Programmable two-stage micropipette puller. INSTRUCTION MANUAL. World Precision Instruments

Transcription

1 Programmable two-stage micropipette puller INSTRUCTION MANUAL Serial No World Precision Instruments

2 Contents Introduction... 1 Specifications... 2 Front Panel... 3 LED Display...3 Keypad...4 Accessories...4 Pulling Area...5 Hardware Setting... 6 Mount the Capillary...6 Set Taper Length...6 Mount the Weight...6 Mount Coil Heater...7 Operation... 8 Basics...8 Turn On Run Stop Pulling Change Heat Setting Alarm Quick Start Transporting the Instrument Replacement Parts Troubleshooting...14 Appendix A: Pulling Principle...16 Appendix B: Program Flow Chart...18 Appendix C: Reference Data...21 Warranty Copyright 2007 by World Precision Instruments, Inc. All rights reserved. No part of this publication may be reproduced or translated into any language, in any form, without prior written permission of World Precision Instruments, Inc. World Precision Instruments 25

3 Introduction PUL-2 is a microprocessor-controlled two-stage puller for making glass micropipettes or microelectrodes used in intracellular recording, patch clamp studies, and microinjection. The glass capillary is heated by a nickel-chromium coil and pulled by gravity. The first pulling stage produces a thin section on the capillary; the second stage breaks the capillary at a lower temperature to form the tip. The puller can produce pipettes with tip diameters from less than 1 micron to tens of microns. PUL-2 is compact, versatile and reliable, and the self-explanatory front panel assures ease of use. Permanent memory allows storage of up to 99 heating settings. World Precision Instruments 1

4 Specifications Capillary...Borosilicate glass, OD: 1.0 mm mm, length > 55 mm. Pulling Force...selectable at 170 g, 250 g or 330 g Taper Length mm Heating Element...Nickel-chromium wire 0.5mm, coil ID 3.0 mm Heating Power Scale , proportional to the voltage on the heater Second Stage Wait Time...6 seconds Auto Shut-off Time...90 seconds Permanent Memory Set...99 Power V, 50/60 Hz, maximum 70 watt Dimensions...21x21x20 cm Weight...15 lb World Precision Instruments 2

5 Front Panel The PUL-2 front panel has an LED display and a self-explanatory keypad (Fig. 1). LED Display Program number, memory status, or heat setting are displayed here. There are several different types of display as listed below, where X means any single digit between 0-9; numbers may be changed with the ^ buttons below the display. PrXX means the permanent memory XX, from which a program loads or to which a program is saved. It is the default LED display after power on or after aborting from most of the other functions. rexx displays when the RECALL key is pressed so a program may be recalled from permanent memory XX to the buffer. Use the ^ keys to select the desired memory register, then press to load it. SAXX displays when the SAVE key is pressed so the heat setting in the buffer may be saved into permanent memory XX. Use the ^ keys to select the desired memory register, then press to complete the save. Figure 1 hxx.x means the heat setting at the first pulling stage. It is shown when pressing key 1. HXX.X means the heat setting at the second pulling stage. It is shown when pressing key 2. _XX.X shows the heating scale, proportional to the output voltage on the heater. World Precision Instruments 3

6 Err is shown for 30 seconds when pulling time exceeds 90 seconds. After 30 seconds, it automatically returns to PrXX state. OOOX shows the delay time count down between the two pulling stages. Keypad POWER Press POWER key to turn on the micropipette puller. Press again to turn power off. Press to abort current action and return to previous setting or PrXX state. RUN Press RUN to start pulling. NUMBER KEY Press the number key 1 to show the heat setting of pulling stage 1, hxx.x. Press the number key 2 to show the heat setting of pulling stage 2, HXX.X. INCREASE KEY ^ Press the ^ key to increase the LED digit above by one. Execute current active function. The function is complete only after pressing. SAVE SAVE key will save the displayed heat setting. Press SAVE to enter SAXX mode. Press key to save the setting. RECALL Press RECALL to enter rexx mode. Press key to load the setting. Accessories A transparent windshield, hinged with industrial strength tape, keeps a consistent pulling environment. The puller comes with two removable magnetic weights, 64 gram and 165 gram. World Precision Instruments 4

