Warner Instruments PLI-10 User manual

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Wa ar rn ne er r
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In ns st tr ru um me en nt ts s
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Pi ic co o- -l li it te er r
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In nj je ec ct to or r
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Mo od de el l
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PL LI I- -1 10
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The PLI-10 Pico-liter Injector is designed to provide controlled delivery of picoliter to
nanoliter volumes of solution through a glass micropipette. The PLI-10 is ideal for
applications such as neuronal tract-tracing, delivery of pharmacological agents in vitro,
and Xenopus oocyte injection.
Features for the PLI-10 include
9Low cost
9Femtoliter to microliter injections
9Digital readouts for injection pressure, time, and count
9Reliable optically encoded circuit for injection time set
9Easy to use
9Same high quality pressure regulator as PLI-90 and PLI-100
THIS EQUIPMENT IS NOT DESIGNED NOR INTENDED
FOR USE ON HUMAN SUBJECTS
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
[phone] •[phone] •Fax [phone]
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Table of Contents
NOMENCLATURE....................................................................................................................................4
Text conventions.....................................................................................................................................4
GENERAL...................................................................................................................................................5
Instrument description...........................................................................................................................5
FRONT AND BACK PANEL CONTROLS AND CONNECTIONS....................................................5
Front panel..............................................................................................................................................5
Rear panel ...............................................................................................................................................6
SETUP AND OPERATION.......................................................................................................................8
Setup........................................................................................................................................................8
Operation ................................................................................................................................................8
ADDITIONAL INFORMATION..............................................................................................................9
Required Auxiliary Equipment for Microinjection................................................................................9
Required Supplies..................................................................................................................................10
Optional Equipment for Microinjection...............................................................................................10
Micropipettes..........................................................................................................................................10
Distinguishing Parameters....................................................................................................................10
Choosing the Right Pipette....................................................................................................................10
General Considerations.........................................................................................................................11
Injection Pressure and Time Pulse.......................................................................................................12
Gas Usage Warning...............................................................................................................................12
Hose Connections..................................................................................................................................12
Volume Calibration Chart.....................................................................................................................13
APPENDIX................................................................................................................................................14
Specifications.........................................................................................................................................14
Ordering Information..........................................................................................................................14
Warranty and Service..........................................................................................................................15
Warranty.............................................................................................................................................15
Service................................................................................................................................................15
Certifications.........................................................................................................................................16
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
[phone] •[phone] •Fax [phone]
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NOMENCLATURE
Text conventions
This manual refers to instrument controls at three functional levels; control blocks, specific controls
within a block, and settings of specific controls. To minimize the potential for confusion, we have
employed several text conventions which are specified below. Since our goal is to provide clarity rather
than complexity, we welcome any feedback you may wish to provide.
¾Warner Instrument product numbers are presented using bold type.
¾References to specific controls within a block are specified using SMALL CAPS.
¾Finally, references to individual control settings are specified in italic type.
¾Special comments and warnings are presented in highlighted text.
Any other formatting should be apparent from context.
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
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GENERAL
Instrument description
The PLI-10 is specifically designed for solution delivery to extracellular locations in tissues and for
intracellular Xenopus oocyte injection. These ‘large volume’ injections (10 pl to greater than 100 pl) are
common for many applications that do not require multiple pressure settings. As such, the PLI-10 is an
effective, low-cost alternative to our more elaborate pressure ejection systems (e.g., the PLI-90 and PLI-
100).
Pressure to the micropipette is controlled precisely through a high-quality multi-turn regulator and is
reported digitally for reproducibility. Injection time is set using an optically encoded circuit which permits
fine and coarse settings from a single dial knob. The system trigger timer can be controlled by the front
panel push button, a foot switch, or an external trigger (TTL pulse to TRIGGER INPUT BNC).
FRONT AND BACK PANEL CONTROLS AND CONNECTIONS
Front panel
A. RESET COUNT pushbutton: Resets the injection count display to zero.
B. INJECTION COUNT display: Records and displays the total number of injections triggered (0 to
9999 injections).
C. INJECT LED: LED indicator remains lit for duration of injection period.
D. INJECTION TIME (s): Displays injection time period in seconds (0 to 99.99 s)
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
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E. INJECTION PRESSURE: Displays injection pressure in either PSI or kPa (maximum 60 PSI; 413
kPa).
F. PRESSURE UNIT toggle switch: Toggle switch controls injection pressure display unit of
measure. Injection pressure can be displayed in either pound-force per square inch (PSI) or
kilopascals (kPa).
G. PRESSURE OUTPUT (Pout) connector: Pressurized gas output line. Output tubing line
connects Pout to injection pipette holder.
H. MANUAL INJECTION TRIGGER: Press-button trigger delivers regulated pressure for the
digitally set period of time at the desired pressure.
I. EXTERNAL INJECTION TRIGGER INPUT BNC: This input is for electrically initiating injection
from an external trigger source. A positive TTL pulse of is required (baseline 0 V, trigger 5 V,
lasting >10 ms).
J. INJECTION TIME SETTING DIAL: This dial controls the injection time setting. Turning the dial
changes the injection time in seconds; pressing the dial in and turning changes the injection time
in fractions of a second.
K. INJECTION PRESSURE REGULATOR: This seven turn control sets the injection pressure over
the range from 0.2 to 60 PSI (1.4 to 413 kPa).
L. POWER switch: This switch turns the instrument on and off.
Rear panel
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
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M. PRESSURE INPUT (P ) connector: This connector is the input for the compressed gas. 75 PSI
in
is the recommended input pressure; maximum input pressure is 105 PSI.
