 000.013 FemtoJet ®100 V – 240 V ± 10 %, 50 – 60 HzFully programmable microinjector with integratedpressure supply. For injection of smallest amountsof liquid into living cells.Delivery package:1 FemtoJet ®1 Power cable1 Interface cable 51711 Universal capillary holder1 Pressure tube, 2 m5247 617.002 1 Hand control for remote control10 Femtotips ® / Femtotips ® II (injection capillaries)10 Microloader20 CELLocates1 Operating ManualAccessories (not standard)5247 623.002 Foot control FemtoJet ®5325 620.007 Interface cable PCfor control and for program updates5247 622.006 Interface cable 5171for connection to the micromanipulator5242 952.008 20 Femtotips ®5242 957.000 20 Femtotips ® II5242 956.003 200 Microloader5246 164.004 1 Pressure tube, 2 m, for universal capillary holder5176 190.002 Universal capillary holder(incl. grip head 0, adapter for Femtotips ® ,clamping piece and spare O-rings)4910 000.018 Eppendorf Reference ® pipette, variable, 0.5 to 10 µl3110 000.021 Eppendorf Research ® pipette, variable, 0.5 to 10 µl5176 210.003 Grip head 0 for universal capillary holder;for microcapillaries with an outer diameterof 1.0 to 1.1 mm5176 212.006 Grip head 1 for universal capillary holder;for microcapillaries with an outer diameterof 1.2 to 1.3 mm5176 214.009 Grip head 2 for universal capillary holder;for microcapillaries with an outer diameterof 1.4 to 1.5 mm41

7 Ordering information5176 207.002 Grip head 3 for universal capillary holderfor microcapillaries with an outer diameterof 0.7 to 0.8 mm5176 196.000 O-ring setfor grip heads 0 to 3 with extraction tool5176 195.004 Service kitfor grip head and capillary holder,consisting of O-ring set, adapter for Femtotip ®and clamping pieceCELLocateGlass coverslips with integrated microgridfor relocating cells in light and electron microscopy,individually sealed, with log pad5245 952.009 Grid size 175 µm, sterile, set of 805245 962.004 Grid size 55 µm, sterile, set of 80Micromanipulation5171 000.019 Micromanipulator 5171Programmable micromanipulator withcoordinate storage for automatic microinjection,microdissection and electrophysiology(in combination with FemtoJet ® ),complete with power unit, module unitand control board5179 000.018 InjectMan ®Micromanipulator for automatic injectionin adherent cells in combination with FemtoJet ® ,complete with power unit, module unitand control board5178 000.014 TransferMan ® NKProportional micromanipulator formicroinjection in suspension cells5177 000.010 TransferMan ®Dynamic micromanipulator formicroinjection in suspension cells5246 621.006 Transmission cable 5246"Y cable" for connection with theinterface cable 51715211 058.007 Foot control for microinjection,connectable to transmission cable 5246 "Y cable"42

8 IndexAAdapter for Femtotips ® 18Application 3, 4CCapillary change 26Clean 8pressure 20function 22Cleaning 29Commands 45Compressor 21, 23Compensation pressure pc 5, 20, 21Connectioncapillary holder 18micromanipulator 25universal capillary holder 18Constantpressure 23, 27working pressure 23working pressure p w 20DDisinfection 29Display 6, 8, 12in hPa 12in psi 12switch on/off 27EErrormessages 33potential 30FFoot control 7, 17Functions 26keys 8menu 15GGrip head 18, 19HHand control 17IInformation on working practices 20Injectionautomatic 14, 23first 24manual 14, 23movement 14, 23parameters 12pressure pi 5, 14, 20, 21time t i 5, 14, 22trial 24Interface 10, 25description 45KKeyAuto/Manu 8, 14Clean 8, 14, 22Count 8, 15Inject 5, 8, 14, 32Menu 8, 26Standby 8, 16Keypad 6, 14LLoudspeaker, switch on/offMMains switch 10Menu 15Mode of operation 12OOperation 12via computer 43, 45Ordering information 41Overview 643

8 IndexPPressure– dewateringpressure accumulator 28– set pressure unit 26tube 9, 18QQuery 45RReset 9, 17function 16SSafety precautions 3, 4Self-test 30complete 31to find leaks 31Servicing 29Short description 8Startup 10Standby without deairing 16Switchon 10off 11System pressure ps 20TTechnical data 40Tension piece 19Troubleshooting 30Tube connection 6, 9UUnitdescription 5rear of 10Universal capillary holder 18VVariable regulator 5, 6, 9, 13, 26WWarnings 3244

Appendix AOperation via a computerCertain commands and queries (Section 6.2) can be transmitted to theunit using the optional cable/PC interface.A.1 Description of interfaceA computer can be connected to the remote control of the FemtoJet ® viaone of the two RS 232 interfaces. Parameters can be entered or read outvia the computer.The computer is connected by the special cable/PC interface cable.The computer to be connected must comply with EN 60950/UL 1950standards.The serial interface of the computer should be set to the following transmissionparameters:Baud rate: 9,600Start bit: 1Dat bits: 8Parity: nonStop bit: 1Only ASCII symbols (incl. CR/LF) which can be represented aretransmitted.Therefore a terminal program, for example, is suitable forremote control.A.2 Commands and queriesThe commands to be sent are defined with the following structure:Commands to enter parameters:"Cxxx=ssssssssss"Commands to execute and to read out parameters:"Cxxx"C: Commandxxx: Code for the executing command=: Separation for the following input parametersssssssssss: Input parametersIn response to any command, the device provides a symbol string of asimilar structure:"Axxx" for answers without output parameter"Axxx=rrrrrrrrrr" for responses with output parameterA: Answer45