7 Pulling Area Warning: The coil can be burned if heating voltage is too high. Do not set heat higher than 55 for a 3-loop coil. Figure 2 Adjusting Screw A positions the upper stage. Green LED B is a pull-position indicator. The PUL-2 is ready to pull when the LED is lit. Capillary Tightening Screw C in upper stage. Upper stage D. Nickel-chromium coil E: wire 0.5 mm, coil ID 3 mm, three loops. Lower stage F. Capillary Tightening Screw G in lower stage. Adjusting Screw H positions the lower stage. World Precision Instruments 5

8 Hardware Setting Mount the Capillary Loosen the capillary tightening screws C and G enough to allow the capillary through the holes. Insert the capillary through holes and the heater coil. Tighten screw C in the upper stage at appropriate capillary length. Be careful when tightening the screw to avoid crushing the capillary. Lift the bottom block up until the green LED lights, then tighten screw G in the lower stage so that the bottom block does not fall. After the capillary is mounted, the green LED should be still on. If the green LED is off, lift the stage until green LED is on and tighten screws C and G. Set Taper Length Two pipettes are made in one pull. Taper length of the upper and lower pipettes may be determined by the respective positions of the millimeter marks on the upper and lower stages (Fig. 2a). The upper stage (and thus the upper pipette taper length) can be set anywhere between 0 and 5 mm. The upper stage can be adjusted with screw A. The lower stage can be anywhere between the 0 and 10 mm marks; taper length of the lower pipette is determined by the difference between the position of the upper and lower stages. The lower stage can be adjusted after loosening screw H by moving the mark block at the right side of the lower stage. For example, to pull both pipettes to a 3 mm taper length: Loosen screw H and move the lower mark block up or down to the 6 mm position from the bottom mark; tighten screw H at that position. Adjust screw A to move the upper stage to 3 mm from the bottom mark. Figure 2a Mount the Weight Pipette tip size is determined by several factors: heating power, taper length, pull strength, capillary material, and capillary OD and ID. Pull strength is conveniently changed by mounting weights on the magnetic disc in the lower stage; it is recommended that increased weight be used to pull larger tips. World Precision Instruments 6

9 Mount Coil Heater Loosen four screws on the electrodes where the heating element E is mounted. Remove the old heating element if one is there. Clean the electrodes. Use tweezers to put the two legs of the new heating element into the electrodes. Mount a capillary to check that it is centered within the heating element s coils. Adjust the position or the angle of the heating element leg, if necessary. The leg can be bent with the tweezers while low heat is on. For example, set the heat scale in the second stage to 10, lift the lower stage with a finger, press RUN to start heating, then use tweezers to push or pull the coil to adjust the leg position. Tighten the four screws on the electrodes after the adjustment. World Precision Instruments 7

10 Operation Basics Permanent Memory Figure 3 Heating power is scaled from 0 to 100, proportional to the output voltage on the heating element. The three-loop coil works best at about and has a typical heating length D ~2.5 mm for borosilicate capillaries (refer to Appendix A). Overheating the coil will reduce coil life or even burn it out immediately. Start with low heating power, such as 40, when a new coil is mounted. Load Save Program Number Buffer Collecting Reference Data for Table 1 The micropipette puller has a permanent memory and a buffer memory as diagrammed in Figure 3. Pulling is processed according to the heat setting in the buffer memory. When the PUL-2 is turned on, the last-used program is loaded into the buffer memory. A previously saved program may be loaded from the permanent memory into the buffer. You may also alter a program that has been loaded in the buffer and then the revised program can be saved in the permanent memory. The program flow chart is in Appendix B. When PUL-2 is used for the first time (or after changing to a new kind of glass capillary or after changing a heating coil), it is recommended to pull a set of capillaries as references, as described below, and record the results in the Tables in Appendix C. If reference data is already available, skip this section and go to Turn On (page 10). Record the date, pulling weight and glass capillary item number in the first column. 1. Set the heating power of the second stage at Loosen the adjusting screw in lower stage (indicated as H in Figure 2) and set the proper taper length. The lower taper setting is set at about 4 mm (from bottom) at the beginning. It will be set for different lengths later for the other tests. Tighten the adjusting screw after setting. Record the taper setting in the Taper Setting column. To make a symmetrical pull, the top taper length is set to about half of the lower taper length by adjusting screw A in Figure 2. World Precision Instruments 8

