PicoInjector PLI100
Contents
1. Bushing Metal Sleeve the holder through its Knurled Nut o ring silicon rubber Glass tbe gasket and its fitting is then tightened Fig 3 PLEPH1 PHIA 1 0 mm OD Glass Pipette Assembly 11 Harvard Apparatus Model PLI 100 Pico Injector User s Manual Using the Pico lnjector Contd PLI PPH Unscrew the end of the holder with the 2 mm diameter hole and place it over the out put hose PLI OH Reattach this fitting securely with the hose inserted as far as it will go The 1 5 mm OD cap illary is similarly attached Hex Nut Output Hose Holder Bushing Metal Sleeve to the other end of the Silicon Rubber Gasket 7 5mm L x 15mm ID holder through its o Knurled Nut ring Glass Tube Attach output and hold Fig 4 PLEPH1 PHIA 1 5 mm OD Glass Pipette Assembly ing hose to the appropri ate front panel gas ports Tighten them securely so that the valves within these ports are open allowing pressure to be controlled in the hose and connecting micropipette Micropipette Filling Filling can be done from the tip of the micropipette or with a few extra steps from the barrel of the pipette Filling from the tip is most suitable if a filling fiber is deliber ately not being used due to irregularities in the balancing see BALANCE below Simply prepare a bolus of injection material on a glass slide and maneuver the tip into it using a micromanipulator Depress the FILL button to apply about 12 psi 822. C into Swiss 3T3 cells Silver R B Proc Nat Acad Sci 83 4302 4306 1986 antibodies into sand dollar embryo de Laat A M and Blaas J PI Sci 50 161 169 1987 cytoplasmic organelles into plant protoplasts
3. Foot Switch es up to four functions 19 Rack Mount Brackets Pipette Holder up to 2 Input Hose Adaptor introduction Harvard Apparatus Model PLI 100 Pico Injector User s Manual The PLI 100 PICO INJECTOR allows small liguid volumes to be delivered precisely through micropipettes by applying a regulated pressure for a digitally set period of time 2 The pressure is applied pneumatically compressed gas to deliver volumes from microliters to femtoliters from the same instrument This digital injection pressure is enhanced by three auxiliary pressures FILL Suction can be applied to the delivery pipette to fill it from the tip HOLD A second internal vacuum pump is available to hold a suspended cell for injection with a second holding pipette BALANCE A lower balance pressure applied to the delivery pipette between injections prevents clogging caused by pipette movement as well as dilution of the injected material by capillary action CLEAR Momentary application of high pressure can be used to clear clogged pipettes Triggering of injection is accomplished three ways panel push button optional foot switch or an externally applied electrical signal TTL pulse The duration of injection can be determined by an internal clock set with a digital switch or can be gated by the duration either of an externally applied electrical pulse or of the depression of an optional foot switch This manual assumes the reader has no previous
4. clear a clogged pipette The pressure applied is the supply pressure If the button is left pushed in for longer than a half second the clearing surge is extended The button remains lit for the duration of the clear FILL pushbutton Push the button to apply suction to the delivery pipette to fill it from the tip The suction is applied as long as this button is held and LED is lit HOLD pushbutton Push this button to apply sufficiently small suction to holding pipette LED is lit to hold a cell for injection Push this a second time to drop this suction to zero PRESSURE display This three digit display gives the gauge pressure selected by the PRESSURE METER SOURCE switch A negative sign indicates suction PRESSURE UNITS pushbutton Leave this button out to read the gauge pressure in the display in pounds per square inch psi Push it in for the pressure in kilopascals kP 1 psi 6 89 kP Gauge pressure is the absolute pressure atmospheric pressure Atmospheric pressure is about 15 psi 103 kP A red light indicates which units are in use RESET pushbutton This button resets the injection count display to zero INJECTION COUNT display This four digit display 0 9999 gives the total number of injections triggered man ually by the panel pushbutton or footswitch or electrically since the last reset Front Panel Connectors and Controls Harvard Apparatus Model PLI 100 Pico Injector User s Manual 10 PRESSURE METE
