KNOWLEDGE · ROBOTICS AND AUTOMATION
Hydraulic and pneumatic microinjectors: set the pressure, or set the volume?
What differs is what pushes the sample from behind the needle. A pneumatic injector pushes with air, a hydraulic one with oil. Air compresses easily; oil hardly compresses at all. So a pneumatic injector sets the pressure, and the needle tip sets the volume that comes out. A hydraulic injector sets the volume, and as much comes out as it pushes.
A microinjector puts nL (10⁻⁹ L) to fL (10⁻¹⁵ L) of liquid into cells or embryos through a glass needle. These amounts are too small to push by hand, so a pneumatic injector sets them with pressure and time, and a hydraulic one with the volume its piston pushes. A hydraulic injector pushed by a motor, with the tubing removed, is a positive displacement injector (Nanoject).
Sources: specifications: Eppendorf 2017 brochure p.11, p.12 · positive displacement: Drummond Nanoject III manual
Try it
Change the needle
Both injectors are set to 1.0 nL with a 10 µm tip. Change the tip and the sample.
-
Pneumatic
Set: injection pressure and injection time
- Volume out0.73 nL (droplet 112 µm)
- Pressure behind the needle×1.00 unchanged
To get back to 1.0 nL, measure a droplet and set the injection time ×1.37
-
Hydraulic (positive displacement)
Set: the volume the plunger pushes
- Volume out1.00 nL (droplet 124 µm)
- Pressure behind the needle×1.37
Nothing to reset when the needle changes
With the tip 10% smaller at 9 µm, the pneumatic injector delivers 0.73 nL (27% less). The positive displacement injector still delivers 1.00 nL, and the pressure behind the needle becomes 1.37 times as high.
The droplets are shown as shot onto mineral oil; the dashed circle is 1.0 nL (124 µm). Treating the tip as a cone, flow goes with the cube of the tip radius and inversely with viscosity. So the injection time the pneumatic injector must reset and the pressure on the positive displacement injector change by the same factor.
Sources: the cube law: this page’s computation (Hagen–Poiseuille, checked with a lumped model) · strongly depends on tip size: WPI μPUMP manual p.16 · cell suspensions have uneven viscosity: Ding et al. 2024 (the 2× is an assumption)
01 Air and oil
Air compresses, oil hardly does
The bulk modulus of air at 1 atm is about 0.1 MPa; that of mineral-oil hydraulic fluid is about 1.4–1.7 GPa. Air compresses over 10,000 times more easily.
To build 100 hPa on a hand-turned injector, 10 mL of trapped air takes 15 turns of the fine knob (turning slowly); oil takes about 0.1 turn.
So air stores the pressure you build, and the tip lets liquid flow to match it. Oil stores almost no pressure, so the volume you push goes out through the tip.
Sources: compressibility of hydraulic fluid: Casey (Machinery Lubrication), Gholizadeh et al. 2011 · the knob: this page’s computation from the CellTram 4r specifications (Eppendorf 2017 p.11)
02 Pneumatic
Sets the pressure and the time
From the pressure source, through two regulators (injection and compensation pressure) and a valve, air pushes the sample in the needle directly.
- Sets
- Injection pressure and injection time. The FemtoJet covers 5–6,000 hPa, with time in steps of 0.01 s.
- Setting up
- Droplet calibration. Every time the needle changes, the volume is measured from the diameter of a droplet shot onto mineral oil (V = πd³/6; 124 µm is 1 nL). Between shots, a compensation pressure keeps medium from being drawn into the needle.
- Limit
- Very short injections. The valve takes 6–10 ms to respond, so WPI advises against injection times below 10–15 ms.
- Triggered by
- Foot pedal, TTL, RS-232. Easy to tie to a robot.
In numbers
A short pulse is cut off before the flow reaches its maximum. Still, a slow rise alone does not reduce the amount, because the pressure falls as late as it rose. The amount comes up short when the valve opens more slowly than it closes. Assuming 6 ms to open and 2 ms to close, a 10 ms pulse delivers only 60%.
The WPI manual says a tip a few percent smaller can change the flow up to tenfold. By the tip’s shape alone, a tenfold change takes a tip 44–68% smaller. It can happen when the injection pressure sits close to a threshold it has to exceed. To release a droplet into oil, the pressure must overcome the water–oil boundary at the tip (20 kPa for a 10 µm tip). With the injection pressure at 1.1 times this threshold, a tip 8% smaller cuts the flow tenfold.
Sources: structure and specifications: Eppendorf 2017 p.6, p.12, WPI μPUMP manual p.1, p.10, p.14, p.17 · droplet calibration: Yuan & Sun 2009, μPUMP manual p.22 · short pulses: Forman et al. 2017 · the pulse amounts, the threshold: this page’s computation (the valve delays are assumed)
03 Hydraulic
Sets the volume
The automated hydraulic injector is the positive displacement injector. A motor and screw push a wire plunger, and the plunger pushes the oil in the needle directly. With no tubing, the volume pushed leaves the tip within 1 ms on a recommended needle.
