Pressure Injector
Contents
1. PROGRAMMABLE MICROINJECTOR PM 2000 USER S INSTRUCTION MANUAL WEHE UIN TABLE OF CONTENTS INTRODUCTION unisce ear ed ee idus 1 SETTING UP THE SYSTEM Connection 3 About the micropipette 3 Testing procedure 4 2 A CLOSER LOOK AT THE INJECTOR Front Panel 5 Rear Panel 6 The Key Pad 6 3 OPERATING AND PROGRAMMING After turn on the power 8 Mode 1 AUTO SEQUENCE 8 Mode 2 PRESSURE MONITOR 10 Mode 3 TIMER 11 Mode 4 COUNTER 12 Mode5 ACTION 12 4 PRINCIPLES AND TECHNIQUES FOR MICROINJECTION Using The Balance Pressure 14 Microinjection for Different Tip Openings 15 Manual Control Actions for a New User 16 Saving Gas 16 5 MAINTENANCE AND TROUBLESHOOTING 6 SPECIFICATIONS 7 ACCESSORIES S WARRANTY 2 ue ee ee MDI 6 01 IMA S W V8 3 14 17 18 19 20 INTRODUCTION A superior device for microinjection and intracytoplasmic sperm injection Theadvantage of semi automatic cell microinjection has changed the way thatthe scientists carry out in biomedical research Theusedtest tube experiments and in vitro fertilization IVF can be performed ina single cell now In the context of proper injection controls modulation of cellular processes can be dissected in an unambiguous and rapid m2. 100 sims Press to select unit second or unit millisecond iae cms al ads VENT during TIMER mode Press to set or cancel a INJ BAN HOLD FILL VENT function in the SEQUENCE mode Press to open orclosea vent hole in the inject port during di ACTION mode An opening vent hole will be display as V nexttothe ACTION onthe LCD display An opening vent hole will provide a The Key Functions channel of atmosphereto theinjection port There fore any balance injection or suck pressurein the gp Presstochange MODE form SEQUENCE injection port will be reduced or bypass by the t to PRESSURE to TIMER to COUNT to venthole See TIPS AND TECHNIQUES for ACTION then going back to SEQUENCE detail application STOP AUTO BVST TST v SEQ 10 SEQ B 100 CLR RELB Press to set the last step and as a sequence repeating stepduring SEQUENCE mode Press totriggeranauto sequence step orinterrupt an action during ACTION mode Press to disable the buzzer during the poweron display Press to display previous pressure read ing memory in the PRESSURE mode Press to select a step down number forthe SEQUENCE mode Press to reset number for TIME mode and COUNT mode Resetting timer is also setting the timerto MANUAL control Press to enable the buzzer again during the power on display Press to select
3. Larger Tip 2200 um or Unpulled Section of a Pipette There is very rare to need a pipette opening larger than 200um because the injected volume is big even with the small injection pressure and is hardly controlled precisely The balance pressure is very low usually 0 1 psi even turning the Balance pressure to minimum is still too high In this case the Ventfunction must be pressed on The Vent function actually opens a vent hole inside the injection channel to bypass some balance pressure and cause the balance pressure can be adjusted to lower than 0 1 psi However with larger tip the pipette is easily filled up just by its capillary action Always turn on balance first the liquid can be sucked by pressing on Fill and liquid can be pushed out by pressing on the Inject If the liquid is pushed out again after filling the balance is too high Ifthe liquid is sucked back again after the injection the balance is too low Usually because the injection pushes the liquid all the way from unpulled section to out of tip of the pipette the pressure balancing the unpulled sectionis too low to keep the balance on the tip then the liquid will be sucked back Therefore perform Fill function or capillary action to suck liquid higher than the unpulled section then set correct balance to keep liquid stable inside the unpulled section Now the liquid can be freely sucked in or pushed out only inthe unpulled section of the pipette by press Fill or Inject Ift
4. Meanwhile the action timer count down is also displayed inthe LCD If a manual interruption happens the actual action time will be displayed A displayed number records the INJECT or FILL acting count User friendly keyboard remote port foot switch and full information LCD Asimpleand straightforward 4x4 keyboard allows the user to preset timer counter and action sequences monitor pressure and vacuum and perform actions An LCD provides full information display suchas pressure vacuum time count sequence steps and actions etc The remote port of a DB9 connector gives users a very useful option to interface the outer control unit There is an optional remote keypad can be plugged in this remote port let a microscope operator can combine to set up a very convenient microinjection work station Another function of the remote portis as acomputer interface port for cell electrophysiology applications 1 SETTING UP THE SYSTEM There are different system setup for different applications An experienced user can setup a perfusion system as wellasaninjection system with this injector The following is only an example of a microinjection system set up Connection Connectthe power cord to an AC outlet with the voltage as specified on the rear panel There is aGASINPUT Connector on the rear panel See REAR PANEL Screw the 1 8 Hose Connector into the Pressure Input Connector and connecttoa gas supply witha 1 8 IDhose Compressed air supply source
5. press again to recover MODE 5 ACTION Press toenter action mode Pressing the MODE key fifth time the injector enters the last mode MODE 5 from MODE 4 The message ACTION will be displayed on the LCD and the input OE pressure is monitored on the right side of the message V In this mode the user can press one of the CLR RELB RELH CLRH INJ ACTION 100 psi BALN HOLD FILL and VENT s ms keys to generate different action The CLR CLRH INJ and FILL functions are determined by the their presetting timer counter and control pressure During their actions the operating pressure is displayed instead of input pressure Therefore instant action pressure adjust mentis possible during action mode If the preset timer is longer than 1 second iis cdd the timer countdown will be displayed on right side of the function name If INJ l or FILL function acting besides timer countdown a number will be displayed on the side of function name The number records the count of function continued PIBE acting The INJ acting resets the FILL counting and the FILL acting resets the INJ V counting A CLR acting resets both two counts ACTION 100 psi The HOLD RELH BALN and RELB functions are manualcontrolandpressure FIL 1 10 55sec adjustable only There are no timer countdown display forthese actions Their actions and CLRH acting will not affect the INJ or FILL counting n