5. experience with microinjection either extracellular or intracellular Those planning on larger volume delivery 100 pL and up can ignore the BALANCE feature Some reference will be made to a less pre cise technigue for smaller volume delivery to highlight the precision determining fea tures of this instrument This technigue3 4 involves pressurizing the gas in a macro syringe attached to a delivery micropipette by advancing the piston of the syringe with a micrometer screw Ejection takes place continuously from the pipette the vol ume delivered depends both on the unregulated over pressure in the syringe and the poorly known time the pipette is left inside the cell This continuous ejection tech nique supposedly reduces the frequency of pipette clogging Reliable microinjection also requires skill in making and using micropipettes Authorities on microinjection state that the most important single factor in microin jection is the micropipette The PICO INJECTOR with its reproducibility simplifies the search for the optimum micropipette 5 Harvard Apparatus Model PLI 100 Pico Injector User s Manual Preliminaries Microinjection Microinjection also involves other skills several other instruments and accessories and various supplies The purpose of this section is to give an overview of these techniques for those new to microinjection some guidance on equipment selection is also sup plied Required Auxiliary Equipment for Micro
6. physiological characteristics of micropressure ejection from pipettes Neuropharmacology y 19 931 938 1980 Hiramoto Y Exp Cell Res 27 416 426 1962 Pneumatic micrometer syringe mercury See Kiehart D P Methods Cell Biol 25 13ff 1982 for a careful explana tion Graessmann A Exp Cell Res 60 373 382 1970 Mario Capecchi private communication Dennis Stacey private communication 10 micron pipettes would be suitable for frog oocytes but there is little need for the sizes between 1 5 and 10 microns Pipettes will have more than one taper angle if the pulling force and or heat change during pulling Two stage pullers give two angles for example Delivery rate depends on the angle nearest the tip Stephens D L et al Easy to use equipment for the accurate microinjection of nanoliter volumes into oocytes Anal Biochem 114 299 309 1981 Brinster R L et al Proc Natl Acad Sci 82 4438 4442 1985 DNA into mouse oocytes Webster D R et al J Cell Biol 105 265 276 1987 tubulin into human fibrob lasts and hamster ovary cells Palmer M R et al in Electrophysiological Techniques in Pharmacology pages 169 187 1986 Alan R Liss Inc Various materials onto mammalian neurons mRNA into Xenopus oocytes see footnote 9 Stacey D W et al Exp Cell Res 171 232 242 1987 ras protein into tumor cells Pasti G et al Nature 324 375 377 1986 protein kinase
7. pressure is needed In general however when a balance pressure is being used such a fiber should not be used sometimes the injection material will run out spontaneously or the pressure needed to balance will abruptly change with time These may be due to par ticles lodging on the fiber 12 Harvard Apparatus Model PLI 100 Pico Injector User s Manual Using the Pico lnjector Contd Balance continued If desired a rough estimate of the volume coming in can be made Focus the micro scope on the liquid meniscus in the loaded pipette while its tip is still in air with the balance pressure set to zero Use an eyepiece reticle to measure the meniscus move ment when liquid is then raised to cover the tip The inflow volume is approximately the difference between the volume of two cones The estimate is more precise for smaller initial volumes in the pipette Clear To clear a clogged micropipette push the CLEAR button momentarily once the tip has been removed from a cell Watch the tip region while this is done to see the brief movement of liquid denoting that the tip is clear Repeat if needed The clearing pres sure pulse has a preset duration of 500 msec to avoid excessive release if the clog clears immediately Repeat as needed Hold To hold a suspended cell for injection attach a suitable holding pipette to the pipette holder holding hose Momentarily attach to the P oyr port Backfill the holding pipette using the fill funct