Appendix AIf the command is faulty, an error message is returned instead of theanswer:"ERR=eee"ERR: Error=: Separationeee: Error numberLower and upper case letters can be used.Command to enterparameters[C]Code Input Meaning ofparameter input parameters[xxx] [ssssssssss]Short acoustic signal 003 1 – 254 Number of signalsLong acoustic signal 004 1 – 254 Number of signalsAutomatic injection(inject at t i )Set injection counter nto 0010 0; 1 – 999 Injection timein 1/10 seconds0: injection time set onthe FemtoJet ®012 – – – No parameter inputPerform Clean 013 1 – 100 Time in 1/10 secondsExecute Standby 014 0 or 1 0 = short1 = longSwitch acoustic signalon/off040 0 or 1 Status:0 = off / 1 = onSet pressure unit 041 0 or 1 Unit:0 = hPa / 1 = psiSet injection time t i 042 1 – 999 Injection timein 1/10 secondsSet compensationpressure p c043 0 – 6,000 / 87 Pressure in hPa/psiSet injection pressure p i 044 0 – 6,000 / 87 Pressure in hPa/psi46

Appendix ACommand to enterparameters[C]Code Input Meaning ofparameter input parameter[xxx] [ssssssssss]Set backgroundillumination045 0 or 1 0 = off1 = onDewater 046 – – – No parameter inputCommand to read outparameters[C]Code Significance ofoutput parameter[xxx][A]Query program version 101 Versions number x.xxOutputparameter[rrrrrrrrrr]Query bleeper 150 Status:0 = off / 1 = onQuery pressure unit 151 Unit:0 = hPa / 1 = psiQuery injection time t i 152 Injection timein 1/10 seconds0 or 10 or 11 – 999Query compensationpressure p cQuery injectionpressure p i153 Pressure in hPa/psi 0 – 6,000 / 87154 Pressure in hPa/psi 0 – 6,000 / 87Query backgroundillumination155 Status:0 = off / 1 = on0 or 147

Appendix AError no.for incorrectoperatingconstructionERR = [eee]CauseSolution060 The first character is nota c or C.061 The 2nd to 4th characteris not a digit.Check entry parameters.Change entry.Check entry parameters.Change entry.062 The 5th character is not =. Check entry parameters.Change entry.063 The parameter is notwithin the permitted range.Check entry parameters.Change entry.064 The command is notknown.Check entry parameters.Change entry.48

Appendix AError no.duringexecutionERR = [eee]CauseSolution070 FemtoJet ® is not in routinemode.Check FemtoJet ® .Bring FemtoJet ® intoroutine operation.Eliminate error.071 FemtoJet ® is not ready. Check FemtoJet ® ormicromanipulator.As necessary, wait oreliminate error.072 The command is not finishedwithin the scheduled time.Check FemtoJet ® ormicromanipulator.Eliminate error.073 This command cannot beexecuted at the moment,because of insufficient storagepressure.See: Warning 001Check FemtoJet ® .Eliminate error.074 The connected micromanipulatordid not respond.See: Warning 003Check micromanipulator.Eliminate error.Other error numbers as well as the error cause and solution are describedin Section 5, "Troubleshooting". During remote control, no error textsappear in the FemtoJet ® display.49

Support and Services DirectoryContact InformationUnited StatesCanadaBusiness Hours: 8:30 a.m. to 6:00 p.m. EST 8:30 a.m. to 6:00 p.m. ESTPhone: 800-645-3050 800-263-8715516-334-7500 905-826-5525Fax: 516-334-7506 905-826-5424Address: Eppendorf North America, Inc. Brinkmann Instruments (Canada) Ltd.One Cantiague Road6670 Campobello RoadWestbury, NY 11590-0207Mississauga, ONT L5N 2L8Website: www.eppendorf.com www.brinkmann.comEmail: info@eppendorf.com canada@brinkmann.comCustomer 800-645-3050, menu option 2 800-263-8715, menu option 1Support: custserv@eppendorf.com custserv@brinkmann.comRepair: 800-645-3050, ext. 2404 800-263-8715, ext. 232service@eppendorf.comservice@brinkmann.comApplications 800-645-3050, ext. 2258 800-645-3050, ext. 2258 (U.S.)Lab: apps@eppendorf.com bioapps@brinkmann.comFor more information contact your Eppendorf North America Sales Representative at 800-645-3050.© 2005 Eppendorf AG. Eppendorf ® is a registered trademark of Eppendorf AG. Add other trademark information here.Product appearance, specifications, and/or prices are subject to change without notice.www.eppendorf.com • Email: info@eppendorf.com • Application hotline: 516-515-2258In the U.S.: Eppendorf North America, Inc. 800-645-3050 • In Canada: Brinkmann Instruments (Canada) Ltd. 800-263-8715