11 Set the first stage heating power at 40* and record this setting in the Heating Power column of Table 1. Use calipers to measure the length of the glass capillary before mounting it onto PUL-2. Mount the capillary and press RUN. Use a stopwatch to record the heating time in the first stage. Record the heating time in the Heating Time column. When the second stage heating starts, open the cover and loosen the bottom tightening screw (G in Fig. 2). Heating turns off automatically when the lower stage drops. Remove the capillary carefully and measure its length. The difference between the capillary before and after pulling is the accurate total taper length. Record the number in the Taper Length column. Increase heating power by 0.5 and repeat steps 4-8 twice with new glass capillaries. Repeat until heating time is less than 15 seconds. Collecting Reference Data for Table 2 The heating power of the second stage and the taper setting are critical parameters in determining the final tip size. The taper setting can be used as a rough means of setting tip size, and heating power can be used to fine tune the tip size. After finishing the previous tests, perform the following tests and record the data as in Table 2 of Appendix C. * This is an arbitrary number which is usually a good starting point. However, if this setting proves too low to melt the capillary sufficiently to allow pulling, increase heat setting by 1 until the starting point is found. 1. Set the first stage heating power to that which corresponds to about 20 seconds on Table 1 and record it in the Heating Power 1st Stage column on Table At the beginning, set the heating power of the second stage 4 lower than the first stage; for example, 40 for the first stage and 36 for the second stage. After running the first test following the steps below, gradually vary the heating power for further testing, keeping it 3 to 5 lower than the first stage. The variation could be as small as 0.1 for optimal results. Record the heating power for the second stage in the Heating Power 2nd Stage column in Table 2. World Precision Instruments 9

12 Set the lower taper at about 4 mm (from bottom) at the beginning. It will be set for the other length later. Record the taper setting in the Taper Setting column in Table 2. To make a symmetrical pull, set the top taper length to about half of the lower taper length by adjusting screw A in figure 2. Mount the capillary and press RUN. Remove the pipettes and use a microscope to measure the tip size, recording the result in the Tip Size column in Table 2. Repeat steps 4 and 5 twice with new glass capillaries. Increase the taper setting 0.5 mm and repeat steps 4 6. Fine changes can be made to the pulled micropipette when the heating power of the second stage is varied. However, it should be kept in mind that other factors also play a role in the final pulling results, such as environmental temperature, humidity, air flow, etc., so it is best to pull many pipettes at one time instead of pulling a few before they re needed. In day-to-day use, the heating power of the second stage can be readjusted slightly to produce the desired pipette; changes to the first stage heating power and the taper setting should be minimized. It is assumed that the ID/OD ratio of the pipette is the same as that of the original capillary. The outside diameter of tips larger than 2 microns can be measured with a microscope. Bubble pressure measurement can be used to measure outside diameters as low as 0.1 microns*. Turn On Press the POWER button to turn the PUL-2 on. The LED display shows PrXX with XX being the last-used memory number (e.g., PrO2, Pr44, etc.). The heat setting in memory XX is loaded into the buffer. * Bubble pressure measurement of micropipet tip outer diameter, S. Mittman, etc., J. of Neuroscience Methods, 22(1987) Run Press the RUN key to start pulling. Heat setting hxx.x is shown during the first pulling stage. After the first pulling stage, the capillary will have a narrow waist, the heater will turn off and the delay countdown 000X will show on the display. Heat setting HXX.X is shown during the second pulling stage. When the pipette breaks and the lower stage drops, the heater turns off and the instrument returns to a readiness state, with PrXX in the display. World Precision Instruments 10