8. solution To easily obtain the desired pressure setting set the time pulse on 1 one second with the injection pressure set at its minimum Trigger the time pulse while viewing under magnifica tion Increase the injection pressure until the solution within the pipette begins to flow out the tip opening The pressure shown on the LCD can now be used as the ini tial injection pressure setting Adjust the injection pressure and timing to obtain the desired injection 3 Holding Pressure Set the holding pressure at the minimum This will prevent possible damage to the cell Fill 3 4 of the pipette s capillary tube with a solution medium This serves as a buffer when the holding function is 2Z preventing a constant vacuum at the pipette tip caused by capillary action Gas Usage Warning To provide finely controllable output pressure the gas regulators are of the bleeding type Such regulators use gas even in the absence of ejections The PICO INJECTOR thus uses gas even when off To eliminate this consumption and as a good safety practice turn off the gas supply at the source when the PICO INJEC TOR is not in use The suction functions HOLD and FILL use much more gas than INJECT and BAL ANCE Plan accordingly Notice that the BALANCE is on continuously once an output hose is attached to the front panel without a micropipette attached even this gas usage can be significant Hose Connections The input output and holding hos
9. 75 2 2272 05 2 27 40 1 75 7 3029 40 3 03 50 1 75 gt 3786 75 3 79 60 1 75 E 4544 10 4 54 Constant includes unit conversion factors and effects of water like viscosity and needle taper for a cone half angle of 10 medium taper Power Entry Module PEM 15 AO Ling Corded 1 0 12 EO 2 PowerEnky koda PEM pare v H 3 u pu z a wo Tate Por Puss Drvo 2 bo Por Pune Raeerbty 2 Figure 5 Power Entry Module PEM and Fuse Drawer 1 With a small flat screwdriver squeeze tabs of fuse drawer and pull out 2 With a small flat screwdriver open tabs of fuse assembly very gentle to avoid breaking the tabs Harvard Apparatus Model PLI 100 Pico Injector User s Manual 3 Rotate fuse assembly to AC Line in use Replace fuse according to fuse rating 100 120 5 Amp Fuse 220 240 25 Amp Fuse 5 Assemble fuse drawer into Power Entry Module PEM until you hear a click ACLine Contact e j 2 aclinoContct o 2 3 u ov 3 a no Assembly for 100 120 Assembly for 220 240 Window boread ACLinousod a MNE Figure 6 Bottom View 16 Harvard Apparatus Model PLI 100 Pico Injector User s Manual Footnotes and References osmom 10 1 12 13 14 15 16 17 McCaman R E etal A pressure system for intracellular and extracellular ejections of microliter volumes Brain Research 142 141 1977 Palmer M R et al Physical and
10. Facilities and Parts Harvard Apparatus stocks replacement and repair parts When ordering please describe parts as completely as possible preferably using our part numbers If practi cal enclose a sample or drawing We offer a complete reconditioning service CAUTION This apparatus is not registered with the FDA and is not for clinical use on human patients A CAUTION Not for clinical use on human patients Specifications Harvard Apparatus Model PLI 100 Pico Injector User s Manual PLI 100 Pico Injector Specifications Input Gas Pressure Injection Pressure Balance Pressure Fill Pressure Hold Pressure Clearing Pressure Injection Time Pressure Display Injection Count Display Duration Mode Trigger Mode Pressure Monitor Out Vacuum Modes AC Power Lines Accessories Supplied Weight Dimensions Optional Accessories 70 105 psi 480 720 kPa 0 2 60 0 psi 413 kPa regulated multi turn control 0 1 9 9 psi 68 9 kPa regulated multi turn control 12 psi 82 kPa Vacuum 0 to 1 25 kPa 12 7 cm of water Vacuum Inlet pressure unregulated 0 01 99 sec 1 99 sec Three and a half digits 0 9999 Internally timed or externally gated Front Panel Foot Switch external TTL pulse Trig in Gate in BNC Connector 10 mV psi Fill Hold 115 VAC 5 amp fuse 220 VAC 250 amp fuse Input hose output hose holding hose and power cord 15 pounds 6 8 kg 15 in x 10 in x 5 in 38 cm x 25 5 cm x 11 cm