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N. POWER INPUT connector: This connector is the input for the external power supply (9V DC @
400 mA; universal input voltage 90-264 VAC).
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
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) as an
P. REMOTE INJECT foot switch input connector: An optional foot switch (PLI-FS) can be
connected to the instrument. Pushing the foot switch triggers an injection for the preset time and
pressure values displayed on the instrument front panel.
O. PRESSURE MONITOR OUTPUT BNC: This connector outputs the injection pressure (Pout
analog signal that can be directed to an external recording device. (10 mV/PSI or 1 mV/kPa).
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SETUP AND OPERATION
The PLI-10 is provided with input and output hoses for gas handling and a foot switch for triggering
injections. Additional requirements include an acrylic pipette holder (see ordering information below) and
glass micropipettes. A regulated air/gas supply and a micromanipulator to hold the injection pipette are
needed to create a complete microinjection system. The setup and operation of the PLI-10 is
straightforward.
Setup
1. Connect the instrument to an electrical outlet using the 9V DC power supply input (Item N).
2. Attach one end of the input hose to a regulated air/gas supply and use the quick-clip connector to
connect the input hose to Pin (Item M) on the back of the instrument.
3. Connect the output hose to the instrument Pout (Item G) using the quick-clip connector. Attach the
other end of the output hose to the pipette holder. Position and secure the pipette holder into a
micromanipulator.
4. Turn the unit on (Item L). The three LED displays (Items B, D, and E) will illuminate.
5. Press the
RESET COUNT button (Item A) to reset the injection count display (Item B) to zero, if
necessary.
6. Turn the
TIME SET DIAL (Item J) to the desired injection time (displayed in Item D). Pressing the TIME
SET DIAL in and turning will allow fine adjustment of injection time in tenths of seconds.
7. Turn the
INJECTION PRESSURE set knob (Item K) to set the desired injection pressure (displayed in Item
E). Pressure can be set and monitored in units of PSI or kPa, set by the TOGGLE SWITCH (Item F). If
the desired pressure is unknown, it can be determined by observing a loaded micropipette under a
microscope. Gradually increase injection pressure setting until solution is flowing from the
micropipette tip.
8. Optional remote triggering of an injection can be achieved using a footswitch or TTL pulse. Insert the
footswitch plug into the REMOTE INJECT jack (Item P). Pressing the footswitch triggers an injection.
Similarly, an analog 5V TTL input to the BNC TRIGGER (Item I) can be used to trigger an injection from
an external device (such as a digital acquisition system).
9. Optional monitoring/recording of injection pressure settings can be achieved by the analog PRESSURE
signal output (Item O). This output is calibrated to 10 mV/PSI or 1 mV/kPa.
Operation
10. The assembled system is ready to use. Turn on the air/gas supply at the source, and slowly increase
the outflow regulator to the desired pressure setting.
NOTE: The input pressure should be at least 7 PSI higher than the desired working/injection pressure
for the pneumatic components to operate properly.
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11. Turn the instrument on, and set the injection pressure and injection time to the desired values. The
POUT tubing line should remain unattached to the control unit at this time to prevent any latent
pressure from building up in the micropipette.
12. Backfill the micropipette with a desired volume of the compound to be injected. Microfill-style syringe
tips can be used to fill the micropipette and avoid solution contaminating the outside of the
micropipette.
13. Insert micropipette into the pipette holder and secure it into place by tightening the acrylic screw.
14. Attach the POUT tubing line to the connector on the control unit.
15. Position the micropipette to the location where the injection bolus is to be delivered. This often
requires a micromanipulator.
16. Trigger an injection by pressing the inject button (Item H). Alternatively, an external trigger or
footswitch can be used to trigger an injection.
17. After injection is complete, the micropipette should be disposed of in an appropriate “sharps
container”. For tract-tracing studies, using a fresh micropipette for each injection is advised. A single
pipette can be used to serially inject multiple oocytes with the same compound if the micropipette tip
remains undamaged.
18. Upon completion of the experiment, remove the pipette from the holder and power the unit off. The
air/gas input should also be turned off at the source.
ADDITIONAL INFORMATION
Note: The following material is excerpted from the user’s manuals for the PLI-90 and PLI-100.
These manuals are available for download on our website for your reference.
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 supplied.
Required Auxiliary Equipment for Microinjection
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 movements of the injection pipette can be produced by an inexpensive mechanically
linked micromanipulator for large cells such as frog oocytes.
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
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Required Supplies
These are compressed gas and glass capillary tubes. Compressed air is suitable for oxygen-
insensitive injection material. Nitrogen is a satisfactory inert gas for the general case. An input
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 holding a cell),
a microgrinder (to bevel the pipette tip to increase the delivery rate without additional cell
damage), and a microincubator (to hold the cells at incubation temperature during microinjection).
Micropipettes
The micropipettes are made by pulling a glass capillary (1-2 mm. outer 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.
Distinguishing Parameters
Two useful distinguishing parameters of the micropipette are the inside diameter of the tip
and the angle of taper to the tip. For a single-stage puller, a smaller taper angle will yield a
longer 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, distinguish 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.)
Choosing the Right 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 that is
required. 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 plasma membrane.
Although even intracellular injection can be done from below with an upright microscope,
most injections are done from the side or from above the cells. Different strategies have been
used to suitably fix the cell in position for successful intracellular injection:
Warner Instruments •1125 Dixwell Avenue, Hamden, CT 06514 USA
[phone] •[phone] •Fax [phone]
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