13 If the maximum pulling time (90 seconds) is exceeded without a completed pull, the instrument automatically stops Err will be shown on LED display and an alarm will sound for 30 seconds. After this warning, the puller returns to a readiness state, with PrXX in the display. Check the Troubleshooting section of this manual for possible causes and remedies. Stop Pulling Press to stop pulling at any time. The heater will shut off immediately and the puller display will return to PrXX. Change Heat Setting Show current heat setting Press Number Key 1 to show the heat setting of the first pulling stage, hxx.x. Press Number Key 2 to show the heat setting of the second pulling stage, HXX.X. Load a new heat setting There are two ways to load a program stored in permanent memory. Press RECALL to enter recall mode rexx will show in the display (with XX shown here to represent stored programs from 1 to 99). Use the Increase Key ^ to change XX. Press to load the desired program into the buffer. Or use the Increase Key ^ to change XX in PrXX mode directly; press to load the desired memory into the buffer. NOTE: Pressing in PrXX mode also saves any changes in the buffer into the permanent memory XX if any setting change has been made. So use RECALL to load a new program if there is any confusion. Change the heat setting Press the Increase Key ^ to change the heating scale in hxx.x or HXX.X. The new value is shown as _XX.X. Press to put the desired value into the buffer. Or press the SAVE key (SAXX will show in the display) and press key to store the value to both the buffer and permanent memory. If any other key is pressed when the display shows _XX.X, the puller will ignore the change and run the new function. NOTE: key does not undo the changes in the buffer. Pressing key in PrXX mode saves both stage settings in buffer into the permanent memory. World Precision Instruments 11

14 Save/Copy the heat setting When LED shows hxx.x or HXX.X, press SAVE key to enter SAYY mode. Use Increase Key ^ to change YY. Press to store the heating value into new permanent memory. Note that this step saves only the setting of the last displayed stage. Press the Number Key to show the other heat setting and repeat the steps above to store the other setting. Alarm An audible alarm is triggered if a pulling stage cannot finish within 90 seconds. The alarm turns off automatically after 30 seconds. Quick Start Turn on the puller. Mount the capillary correctly so that the green LED lights. Close the windshield. Press key 1 to check the heat setting of the first pulling stage. A typical heat setting for the first pulling stage is between 40~45 for the three-loop heating coil. If the displayed setting is too far from this value, use the Increase keys ^^^ to change the setting. Use the heat setting in Appendix C as a reference. Save the setting into the buffer by pressing or save into both buffer and permanent memory by first pressing SAVE and then pressing. Press key 2 to check the heat setting of the second pulling stage. A typical heat setting for the second pulling stage is between 35~40. If the displayed setting is too far from this value, use the Increase keys to change the setting to about 4 below the setting in the first stage. Then press RUN to start pulling. The heater heats up in seconds. The capillary is pulled to have a taper in the first stage. After a 6-second delay, the heater heats up for the second pulling stage. The second pulling should be finished in seconds. Transporting the Instrument Make sure to remove the movable magnet weight and wrap it with soft material before packing the puller. The top stage and the bottom stage should be tied firmly. Close the windshield door and seal with tape or wrap with plastic film. World Precision Instruments 12

15 Replacement Parts Heating Coil, Nickel-Chromium There are no other user-replaceable parts. If repairs are required, return the instrument to the factory: call WPI to obtain an RMA number (Return Merchandise Authorization) or enter in your web browser. World Precision Instruments 13