11. LIH to the rear panel input connector of the PICO INJECTOR Connect the other end with optional input hose adapter if needed to the gas supply Turn on the POWER switch and verify the input gas pressure with the digital meter by setting the PRESSURE METER SOURCE switch to P cr p Practice with the inject and balance pressure controls by first turning the METER SELECT switch and then adjusting each in turn With non zero values of inject and balance pressure set the PRESSURE METER SOURCE switch to Pour NOTE The PRESSURE METER SELECT switch must be set to P gur to read changes in pressure on the display or MONITOR output Set the inject time to five seconds and push the panel INJECT switch to see the temporary change in pressure from balance to inject and back A buzzer will sound during injec tion If this is not wanted turn off the switch on the rear panel Two output hoses PLI OH 8 PLI HH are supplied with the PICO INJECTOR These are designed for any of the three optional pipette holders PLI PH1 PLI PH1A PLI PPH described in the PRICE LIST Connect the PLI OH PLI HH hoses to the chosen holder s as follows PLI PH1 PLI PHIA Unscrew the end of the holder with a 2 mm diameter hole This end hex piece should be placed over the tube end Thread the hex piece on the holder and tighten firmly The 1 mm Hex Nut Output Hose Holder OD capillary is inserted Silicon Rubber Gasket 5mm L x 1mm ID in the opposite end of
12. Model PLI 100 Pico Injector User s Manual PLI 100 Basic Pico Injector 65 0001 PLI 100 Plus Pico Injector 65 0002 PLI 100 Deluxe Pico Injector 65 0003 HARVARD APPARATUS Harvard Apparatus Model PLI 100 Pico Injector User s Manual Table of Contents Warranty and Repair Information 2 Specifications uno Sea a kk 3 INFOdU NON sniper oa a oe oken 4 Preliminaries nnnnaannnnnnanaaaa 5 6 Front Panel Controls and Connections 7 8 Rear Panel Connectors and Attachments 9 Using the Pico lnjector 10 12 Micro Injection Substances Cell Types 13 Setting Pressure and Time Pulse 13 Gas Usage Warning 0000 anna 13 Hose Connections aaa aaa 13 Volume Calibration Chart ooo aaa nnnnnnnna 14 Power Entry Module aaaanna aaa nakana 15 Footnotes and References aaa nana aaa aaa aaa aa nana 16 Warranty and Repair Information Harvard Apparatus Model PLI 100 Pico Injector User s Manual Serial Numbers All inquires concerning our product should refer to the serial number of the unit Serial numbers are located on the rear of the chassis Calibrations All electrical apparatus is calibrated at rated voltage and frequency Warranty Harvard Apparatus warranties this instrument for a period of one year from date of purchase At its option Harvard Apparatus will repair or replace the unit if it is foun
13. R SOURCE switch This switch selects various places inside the PICO INJECTOR for reading the pressure It does not change the pressure applied to the output PINJ is the pres sure applied to pressure applied at the output during injection PBAJ is the pres sure applied to the pipette when injection is not taking place PCI is the pres sure externally supplied to the input on the rear panel and used the CLEAR mode Pryyy is the negative pressure applied to fill the injection pipette from the tip Pour is the pressure currently applied to the output port 11 P INJ regulator This seven turn control is used to set the injection pressure over the range from about 0 4 to 60 0 psi about 2 8 413 kP Clockwise rotation increases the pres sure Locking nut should be loose to avoid damaging the shaft s threads if chang ing pressure constantly 12 POWER pushbutton Push this button once to apply AC power to the unit The button will light Push it again to turn the instrument off 13 TRIG IN connector This BNC connector is for electrically initiating injection A positive TTL level pulse is required baseline 0 0 V trigger 5 V lasting at least 50 microseconds 14 GATE IN connector This BNC connector is for external timing of the duration of injection A positive TTL level pulse see 13 is reguired with the duration of injection determined by the duration of the applied pulse 15 TRIG GATE OUT connector This TTL output see 13 can be used t
14. d to be defective as to workmanship or material This warranty does not extend to damage resulting from misuse neglect or abuse nor mal wear and tear or accident This warranty extends only to the original customer purchaser IN NO EVENT SHALL HARVARD APPARATUS BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the above limitation may not apply to you If a defect arises within the one year warranty period promptly contact Harvard Apparatus Inc 84 October Hill Road Building 7 Holliston Massachusetts 01746 1371 using our toll free number 1 800 272 2775 Goods will not be accepted for return unless an RMA returned materials authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replace ment and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the purchase of the original device This warranty gives you specific rights and you may also have other rights which vary from state to state Repair