6. Indicating LEDs Indicate CLEARing CLEARing HOLD HOLDing BALANCing FILLing and INJECTing actions 14 INJ Foot Switch Jack Port Connect a foot switch or other handy switch witha 3 5mm plug into this jack port to perform the same function as pressing the INJ key on the key board 15 AUTO Foot Switch Jack Port Connect a foot switch or other handy switch witha 3 5mm plug into this jack port to perform the same function as pressing the AUTO key on the key board 16 REMOTE Key Board PORT DB9 Connector Connect an optional remote keyboard to this port to perform convenientremote control REAR PANEL D 1 AC POWER INPUT Connector 3 Fuse Box Connect the power supply cord and plug in an AC Replace with the same rating and size fuse only power outlet same as the specified voltage 4 Gas Filter View Hole Check filter bowl from this hole 5 Filter Drain Hole On the bottom panel as indicated If accumulated 2 GAS INPUT Port Connectthe 1 8 tubing connector for compressed gas input liquid is found in the filter bowl press the drain valve through the hole to drain out liquid The Key Pad a N on Press toturn offinput pressure and halt the mode ory pon ae ts off press again torecover mode and pressure PYST ST N SEQ ASEQ8 0 1 10
7. a step up number for the SEQUENCE mode Press to add 1 tothe number for TIME mode and COUNT mode Press to select a sequence down number for the SEQUENCE mode Press to add 10 to the number for TIME mode and COUNT mode Press to save pressure reading to the memory in the PRESSURE mode Press to select a se quence up number for the SEQUENCE mode Press to add 100 to the number for TIME mode and COUNT mode Press to select CLEAR function for SE QUENCE TIME and COUNT mode Press to display input pressure from PRESSURE mode Press to generate the CLEAR function during ACTION mode This function will clear the injection pipette input source pressure is directly used and controlled by its timer and counter This function will help to avoid the injection pipette from being clogged Press to select RELEASE BALANCE function for SEQUENCE mode Press to turn off the Balance function during ACTION mode This function will shut off the injection port from the BALANCE pressure Press again will open balance pressure fora pulse of 9 millisecond then closing again RELH CLRH INJ BALN FILL Press to select RELEASE HOLD function for SEQUENCE mode Press to turn off the Hold function during ACTION mode This function will turnoff theholdcell vacuum from cell hold port and release the held cell Ifthe cellis still attaching the holding pipette evenafterre
8. modifications to the equipment not approved in writing by MDI 3 GEOGRAPHICAL LIMITATION NONTRANSFERABILITY AND INCONSISTENT LAN GUAGE Inthe case of equipment located outside of the 50 states the District of Columbia and the Commonwealth of Puerto Rico which is returned in whole or in part to MDI for warranty service the transportation costs incurred in such return shall be at buyer s expense This warranty is not transferrable and maynot be supplemented or amended except in writing referring specifi cally hereto and signed by buyer and MDI Without limiting the generality of the foregoing any inconsistent language contained in requests for quotation buyer s purchase orders shipping instructions or similar documents is specifically rejected by MDI VE Pt MicroData Instrument Inc 1207 Hogan Drive South Plainfield NJ 07080 U S A Tel 908 222 1717 Fax 908 222 1365 e mail info microdatamdi com http www microdatamdi com 20
9. the timer to MANUALO The action time will depend on the time of pressing and holding the key Or press s ms VENT key to display sec then press key Press to enter timer mode LL LS qe V T CLR 0 01sec CLRH 0 01sec FILL 0 01sec INJ 0 01sec Presstoreset timer VST 0 V T CLR 0 01sec CLRH 0 01sec FILL 0 01sec INJ IMANUAL Os acl Press 4 times to maximum SEQS 100 V T CLR 0 01sec CLRH 0 01sec FILL 0 01sec INJ CONTINUESs 100 four times to display CONTINUES The action will be continue until pressing the STOP AUTO key to interrupt the action Press to T CLR 0 01sec purines CLRH 0 01sec eig Press to next mode FILL 0 01sec FILL X INJ CONTINUES MODE Press to change V T CLR 10msec timeunit Counter mode CLRH 10msec f FILL 10msec sms INJ CONTINUEms XEM Keys for Mode 3 CLR CLRH INJ FILL Press to select one s ms Press to select unit second of four action timers VENT or unit millisecond S AC P O Press to mode 4 PYST ST V SEQ ASEQ8 Press to add number pon Press to turn off input pressure and 0 1 10 100 lon the timer off halt the mode pre
10. to MANUAL Os m the action only performs with the finger pressing the key and turn off when the finger leaving the key Pressing the MODE key one more time the program will jump back to MODE Vent closed ACTION 100 psi ii Press Press open sms sims lt 3 toclose vent VENT VENT vent y ACTION V 100 psi Vent opened Press to trigger the step function of the sequence 1 Z V STOP AUTO OT Press back to Press to stop in V eee first mode i terrupt the action ACTION 30 psi Dn age ACTION lt 100 psi ms INJ 1 0 85sec STOP a a AUTO SEQUENCE lt 4 0 9 7 AUTO SEQUENCE V STEP 2 REL BAL POEM STEP 1 INJECT Sequence mode Keys for Mode 5 CLR Rete RELH CLRH INU learn Hop FILL sims Press one of function VENT keys to generate an ac net Press back to mode 1 STOP Press to trigger an auto sequence pon Press totom off input pressure and c AUTO step or interrupt a manual action off halt the mode press again to recover 13 4 PRINCIPLES AND TECHNIQUES FOR MICROINJECTION USING THE BALANCE PRESSURE There are two purposes whena balance pressure is applied to the injection pipette The first purpose is to restrict the inject pressure or suction vacuum to be buffered in the p