16 Troubleshooting No Power Check power cable connection. Check the fuse, which is in the power input module. If the fuse is blown, replace with a new 1A fuse. Reset Parameters In some rare cases, the PUL-2 may lose its manufacture fixed parameters because of shaking during transportation or power spike. The user can recover the parameters following instructions. a. Press RECALL while turning on the power. b. After power is on, release the RECALL. The puller LED display should show a countdown in the display. If no countdown occurs, turn off the power and repeat step a) until a countdown shows on the LED display. c. Press and hold RECALL until countdown finishes. When the countdown finishes, the LED shows the delay time. d. Use ^^^ keys to adjust the number to the required delay time. The last digit is seconds. The factory default setting is 6 seconds (the last digit is 6 and all others set to 0). e. Press to finish update. The alarm time for maximum heating time shows up after this. The factory default is 99 seconds. To keep the default value, press key again. You may wish to change it by using ^^^ keys. Press key after change. The last pressing of stored the values in memory, so the puller is now ready to use. Will Not Pull The most likely reason for this problem is that the sensor does not detect the load. Check the marks in the upper stage (D, Fig. 2) and in the lower stage (F). If they are far from center, adjust screw A to move the upper stage or loosen screw H and move the stop block on the right side by hand. Tighten screw H after moving. Make sure the green LED lights after the capillary is properly mounted. World Precision Instruments 14

17 Overheating and Underheating The heat setting default is 40 in the first stage and 36 in the second stage. The default heater is a three-loop nickel-chromium coil. The heat settings may need to be modified for another type of heater or different kinds of capillaries. Overheating can damage the heater, so use a heat setting just high enough to make the desired pipette. Start low and increment slowly. Alarm If the heating time exceeds 90 seconds, an audible alarm will be triggered and LED display will show Err. The alarm automatically stops after 30 seconds. If the alarm occurs during the first stage, check whether anything is blocking the movement of the lower stage; and also check whether the weight is enough. The other possible reason is underheating that is, the heat is not high enough to soften the glass and allow the capillary to be pulled. In this case, increase the heat setting of the first stage. Contact WPI technical support if the above suggestions do not solve your problem. World Precision Instruments 15

18 Appendix A: Pulling Principle When a portion of a glass pipette is heated to the transition temperature, the pulling force pulls the capillary apart and the heated portion of the capillary becomes narrower. Outside the heated region, the glass quickly cools below the transition temperature. It is assumed the length of the portion heated above the transition temperature is a constant, denoted as D. This length D is determined by the heating power, the heating time, the glass property and the pulling force. The final waist size of the thin wall capillary after pulling is empirically guided by: z Φ = Φ0 exp( ) D where Φ, Φ 0 are the waist diameter after and before pulling, and z is the total taper length. In the two-stage puller PUL-2, the taper length z in the first stage is set mechanically by the lower stage adjusting screw labeled as H in Figure 2. It is set between 2 to 6 mm and typically pulls the capillary 2 to 10 times narrower in waist. In the second stage, the taper length is determined by how the capillary breaks. If the temperature is below the transition point and the waist becomes thin enough, the pulling force will break the capillary to form a tip. The pulling force, the heating intensity, the heating time, and the environment all have influence on the breaking of the capillary. World Precision Instruments 16

19 Appendix B: Program Flow Chart The following charts show the programming choices available through the front panel controls and the LED display screens which result from each keypad command. (X and Y in the display represent numbers O to 9.) RUN Abort RECALL Save heat setting of buffer into current program. Load heat setting of YY into buffer. Same as the recall program. SAVE 1 No action, just beep Stage 1 heating setting Abort change YY is lost and return to XX if pressing any other key. 2 Stage 2 heating setting World Precision Instruments 17

20 RECALL SAVE RUN No action, just beep Make program change 1 Abort change 2 Pressing other key invokes the function as PrXX does. YY is lost. World Precision Instruments 18

21 Keep old value Save YY into buffer RUN SAVE RECALL 1 2 SAVE No change in XX memory Change hxxx in XX memory Keep old value New value No change in XX memory Change hxxx in YY memory World Precision Instruments 19