15. es should be attached to their respective connec tors If each connector s needle valve located in the micro injector body is not fully opened the airflow will be restricted or blocked To prevent this from happening check each connector for tightness by turning clockwise This will ensure needle valve depression Volume Calibration Chart Harvard Apparatus Model PLI 100 Pico Injector User s Manual Formula Volume in Nanoliters 17952 x tip I D in micron x Pressure in psi x Time in sec Example Volume 17952 x 5 X 10 X 1 Nanoliters 224 40 Nanoliters Pressure Time Pipette Tip p s i sec I D um Femtoliters Picoliter Nanoliter Microliter Milliliter 1 1 0 1 179 52 0 18 10 1 0 1 1795 20 1 80 gt 20 1 0 1 3590 40 3 59 30 1 0 1 5385 60 5 39 Z 40 1 0 1 7180 80 7 18 50 1 0 1 8976 00 8 98 z 60 1 0 1 10771 20 10 77 1 1 1 179 52 0 18 10 1 1 1795 20 1 80 20 1 1 3590 40 3 59 30 1 1 5385 60 5 39 40 1 1 7180 80 7 18 50 1 1 8976 00 8 98 60 1 1 10771220 10 77 1 1 5 z 22 44 0 02 10 1 5 224 40 0 22 20 1 5 448 80 0 45 30 1 5 673 20 0 67 40 1 5 897 60 0 90 50 1 5 1122 00 kle 60 1 5 1346 40 1 35 1 1 10 179 52 0 18 10 1 10 1795 20 1 80 20 1 10 gt gt 3590 20 3 59 30 1 10 5385 60 5 39 40 1 10 2 2 7180 80 7 18 50 1 10 k E 8976 00 8 98 60 1 10 2 s 10771 20 10 77 1 1 75 g 75 74 0 08 10 1 75 z 757 35 0 76 20 1 75 s 1514 70 1 51 30 1
16. h to the internal timer for subsequent injections Electrical Inputs Outputs 13 16 in Figure 1 These connectors allow additional control especially when synchronizing injection to other stimulations or recording See items 13 16 on pages 8 and 9 for details In par ticular the PRESSURE MONITOR output signal is a precise measure of relative injec tion volume for a given pipette Since the volume delivered is linear in the net injec tion pressure P INJ P pay and injection time the area under a graph of this signal ver sus time is a measure of the relative volume Injection Volume Adjustment Adjust injection volume by changing either injection pressure injection duration or the micropipette inside tip diameter or taper The dependence on net injection pressure duration and pipette taper angle is linear the dependence on tip diameter is cubic 13 Harvard Apparatus Model PLI 100 Pico Injector User s Manual General Information Micro Injection Substances Cell Types A wide range of substances have been micro injected onto or into a large number of cell types References 10 17 on page 16 give some recent examples Setting Pressures and Time Pulses 1 Balance Pressure Set the balance pressure while viewing the pipette under magnification This method will help the user to stabilize the solution within the pipette easily 2 Injection Pressure and Time Pulse Setting the initial injection pressure low prevents the loss of
17. ics are straightforward because the pipettes remain in focus as they are advanced For cells that can be or are attached to a surface in a closely packed layer straight injec tion pipettes can also be used In this case the pipette axis slopes slightly down from the horizontal The tendency of the cells to slide when the pipette enters is resisted by the extracellular environment or attachment to the culture surface The microscope should be focused on the cell s surface The pipette tip then comes into focus just before injection If the cell is nearly spherical the hardest case the pipette should again enter the cell membrane at right angles to avoid shearing Non spherical cells for example cultured fibroblasts have a more robust cytoskeletal structure so the pipette can be pushed in even if not perpendicular to the membrane surface For less firmly attached cells the injection pipette can be bent near the tip after pulling The pipette s main axis slopes slightly down from the horizontal The angle of bend should allow the tip to point straight down With an inverted microscope the tip is viewed through the cell as it is lowered for injection The microscope is focused on the cell s top surface and the tip comes into focus just bef_re insertion Again shearing forces are avoided Suitable bends can be made with a microforge a simple way to do this is to move the pipette near a hot filament at the position of desired bend The tip wil