11. to add up calibrated volumes Up to 12 programmable steps in each sequence give the user versatile sequential injection or suction cycle By pressing the footswitch AUTO key or input an external signal to the TRIGGER BNC during action mode the PM2000 will perform sequential preset action step by step The sequence cycle can be constantly repeated and the user can manually interrupt the sequence atany step This auto sequence function will giveauser many convenience in controlling different microinjection cycles and ensure that all cycle conditions are absolutely identical Users will find this function is also very useful for multi cell injection or suction Pressure vacuum counting and action time monitoring The PM2000 performs real time data acquisition for pressure and vacuum monitoring In the pressure mode user canimmediately see display of Fill vacuum generator pressure Injection pressure Balance pressure and Hold vacuum or monitor the input pressure During the pressure mode user can adjust four regulators to preset four pressures vacuums There is memory for saving all four preset pressure readings in each sequence By pressing akey todisplay previous pressure settings the user can compare present setting with the previous ones By pressing another key the present pressure readings will update the memory and be saved During operations of action mode the operating pressure is monitored and is real time displayed in the corner ofthe LCD
12. 0 psi pressure input gas is the only gas source required for the PM2000 to produce a wide range of pressure and vacuum outputs There are two output ports on the PM2000 front panel one for a cell holding pipette and another fora cell injection or suction pipette The PM2000 has been equipped with not only injection and balance pressure regulators but also hold and fill suck vacuum regulators Both pressure and vacuumare individually controllable There are atotal of eight different functions in the PM2000 Four of eight functions clear injection pipette clear holding pipette suction in the inject pipette and injection in the inject pipette are programmable Two functions control cell holding vacuum for holding cell stop vacuum forreleasing a cell and other two functions for balancing capillary action of injection pipette on off the pressure for the balance The clearing pipette applies input gas pressure and activate programmable timer and counter parameters to clear injection pipette The Clear Hold function applies adjustable hold pressure to push a cell out of the holding pipette or just clear the Hold Pipette Fill function suction generates aregulated vacuum and activates its programmable timer and counter parameters to fill the injection pipette from the tip The inject function applies regulated pressure and programmable timer and counter parameters to perform precise ejection All four programmable timing functions can be manually con
13. DE key second time the injector enters MODE 2 from MODE 1 The fill vacuum generator pressure injection pressure balance pressure and holding vacuum are simultaneously displayed on the LCD FILL Monitors the supply pressure for the fill vacuum generator real time displays the change while adjusting Fill supply pressure control Refer to the Fill Vacuum Flow Characteristics for actual vacuum conver sion INJ Monitors the injection pressure real time displays the change while adjusting the Inject pressure control BALN Monitors the balance pressure real time displays the change while adjusting the Balance pressure control HOLD Monitors the holding vacuum real time displays the change while adjust the Hold vacuum control The actual vacuum will be displayed while the holding action is on The actual vacuum will be more negative than the displayed number when the displayed number is larger than 3 1H2O If the holding action is off the displayed vacuum will be more negative than the actual vacuum Pressing the 0 down ST key the previous saved pressure readings will be displayed on the right side of the monitored pressure With the comparison of previous memory reading the user can adjust the pressure controls to the same number as before to ensure the identical pressure condition If auser wantto up date the present pressure setting to the memory just simply pressing the 100 up SEQ key all current pressure readin
14. E INJECTION PRESSURE BALANCEPRESSURE FILL VACUUM HOLD VACUUM REPEATABILITY DISPLAY ACCURACY TIMERSETTINGRANGE COUNTER SETTINGRANGE TOTALSAVABLESEQUENCES PROGRAMMABLESTEPS PER SEQUENCE REMOTECONTROL PORTS OUTPUT SIGNAL PORTS WORKINGTEMPERATURE 100 120 or 240 VAC 35 Watts 60 100 psi Same as input pressure 0 3 psi regulated by the Hold regulator 0 1 60 psi regulated 0 10 psi regulated 0 24 Hg regulated Q 25 of H20 regulated 0 05 psi 0 05 psi for pressure 0 1 H20 for holding vacuum 0 1 8 10 msec to 327 67 sec 10 msec resolution for whole range or MANUAL control 1 255 63 sequences 12 steps Remote DB9 connector foot switch jack andTrigger BNC fornegative TTL input Two BNCs for INJECT and FILL positive TTL signalsoutput 4 C 43 C 39 F 105 F Fill Vacuum Generated from Displaying Pressure m m A MN e ak oO ak coo N ait Generated Vacuum inch Hg 0 15 30 45 60 75 90 Supply air pressure psi 7 ACCESSORIES Accessories With The Injector Quantity Part Description 1 Power Cord 1 FSW FootSwitch 1 IP 1 Insert Connector with 1 8 I D Tubing for gas input 2 OP 2 Insert Connector with 2mm O D 1 2mm I D Teflon Tubing for pressure gas output Optional Accessories Part Description PM KP Remote Key Pad with 9 pin Connector MOUSE 2 Remote mouse swi