22 Keep old value Save YY into buffer RUN SAVE RECALL 1 2 SAVE No change in XX memory Change hxxx in XX memory Keep old value New value No change in XX memory Change HXXX in YY memory World Precision Instruments 20

23 Appendix C: Reference Data Date, Weight, Glass Capillary Heating Power Heating Time Taper Setting Taper Length Table 1 First stage, comparing heating power and heating time. World Precision Instruments 21

24 Date, Weight, Glass Capillary Heating Power 1st Stage Heating Power 2nd Stage Taper Setting Tip Size Table 2 Two stages. World Precision Instruments 22

25 * Electrodes, batteries and other consumable parts are warranted for 30 days only from the date on which the customer receives these items. Warranty WPI (World Precision Instruments, Inc.) warrants to the original purchaser that this equipment, including its components and parts, shall be free from defects in material and workmanship for a period of one year* from the date of receipt. WPI s obligation under this warranty shall be limited to repair or replacement, at WPI s option, of the equipment or defective components or parts upon receipt thereof f.o.b. WPI, Sarasota, Florida U.S.A. Return of a repaired instrument shall be f.o.b. Sarasota. The above warranty is contingent upon normal usage and does not cover products which have been modified without WPI s approval or which have been subjected to unusual physical or electrical stress or on which the original identification marks have been removed or altered. The above warranty will not apply if adjustment, repair or parts replacement is required because of accident, neglect, misuse, failure of electric power, air conditioning, humidity control, or causes other than normal and ordinary usage. To the extent that any of its equipment is furnished by a manufacturer other than WPI, the foregoing warranty shall be applicable only to the extent of the warranty furnished by such other manufacturer. This warranty will not apply to appearance terms, such as knobs, handles, dials or the like. WPI makes no warranty of any kind, express or implied or statutory, including without limitation any warranties of merchantability and/or fitness for a particular purpose. WPI shall not be liable for any damages, whether direct, indirect, special or consequential arising from a failure of this product to operate in the manner desired by the user. WPI shall not be liable for any damage to data or property that may be caused directly or indirectly by use of this product. Claims and Returns Inspect all shipments upon receipt. Missing cartons or obvious damage to cartons should be noted on the delivery receipt before signing. Concealed loss or damage should be reported at once to the carrier and an inspection requested. All claims for shortage or damage must be made within 10 days after receipt of shipment. Claims for lost shipments must be made within 30 days of invoice or other notification of shipment. Please save damaged or pilfered cartons until claim settles. In some instances, photographic documentation may be required. Some items are time sensitive; WPI assumes no extended warranty or any liability for use beyond the date specified on the container. WPI cannot be held responsible for items damaged in shipment en route to us. Please enclose merchandise in its original shipping container to avoid damage from handling. We recommend that you insure merchandise when shipping. The customer is responsible for paying shipping expenses including adequate insurance on all items returned. Do not return any goods to WPI without obtaining prior approval and instructions (RMA#) from our returns department. Goods returned unauthorized or by collect freight may be refused. The RMA# must be clearly displayed on the outside of the box, or the package will not be accepted. Please contact the RMA department for a request form. Goods returned for repair must be reasonably clean and free of hazardous materials. A handling fee is charged for goods returned for exchange or credit. This fee may add up to 25% of the sale price depending on the condition of the item. Goods ordered in error are also subject to the handling fee. Equipment which was built as a special order cannot be returned. Always refer to the RMA# when contacting WPI to obtain a status of your returned item. For any other issues regarding a claim or return, please contact the RMA department Warning: This equipment is not designed or intended for use on humans. World Precision Instruments, Inc. International Trade Center, 175 Sarasota Center Blvd., Sarasota FL Tel: Fax: sales@wpiinc.com UK: Astonbury World Farm Precision Business Centre Instruments Aston, Stevenage, Hertfordshire SG2 7EG Tel: Fax: wpiuk@wpi-europe.com 23 Germany: Liegnitzer Str. 15, D Berlin Tel: Fax: wpide@wpi-europe.com