18. injection The required equipment includes a pipette puller and micromanipulator s Ideally the puller should be capable of making pipettes with tip diameters in the 0 2 to 1 57 micron range with a short enough taper length for both mechanical strength and low flow resistance If most injections are to be extracellular then a puller suitable for extracellular patch clamp pipettes is satisfactory For intracellular injections some magnetic pullers may be suitable Alternatively a two stage gravity puller with variable weights can be satisfactory over the entire range The required three dimensional movement can be produced by an inexpensive mechanically linked micromanipulator for large cells such as frog oocytes More commonly a hydraulic one for fine vibration free movement is mounted on a coarse mechanical manipulator Suitable equipment is available from Harvard Apparatus Inc Required Supplies These are compressed gas and microcapillaries Compressed air is suitable for oxygen insensitive injection material Nitrogen is a satisfactory inert gas for the general case A pressure of 105 psi is sufficient a regulator will be needed if supplied from a bottle of compressed gas Optional Equipment for Microinjection This includes a microforge to bend a micropipette or to polish a pipette tip for hold ing a cell a microgrinder to bevel the pipette tip to increase the delivery rate with out additional cell damage and an micro incubator t
19. ion switch with fluid so that tube length is half to three quarters filled Disconnect the holding hose connector from the P oyr port and attach to the P Hopp Port Set the hold pressure control to minimum and move the micropipette to the cell using a micromanipulator Turn on the HOLD panel switch and increase the hold pressure as needed When finished with that cell turn off the hold pressure with the panel or footswitch If more cells of the same kind are being inject ed then subsequent holds require only the ON OFF control of the switch If the cell doesn t drop off the holding pipette tip when hold is turned off a brief gentle squeeze of the rubber bulb located on the holding output hose should release it Note Be careful not to overfill the bose to avoid liquid going into the pneumatic system inside the unit Footswitch es Optional footswitches can be used for inject fill and turning on and off the hold func tion These switches are plugged into the rear panel each is wired in parallel with the corresponding front panel switch See the instructions for each panel switch to see how they operate When a footswitch is used with the GATE connector the duration of injection is manually determined by the duration of pressing the footswitch In some applications users may prefer to set up the duration of injection manually con nect the MONITOR output to an oscilloscope or fast enough strip chart recorder to measure this time and then switc
20. kP of suction continuously as long as needed If evaporation of a small bolus is a concern it can be prepared under oil and the micropipette tip similarly moved into it Filling can be done from the back end barrel of the pipette using a syringe with a thin hypodermic needle inserted so its tip is down in the tapered section If the capil lary has a filling fiber attached to the inner wall the pipette tip will then fill by capil lary action without air bubbles at least for larger tips Variations on this procedure involve filling a smaller capillary first and inserting it for the back fill Balance The inflow into the pipette caused by capillary forces prior to or in between injec tions should be a small percentage of that being injected As the volume desired gets smaller the relative inflow gets larger even faster due to the smaller tip size being used To avoid this problem set the balance pressure before placing the pipette in the cell s external medium Ten percent of the injection pressure is a good starting value Exact balance is difficult to determine often the fastest way to handle this is to set the bal ance high enough that slight outflow is observed Balance is assessed by watching the movement of the liquid meniscus in the pipette The outflow left is still small com pared to the continuous streaming used in the continuous ejection technique If a filling fiber is used the capillary inflow is larger so a higher balance