15. EN 8 E 40 z d Y J 1 HOLD Vacuum Control Used to adjust and set HOLD vacuum for HOLD OUTPUT port 2 BALANCE Pressure Control Used to adjust and set BALANCE pressure for INJ OUTPUT port 3 INJECT Pressure Control Used to adjust and set INJECT pressure for INJ OUTPUT port 4 FILL Vacuum Control Used to adjust and set supply pressure of FILL vacuum for INJ OUTPUT port 5 LCD Display Window Displays real time pressure vacuum time count and total information for programming 6 Control Keyboard Used to program and control the microinjector 7 Power Switch and Indicator Press the switch to turn power on press again to trun power off The power indicator LED displays the power onor off 8 HOLD OUTPUT Port Connector Connect the tubbing insert Holding vacuum and clear pressure for the holding pipette are delivered from this port 9 FILL INJ OUTPUT Port Connector Connectthe tubbing insert The injection balance fill andclearpressures forthe injection pipette are delivered from this port Produces negative synchronous TTL signal output during injection atthe FILL INJ OUTPUT port 11 FILLSYN OUTPUT Signal Output BNC Produce negative synchronous TTL signal output during suction atthe FILL INJ OUTPUT port 12 TRIGGER INPUT Signal Input BNC Inputanegative TTL signal to this BNC as a sequen tial step trigger It performs the same function as AUTO key on the keyboard and the same function as the foot switch 13 Action
16. ameter 10 will change fluid conductance by 30 Therefore larger tip will deliver more material but easy damage to the cell Smaller tip need higher pressure to deliver material and easy to be clogged A pipette with smaller tip can inject few picoliters the smallertipthe more precise control For cell holding the tip ofapulled micropipette must be polished by amicroforge 3 Testing procedure Thepowercord mustbe plugged into an AC outlet with an identical voltage as specified on the rear panel After turn on the power the power indicating light must be on and the LCD display will display the software version number The followings are steps to use the injector A Check input pressure no higher than 102 psi let the buzzer on then press the MODE key B Itis Sequences mode now ignore this mode in the first testing press the MODE key again C Itis Pressure mode now Check and adjust pressures adjust Fill pressure to about 60 psi Inject about 30 psi Balance about 0 3 psi Hold about 15 1H2O 12 inch of water then press the MODE key again D Check and preset timers Set Clear and Clear Hold 10 20ms Inject 100ms Fill 500ms then press the MODE key again E Check and preset counters Set all counter to 1 then press the MODE key again F Itisready foractions now Before connecting pipettes to pipette holders be surethe pipette ortubbing opening does not point toa person UseClear CLR and Clear Hold CLRH function to blow tu
17. anner notonly accelerating the understanding of the molecular control of cell physiology but also clarifying the ever present problem of cause and effect in the complexity of biological systems The microinjection has been acost effective simple and powerful new technology The use of microinjection has rapidly grown andis expected to become amajor molecular and cellular technique in near future Equipped with an advanced microcomputer and precise pneumatic components the PM2000 MICROINJECTOR is just designed to satisfy scientists every requirement during microinjection Itis a superior device for cell microinjection intracytoplasmic sperm injection ICSI and cell electrophysiology For the Cell Microinjection the PM2000 can be used to inject any type of molecule orcombination of molecules into the cytoplasm ornucleus Forexample RNA DNA proteins antibodies labelled dyes synthetic molecules peptides and cell organelles all can be injected Cell holding transferring and even chromosome dissection can also be performed The PM2000 can deliver from picolitter to unlimited wide range of liquid toatarget For the Intracytoplasmic Sperm Injection ICSI the PM2000 can be controlled precisely to pickup and transfer sperm With a smooth and very fine adjustable suction ejection pressure and a computerize timing valve even a single sperm can be handled conveniently and precisely Complete capabilities for cell microinjection and suction A60 10
18. bbing few times Then connecting inject pipette to inject pipette holder connect cell hold pipette to cell hold pipette holder Insert injection micropipette to some liquid water don t forget press BALANCE key first to output balance pressure to the injection pipette Slowly increase the balance pressure to push the liquid out of the pipette until all the liquid just out the pipette tip The air should be stable in the pipette tip See Fig 1 If increasing the balance pressure doesn t push the liquid inside the pipette check any leakage at all connections of output port to tubbing tubbing to pipette holder and pipette holder to pipette Now the INJ key can be pressed totry an injection A small air bubble can be injected during the short time inject period If the air is continue injected even the inject action indicator LED turns off be sure the Balance or the Vent function must be turned on before injection For detail Balance and Vent function principle see USING THE BALANCE PRESSURE If the injection is correct then continue totry Vent Fill Clear Clear Hold Hold cell functions to get used to the microinjector Increasing Balance Pressure to push gt gt gt pl Capillary Action Balancing Injecting o Fig 1 2 A CLOSER LOOK AT THE INJECTOR FRONT PANEL PM2000 Microinjector SYN FILL SYN TRIGGER INPUT 10 INJ SYN OUTPUT Signal Output BNC 7 l AA gue ai 2A N lai PE N
19. ferent valve action forthe pipette above Suction from the pipette tip Ejection from the pipette tip Turn on FILL Turn on INJECT valve valve Turn on BALANCE and VENT valves Turn on BALANCE valve only Turn on VENT valve only Output port e pressure 20 Hg 0 0 2 psi 4 5 psi 10 psi 20 psi Adjustable range of 5S Capillary action balance point ejection or suction at 10um tip opening SUCTION AND EJECTION CHARACTERISTIC OF A PIPETTE TIP 14 MICROINJECTION FOR DIFFERENT TIP OPENINGS Thereare different control methods when using pipettes with differenttip opening size The following Table 1 measures balance pressures with different tip opening or taper I D in water from 0 06 psi to 50 psi TAPERID or BALANCE TIPOPENING PRESSURE COMMENL Vent should be turned on as well as setting Unpulled 0 6mm 0 06 psi bal ne Tip alance to minimum ME poA 180um 0 18psi n EE Unpulled Taper Tip 100um 0 35 psi section section section 10 dus Adjusting balance manually will back and for m PSI ward the solution inside the taper vsum 10 25 psi Good for quantitative injection snm 28 90 psi For very sensitive agent test or injection TABLE1 The different microinjector controlling methods and applications for different tip openings are discussed as following