21. l spontaneously bend away In all of the above techniques a three dimensional micromanipulator controls the movement of the injection pipette If this straight pipette is instead attached to a con denser mount inverted scope then a one dimensional manipulator can be used The remaining two directions of manipulation are done with stage micrometers moving the vertical injection pipette over each cell in turn If the vibrations transmitted with the condenser mounting are manageable then this approach gives the fastest rate of cell injection Harvard Apparatus Model PLI 100 Pico Injector User s Manual Front Panel Connectors and Controls 13 INJECTION MAM PRESSURE kPa COUNT o I PRESSURE METER IMJECT MEET J FILL X CO x 10 msec po We lt lt ier aed o mine A ma bomo CD df ali eae Figure 1 Front Panel Refer to Figure 1 for the location of controls and connectors Numbers refer to the order of listing below 1 INJECT pushbutton Push this to manually trigger the injection pressure for a time set by the internal timer The switch remains lit for the duration of the injection INJECT TIME pushbutton This determines the time multiplier for the internal injection timer 10 msec and 1 0 sec multipliers are available CLEAR pushbutton Push this briefly to deliver a half second pressure surge to
22. m case Audio Indicator Switch When ON an audibile tone sounds for the duration of injection Turn it OFF if this audible monitor is not desired Filter Window Allows for easy viewing of filter to determine when it needs to be drained 10 Using the Pico lnjector Harvard Apparatus Model PLI 100 Pico Injector User s Manual General Considerations It is much easier to reliably inject large volumes than small ones For large volumes the balance pressure capability is not needed Pipettes seldom clog so the clear capa bility is also not needed The dividing line between large and small is not rigid it depends on how guantita tive a delivery is reguired That volume line would typically be in the 10 100 pLiter range For convenient visualization and approximate geometric measurement 1 fLiter is a cube 1 micron on a side or a sphere 1 24 microns in diameter 1 pLiter is a cube 10 microns on a side or a sphere 12 4 microns in diameter while 1 nLiter is a cube 100 microns on a side or a sphere 124 microns in diameter Because volume goes as the cube of linear dimensions such geometric volumes are imprecise but often useful Extracellular delivery is nearly always large Intracellular is often of small volumes but not for frog oocytes A more quantitative way to distinguish between large and small is given in the balance section below Interconnections and Initial Set Up Connect the gas input hose PL
23. o hold the cells at incubation temperatures during microinjection Suitable equipment is available from Harvard Apparatus Inc Accessories These are also available to enhance your PICO INJECTOR see Price List Micropipettes The micropipettes are made with a pipette puller from a microcapillary 1 2 mm in diameter by heating some 3 10 mm of its length with a concentric heater while applying a force gravitational or magnetic to pull both ends of the capillary apart Two micropipettes are produced per capillary 1 Distinguishing Parameters Two useful distinguishing parameters of the micropipette are the inside diameter of its tip and the angle of taper to the tip The smaller this angle the longer the tapered region The larger the tip the more material is delivered for the same applied pressure and time Just a 10 decrease in diameter decreases the delivery rate by over 30 A 10 decrease in taper angle longer taper would decrease the delivery rate about 10 The extreme sensitivity of delivery rate on tip diameter makes it important to have a reproducible pipette puller If you use published tip sizes as a starting point dis tinguish between the relevant inside diameter and the more visible outside diameter The ratio of the two is the same at the tip as for the original capillary glass 6 Harvard Apparatus Model PLI 100 Pico Injector User s Manual Preliminaries Contd Micropipettes continued 2 Choosing the Righ