20. gh the Filter Drain Hole to drain out liquid Do not input gas pressure higher than 100 psi Before turning off the microinjector use CLEAR and CLEAR HOLD functions to clear up Injection Output Port and Hold Output Port To preventrisk of electric shock do not open the unit no user serviceable parts inside Refer servicing to qualified service personnel Trouble Shooting After injection stop air still goes out from the injection pipette Turn on Balance or Vent function before injection and adjust to the capillary action balance point The liquid can not be held inside the pipette Check the pipette tubing and connector possible leakage Adjust Balance pressure Replace the leaking output connector and tubing There is input pressure but no output pressure or vacuum when press the function keys Make sure the injectoris in the ACTION mode and the action timers and counts are correctly set Make sure the output connectors are completely screwed in the output ports The cell still attaches the holding pipette even press the RELH to release Hold Press the CLRH to push the cell out of the holding pipette Ifthe CLRH shoot the cell too faraway reduce the Hold pressure Hold pressure regulator controls the Hold vacuum as well as Clear Hold pressure the higher Holding vacuum and the higher Clear Hold pressure will be 17 6 SPECIFICATIONS LINEVOLTAGE POWER CONSUMPTION INPUTGAS PRESSURE CLEARING PRESSURE CLEAR HOLD PRESSUR
21. gs will be stored into the current sequence memory Pressing CLR keys while in the MODE 2 the LCD will display INPUT CLEAR pressure Pressing one of the FILL INJ BALN and HOLD keys the LCD will back to four pressure monitor above Press to enter pressure mode ASS fig P FILL 60 0 psi INJ 20 5 psi BALN 2 5 psi HOLD 3 2 iH20 Press to display pressure memory RV ST 0 V P FILL 60 0 M 50 0 INJ 20 5 M 10 0 BALN 2 5 M 1 0 HOLD 3 2 M 5 0 Press to save pres sure measurement SEQS 100 V P FILL 60 0 M 60 0 INJ 20 5 M 20 5 BALN 2 5 M 2 5 HOLD 3 2 M 3 2 Pressing the CLRH key will display Clear Hold pressure as well as Holding l Press to display input vacuum They are controlled by the same Hold regulator The higher Holding clea preseure vacuum is set the higher Clear Hold pressure will be Press to dis CLR Press to next mode play other V V7 P FILL 60 0 psi Um INPUT CLEAR fier INJ 205 psi inis PRESSURE V BALN 2 5 psi 9 100 psi HOLD 3 2 iH20 Timer mode Keys for Mode 2 py ST Press to display previous ASEQB Press to store present pressure 0 pressure reading memory 100 reading to the memory Won Press to display in Press to turn off input pressure and ANODE pray on put p LT Press t
22. h actions and can repeat the action step cycle without end Press to Press to pre SEQUENCE 1 pe cts SEQUENCE 1 Moa SEQUENCE 1 STEP 1 CLEAR RST STEP 1 CLEAR BYST STEP 1 CLEAR STEP 2 CLR HOLD 1 1 STEP 2 CLR HOLD gt STEP 2 CLR HOLD STEP 3 FILL STEP 3 FILL STEP 1 CLEAR Press to Press to set Press to set Inject INJ Clear Hold gt CLRH set Vent 7 vet V V V SEQUENCE 1 SEQUENCE 1 SEQUENCE 1 STEP 1 INJ STEP 1 CLEAR STEP 1 CLEAR STEP 2 CLR HOLD STEP 2 STEP 2 CLR HOLD STEP 3 FILL STEP 3 FILL STEP 1 VENT Press to nex tl gt ne mode V Pressure mode Keys for Mode 1 FILL sims STOP Press to preset a function VENT AUTO on the step press the same V SEQ SEQ Press to select EYST ST Press to select key will cancel the function 10 1100 a sequence 0 1 a step CLR RELB RELH CLRH INJ BALN HOLD pon Press to turn off input pressure and off halt the mode press again to recover K Press to mode 2 ML MODE 2 PRESSURE MONITOR By pressing the MO
23. he small amount liquid can only be played around inside the taper and tip section turning the balance control knob manually only will move the liquid back and forward from the tip Medium Tip 10 100 um The suction and injection principles are the same as the larger tip pipette except the balance pressure at the tipis higher 4 5 0 35 psi This balance range is covered by the Balance adjustment without turnon the Vent However the balance pressure for the unpulled section is still very low lt 0 1 psi the Vent should be turned on too The Inject and Fill functions still work only for the liquid inside the unpulled section But playing the liquid inside the taper and tip section can be performed with turning Balance knob without turning on the Vent Unpulled section Taper section Holding Pipette Tip section Injection Pipette Mediadrop with target cell Solution or agent drop to be sucked then injected 15 function Forinstance in the application of Intracytoplasmic Sperm Injection ICSD with along and fine pipette taper and tip 2 10um1 D andjustturning the balance pressure adjustment knob a sperm can be picked up and ejected out of the tip precisely The pipette with 20 50 um I D opening can also be used to produce a holding pipette To forma holding pipette the pipette tip is polished by a microforge until 10 20umI D and 70 120um O D for the tip then itis bent to about 30 40 degree angle by a microforge or
24. ing memory different setting timers and counters which will be reflected on other modes In other words the injector can store up to 63 complete different settings which include 63 sets of 12 action steps 63 pressure readings 63 timers and 63 counters There are up to 12 programmable steps ina sequential cycle The LCD displays the first 3 steps in the beginning of this mode The cursor blinks on first step row to indicate the current edited step row Press key 0 down ST to go down the edited step number Press key 1 up ST to go up the edited step number Press key 10 down SEQ to go down the sequence number Press key 100 up SEQ to go up the sequence number Press one of the CLR RELB RELH CLRH INJ BALN HOLD FILL and VENT s ms keys to preset corresponding function in the current edited step Press the same key again will cancel the setting on that step Pressing the VENT s ms key will preset the VENT function step First VENT step will open the vent hole then the second VENT step will close the vent hole toggle key operating Press key AUTO to preset return function in the current step This function will cause the first step to become the next step and repeat the sequential step cycle Therefore the step AUTO must be the last step of the sequence Whenever finishing the 12th step during the action mode the first step will be automatically become the next step automatic repeat cycle Therefore a user can set all 12 steps wit
25. ipette after an action Another purpose is to compensate the suction caused by amicropipette capillary action The following two characteristics compare the injection output with without balance pressure INJECTING INJECTING PRESSURE PRESSURE PRESET PRESET PRESSURE PRESSURE BALANCE BALANCE PRESSURE PRESSURE TIME TIME INJECTION INJECTION PRESSURE PRESETTIME PRESETTIME BUFFEREDTIME Injection with balance pressure will Injection without balance pressure will continue inject turn off at the correct timing for some time even the injection valve is turned off The smaller ID of apipette the strongercapillary action ithas There are different capillary action forces inside a pulled micropipette because there are different IDs fromthe tip to unpulled portion ofa pipette Therefore to keep liquid inside a pipette closer to the tip higher balance pressure must be applied Other factors to effect the applied balance pressure are the surface tension of the liquid and the depth of tip inserting in the liquid An example of balance pressure applied toa pulled pipette as following Balance Pressure 0 06psi 0 06 psi 035psi lpsi LSpsi 3psi irs 0 58mmLD y 5 z 1 mmO D pipette b SR The liquid boundary 10 um tip opening The pulled pipette can be precisely controlled to suck or inject by the microinjector The following characteristic chartshows the princeple of suction orejection corresponding to dif
26. is suitable for oxygen insensitive injection material Inert gas nitrogen is suitable for air sensitive material A pressure of 60 100 psiis sufficient An input pressure of higherthan 110 psi will damage the device Any very active flammable or corrosive gas isnot recommended for the input gas source There are two gas output ports on the front panel See FRONT PANEL There are two inserts with 1 16 ID or other smaller ID hard wall tubbing which will prevent pressure be buffered to the pipettes Put one of inserts into the Hold Output port and connectits tubing to the holding pipette holder Put another insert into the Injection Output port and connect its tubing to the injection pipette holder Don t connect micropipettes to the pipette holders before blowing the tubing and holder Never let the injection tubing pipette holder holding pipette or injection pipette point to a person Connectto apressure Connect the power cord to a AC Nec source 50 100 psi power outlet as specified voltage Pipette Holders Three Axis Hy draulic Micro manipulator Joystick Hydraulic Micromanipulator C em 777 Inverted Microscope Optional Remote Key Pad Foot Switch Injection Pipette Cell Holding Pipette About the micropipettes Microinjectiontechnique alsorequires skills in making andusing micropipettes Micropipettes ranging inside tip diameter from 0 20 5 0 um can be formed by a pipette puller Change in inside tip di
27. leasing holding vacuum the CLRH key can be press to push the cell away The pushing pressure can be adjusted from the HOLD pressure regulator and controlled by its timer and counter The CLEAR HOLD function also canbe used to clear the holding pipette during ACTION mode Press to se lect CLEAR HOLD function for the SE QUENCE TIME and COUNT mode Press todisplay Hold pressure vacuum from PRES SURE mode Press to select INJECT function for SE QUENCE TIME and COUNT mode Dur ing ACTION mode press to generate an injection which is controlled by its timer and counter the adjustable INJECT pressure is delivered to the injection pipette Press to select BALANCE function for SE QUENCE mode During ACTION mode press to deliver an adjustable BALANCE pressure to the injection pipette to balance capillary action Press to select HOLD function for SE QUENCE mode During ACTION mode press to generate an adjustable hold cell vacuum to the holding pipette to graspacell on the polished tip Press toselect FILL function for SEQUENCE TIME and COUNT mode During ACTION mode press to generate a suction vacuum whichis controlled by its timer and counter The adjustable suction vacuumis delivered to FILL the injection pipette 3 OPERATING AND PROGRAMMING After turn on the power After turning on the power switch the LED power indicator must be on and the LCD displays the software version number The following message will be di
28. o mode 3 CLR CLRH put clear pressure Pott halt the mode press again to recover 10 MODE3 TIMER By pressing the MODE key third time the injector enters MODE 3 from MODE 2 The LCD displays all four timers Press one of the CLER CLRH INJ and FILL keys to move the cursor on corresponding timer row The timer will determine the action time Press s ms VENT key to select unit second or unit 10 millisecond Press key 0 to reset the timer and the reset timer also means the action can be controlled manually by pressing the action key without timer control which is displayed as MANUAL 0s for second unit and MANUAL 0m for 10 millisecond unit Press key 1 to add 1 into the timer Press key 10 to add 10 into the timer Press key 100 to add 100 into the timer The maximum time setting for each timer is 327 66 seconds More than 327 66 seconds setting will turn the timer to be CONTINUE displayed as CONTINUEs m which means that action will be acting continually until the STOP AUTO key being pressed Example of setting 125 38 sec for CLEAR Press CLR key to move the cursor on CLR row Press s ms VENT key to display sec Press 0 key to reset the timer Press 100 key one time Press 10 key two times Press key 1 five times Then press s ms VENT key again convert to msc unit Press 100 key three times Press 10 key eight times If manual controlled action time is needed just press 0 key to reset