24. o trigger or synchronize other electrical instruments Its duration is that of the injection 16 PRESSURE MONITOR out This BNC connector delivers an electrical level whose amplitude gives the actual pressure applied to the meter The conversion factor is 10 mV psi Set the PRES SURE METER SOURCE switch to Pour to see changes in pressure during injec tion and filling 17 INJECT TIME digiswitch This two digit switch is used to set the duration of injection when timed internal ly The units 10 msec or 1 sec are set by the INJECT TIME switch 18 Pour connector This connector is attached to the injection pipette using the supplied output hose PLI OH Without the hose attached the port is closed In use the injection balance fill and clear pressures are delivered here 19 PHOLD regulator This regulator sets the low suction pressure applied to the Pgorp Connector counterclockwise increases the pressure Like the other pressure controls the current rotational position is uniquely related to the pressure The range is 0 0 5 kP 5 cm height of water 20 PHOLD Connector The supplied holding hose is attached to this connnector The suction set by the PHOLD regulator is applied here to hold a suspended cell for injection 21 Pgay regulator This regulator sets the balance pressure over the range from 0 1 to 10 psi about 7 70 kP Clockwise rotation increases the pressure Harvard Apparatus Model PLI 100 Pico Injector User
25. s Manual Rear Panel Connectors and Attachments INPUT 70 105psi AUDIO INDICATOR Research Products on O OFF FOOT SWITCH INPUTS FILTER IWJECT GATE Figure 2 Rear Panel Foot Switch Inputs Connectors An optional footswitch PLLFS can be connected to any one of these connec tors When connected to HOLD pushing the footswitch once turns on the suc tion for holding a cell Pushing it a second time turns that suction off When con nected to INJ pushing the footswitch starts injection When connected to FILL the filling suction is applied as long as the footswitch is depressed When con nected to GATING injection pressure continues as long as the footswitch is depressed Pressure Input Connector To have all the functions working the instrument needs 70 to 105 psi To have only injection 70 psi is needed This connector is the input for the compressed gas See PRELIMINARIES for recommendations on the gas A maximum of 105 psi can be safely applied 100 psi is optimal At lower pressure gas will be used at a slower rate Avoid input pressures less than 100 psi if the FILL function is being used Input Filter The PICO INJECTOR is supplied with an input filter It traps particles larger than 0 5 micron as well as the liquid often present in a building s compressed air lines Drain by pushing the button on the underside of its transparent case Through a hole located in the botto
26. t Pipette Choosing a pipette size and shape for intracellular injection is difficult Larger tips deliver more material but increase the risk of cell damage caused by leakage around the pipette while in the cell or later by incomplete sealing The smaller the cell the smaller the pipette tip it will tolerate The smaller the tip the more likely it is to clog For reference intracellular electrophysiologists routinely record for an hour or so from cells of 10 micron diameter with pipette tips of 0 1 micron inside diameter Larger tips can therefore be used for brief injection in such cells For nuclear injections a smaller taper angle is needed to avoid leakage further up the shank of the pipette at the plas ma membrane Although even intracellular injection can be done from below with an upright micro scope most injections are done from the side or from above the cells Four different strategies have been used to suitably fix the cell in position for successful intracellular injection For suspended cells a second larger pipette is used to hold the cell This pipette s tip is first polished with a microforge done by placing the pipette within 5 microns of a hot filament for a few seconds With its axis horizontal it is moved to hold the cell with applied suction The injection pipette is also straight and is inserted horizontally from the opposite side This geometry avoids damage to the cell membrane caused by shearing forces The opt
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