29. quence Mode but check the Pressure Mode Adjust the Balance pressure to minimum Injection to 25 psi Fill to gt 30 psi Then go to Time mode set Clear to 0 01 0 03 sec Clear Hold to 0 1 0 3 sec Press 0 key to reset Injection and Fill to MANUAL 0 control Then press Mode key to Count Mode Set all action counts to 1 Press Mode key again to the Action Mode After connecting output tubing holder and micropipette correctly always press on the balance first According the tip size described above adjusta correct balance pressure by looking the liquid movementinside the pipette through a microscope Then try to press Injection key foot switch ora remote mouse injection key or filland control the timing manually SAVING GAS Although the regulators inside this microinjector consume less gas than other model of injector saving gasis needed during slack time Pressing the P O Pressure On Off key during any one of the five modes will shut off the input gas source and halt the mode but save the mode information and all presetting Pressing the P O key again will turn the input gas source on and send the microinjector back to the last mode 16 MAINTENANCE AND TROUBLE SHOOTING Maintenance Alwaysuseaclean dry gas source as the input gas Every time after using the microinjector turn off gas supply at the source and check the filter bowl from two Gas Filter View Holes If accumulated liquid is found in the filter bowl push the drain valve throu
30. se sequences let the user activate functions without pressing different keys and repeat the action sequence cycle conveniently Besides 12 action steps four timers four counters and pressure reading can be saved in the memory for each sequence The pressure reading is on the next mode The programming of timers and counters will be described in third and fourth modes Press to upper sequence SEQUENCE 1 SEQ i SEQUENCE 2 STEP 1 CLEAR 100 STEP 1 INJ STEP 2 CLR HOLD lt a STEP 2 INJ STEP 3 FILL A STEP 3 INJ Press to lower sequence Presetting an action on each step will let a user activate actions step by step with just press foot switch or press AUTOkey in lateraction mode Ifa user plans to control actions by manually pressing different individual action key presetting action step is unnecessary However if a user wants to control some actions repeatedly the user will find out that using AUTO SEQUENCE function is very convenient 8 When the power is turned on the unit will recover the previous sequence which performed before the unit power was off If a user wants to edit or use other sequence setting key 100 up SEQ can be pressed to go up the sequence number or key 10 down SEQ to go down the sequence number Changing a sequence will not only give the user a different action steps but also will give the user different pressure read
31. splay to monitor input pressure Press to turn off buzzer Press to enter mode 1 t INPUT CLEAR WS gt INPUT CLEAR m PRESSURE EM PRESSURE St 100 psi lt na 100 psi I BUZZERON BUZZER OFF Sequence mode Press to turn on buzzer If input pressure is less than 60 psi adjust or change the input gas source to meet the requirement 100 psi is the maximum The input pressure also is the CLEAR and CLEAR HOLD pressure BUZZER ON indicates that the buzzer will beep when one of the CLEAR CLEAR HOLD INJECT or FILL function is activated The user can turn the buzzer off by pressing the key 0 or on again by pressing key 1 There are five MODEs to operate and program the IM300 Press MODE key on the keyboard to select one of five MODEsas the following sequence PROGRAMMING SEQUENCE DISPLA Y PRESSURE SET TIMER SET COUNTER ACTION back to beginning Keys for power just on condition PYST Press to disable fST Press to enable 0 the buzzer 1 the buzzer MP MODE Press to mode 1 C_T Mode 1 Programming Sequences After pressing the MODE key one time the IM300 enters MODE 1 the Automatic Action Sequence mode This mode lets the user review and select one of 63 sequences or preset actions up to 12 steps for each sequence In the later action mode the
32. ss again to recover 11 MODE 4 COUNTER By pressing the MODE key fourth time the injector enters MODE 4 from Presstoentercountermode MODE 3 The LCD displays all four counters Press one of the CLER CLRH INJ and FILL keys to move the cursor on fing corresponding function row Thecounter will determine how many times the V action occurred in each action trigger manual and sequential If the user calibrates the unit delivery forinjection or suction under certain pressureand COUNT CLR 1 time the counter can be easily used to calculate more delivery CLRH 1 Press key 0 to reset the counter FILL 1 Press key 1 to add 1 into the counter INJ 1 Press key 10 to add 10 into the counter Press key 100 to add 100 into the counter Press to add one The maximum count setting for each counter is 255 Y 1 Press to next mode Press to Fill V Y COUNT CLR 1 NN COUNT CLR 1 Cones CLRH 1 CLRH 1 i FLL m lt PL FILL 1 Action mode me n 2 Keys for Mode 4 CLR CLRH INJ FILL Press to select one Ry ST ST SEQ SEQB Press to add num of four action LO 1 101 100 ber on the counter on Press to turn off input HFassure and ADE Pott h Ve 7 Press to mode 5 alt the mode
33. tches control INJ FILL and AUTO OP 1 Output Tubing with Perfusion Vial and Connector IP 1 xx Input Connector with Selectable Length Tubing OP 2 xx Output Connector with Selectable Length Tubing RMK Rack Mounting Kits with Handles OFC Oil Free Diaphragm Compressor 19 Warranty The following warranty is in place of all other warranties expressed or implied and all other warranties including warranties as to merchantability or fitness are expressly excluded 1 SYSTEM PARTS ANDLABOR MDI MicroData Instrument Inc warrants purchased equip ment to be free of defects in material and workmanship under normal use and maintenance from the date of shipment for a period of one year 90 days in the case of a fuses light emitting diodes and b separately purchased replacement parts Consumable supplies and cables are warranted to be free of defects in material and workmanship at the time of shipment Labor in voiced in connection with repairs performed at MDI s facility is warranted for a period of 90 days from the day of shipment of the repaired equipment 2 LIMITATION OF REMEDY MDI shall have no liability for any direct incidental or consequen tial damages resulting from breach of warranty from the breach of nonperformance of any term This limited warranty does not include service to repair damage from improper installation im proper connections with peripherals external electrical fault accident disaster misuse abuse or
34. the flame of a small burner Small Tip 1 2 5 um This is a very practical pipette tip size for the microinjection Since the tip is small the tip fiction is high and become the major factor to effect the liquid in and out of the tip If the tip is emerged to the liquid the liquid sucked in by capillary actionis notas highas the pipette with bigger tip opening To see the first air bubble producing on the tip balance pressure at the tip the pressure must be added to around 8 12 psi excess some balance adjustment range However since the tip fiction is high the balance pressure can be just set about 0 3 2 psi which will stably keep the liquid inside the pipette from taper section to the whole unpulled section The liquid is less sensitive to the balance pressure than the pipette with larger opening Therefore with the rough balance pressure setting around 0 3 2 psi the injection 15 25 psi or fill function 35 50 psi can push or suck the liquid freely from the taper section to all the unpulled section Itis very controllable except the tip section Since the tip section needs balance pressure gt 10 psi higher than the maximum of balance can be adjusted injection of that tip section will cause back fill Therefore the application of that tiny tip section should not be considered If the user still needs to clear the tip section just turn off the balance and inject a pulse to push all the liquid out Tiny Tip lt 1 um Now weare talking abo
35. trolled or interrupted by pressing AUTO key foot switch or input an external TTL signal during their actions A precise actual action time will be displayed after an interruption and let the user to calibrate liquid volume easily Three BNC connectors corresponding INJECT FILL actions and auto sequence TRIGGER provide electrical TTL outputs and input forexternal synchronizing and recording 1 Programmable sequences for pressure memory time count and action steps The PM2000allows the user to program custom designed action sequences Within each sequence a user can save 4 preset pressure readings preset4 timers and 4 counters for corresponding actions There are 12 programmable action steps in each sequence and total 63 programmable sequences All 63 sequences will be saved even turn off the power The lastusing sequence will be recovered automatically when turn on the power again Therefore a user can program all different pressures timers counters and action steps for differentexperiments up to 63 The user canjust turn on the power tocontinue the lastexperiment or simply select asequence number any one of 63 different experiments can be precisely repeated on any time Ineach sequence the four timers and four counters are preset individually forfour actions CLEAR CLEAR HOLD FILL and INJECTION The time range can be set from 10 millisecond to 327 second in 10 msecond resolution for the whole range The counter function provides a way
36. umber 12 Pressing the VENT s ms key will open a vent hole inside the injection port to atmosphere a small bypassing way to the air Pressing the VENT s ms key again will close the hole A V displayed after the ACTION on the LCD indicates the vent hole is opened and a indicates the vent hole is closed There are two purposes by pressing the AUTO key or step on the foot switch or input a negative TTL to the Trigger BNC A The AUTO key is atrigger fora sequence step during Action Mode If many identical cell injections are needed or eyes are busy on the scope the user can just press AUTO key to follow the preset sequential action steps Press AUTO key one time for one step Or just step on a foot switch which is connected to the AUTOFOOTSWITCH jack It will perform the same function asthe AUTO key Any action key can be pressed to insert a function between two sequential steps The control will be back to the sequence by pressing AUTO key or foot switch again Ifan actionis one ofthe CLR CLRH INJ and FILL functions their timer countdown will be displayed on right side of the function name B During timer action pressing the AUTO key will interrupt and stop the action immediately Meanwhile the actual action time is displayed instead of time count down Setting the timer to CONTINUEs m orlong enough time a user can manually control action time by pressing the AUTO key to break the action When the timer is set
37. uta pipette with the smallest tip The fiction in the tipis even higher than the small tip above Using the Fill function to suck liquid to the higher taper section becomes impracticable To fill the injection pipette ahand held 1 ml pipette or syringe is used to back load the solution to the unpulled section of the injecting pipette Then insert the injection pipette back to the pipette holder Turn on the balance pressure 0 3 2 psi Set the injection pressure about 20 35 psi with short injection time 10 100 ms The injection can deliver picolitter volume with this tiny tip However this kind of tips is very easy to be clogged and is hardly to be cleared up because it takes gt 50 psi pressure to clear the tip and even the taper section Itmust be very cautious when taking out the pipette after finish the injection Make sure all actions must be off except turn on the Vent function to release all buffered pressure inside the holder and tubing before unloading the sharp glass pipette from the holder and don t point the tubing holder or pipette to a person MANUAL CONTROLLING ACTIONS FOR A NEW USER Forthe first time microinjector user manual control injection and fillis always recommended in the beginning Anew user ismore likely to familiar the microinjection by manually controlling the injection or fill time When turn on the microinjector first time and an input pressure gt 30 psi but 100 psi is displayed Press the MODE key ignore the Se
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