- Sets
- The volume the plunger pushes. The Nanoject III injects 0.6–999.9 nL in steps of 0.1 nL (0.55 µm of plunger travel).
- Setting up
- Filling with oil. The back of the needle is filled with mineral or silicone oil and cleared of air bubbles; with a bubble inside, it does not inject accurately. No droplet calibration is needed.
- Limit
- Small amounts and fine tips. It cannot inject less than 0.6 nL, and tips of 10–30 µm are recommended.
- Triggered by
- Foot pedal, TTL. Also used for automated injection.
The hand-turned oil injector
A knob pushes a piston, which pushes through an oil-filled tube (CellTram 4r Oil, Narishige IM-21). The tube swells, so on a fine ICSI needle the volume pushed takes 13–32 minutes to leave the tip. So it is closer to a tool that makes large pressures from the same hand movement.
How the hand-turned oil injector is built
Sources: Nanoject III brochure and manual pp.6–9, p.14 · triggering: Forman et al. 2017 Table S1 · automated injection: Guo et al. 2024 · hand-turned oil injector: Eppendorf 2017 p.11, Narishige IM-21 · time to leave the tip: this page’s computation (the tube’s elasticity is assumed)
04 Uses
Jobs that set the pressure, jobs that set the volume
Pneumatic: the same amount into many specimens
- Zebrafish embryos: 1–3 nL of mRNA or morpholino at the one-cell stage. An automated system injected 15 a minute.
- Zebrafish larva xenografts: a robot injects cancer cells.
- Adherent cells, C. elegans, fruit flies: fL to 100 pL (FemtoJet 4i, 0.5 µm inner tip diameter).
- Pronuclear injection in mice, and drug puffs onto nerve tissue.
One needle repeats the same injection, so only that needle needs calibrating.
Hydraulic: a single cell, or a set volume
- ICSI, nuclear transfer, ES cell injection, biopsy: a single sperm, nucleus or cell is drawn into the needle, held still and pushed out. Injection and enucleation use oil injectors.
- Plant cells: they need high pressure (a hand-turned oil injector reaches 20,000 hPa).
- A set number of nL, again and again: one automated zebrafish system used a Nanoject III.
On the holding side (the holding pipette), air is enough. The manufacturer’s workstations also put an air injector there. Air sets small pressures finely and stores them, so the hold stays steady.
Sources: zebrafish embryos: Yuan & Sun 2009, Wang et al. 2007 · xenografts: Ding et al. 2024 · adherent cells, C. elegans, fruit flies and workstations: Eppendorf 2017 p.7, p.12 · pronuclear injection: Liu et al. 2013 · drug puffs: Forman et al. 2017 · ICSI and nuclear transfer: Ogura 2017, Eppendorf 2017 p.7, p.11 · plant cells: Eppendorf 2017 p.8, p.11 · automated positive displacement injection: Guo et al. 2024 · the holding side: Ogura 2017, Eppendorf 2017 p.7
Summary
Set the pressure, or set the volume
| Pneumatic | Hydraulic | |
|---|---|---|
| Pushed by | Air | Oil |
| Sets | Pressure and time | The volume pushed |
| When the needle changes | The volume changes | The pressure it takes changes |
| Setting up | Droplet calibration for each needle | Filling with oil, removing air bubbles |
| Smallest amount | fL | 0.6 nL (positive displacement) |
| Max pressure | About 6,000 hPa | 20,000 hPa (hand-turned oil injector) |
| Triggered by | Foot pedal, TTL, RS-232 | Hand; positive displacement: foot pedal and TTL |
| Uses | The same amount into many specimens | A single cell, a set number of nL |
| Typical devices | FemtoJet 4i/4x, WPI μPUMP·PV820, Narishige IM-401 | CellTram 4r Oil, Narishige IM-21, Nanoject III |
MythAn oil injector is always more precise than an air injector.
In factThey set different things. Turning the knob 0.5° changes about 150 Pa with oil and 1–10 Pa with air. Oil builds large pressures quickly; air sets small pressures finely. What delivers a set volume as it is, is the positive displacement injector, which has no tubing.
Confirmed · Hypothesis · Unknown
- ConfirmedThe structures and specifications were checked against the manufacturers’ documents (Eppendorf, WPI, Drummond, Narishige), and the uses against papers and protocols. The cube law, the knob’s pressures, the time to leave the tip and the threshold were computed with a separately built lumped model.
- HypothesisThe tube’s elasticity and the needle’s cone angle (the hand-turned oil injector’s delivery time depends on them), the valve’s opening and closing times, the 2× viscosity of cell suspensions, the reading that WPI’s “tenfold for a few percent” is the case near a threshold, and the two reasons in 04 (only one needle needs calibrating; air keeps the hold steady).
- UnknownThe pressure inside zebrafish embryos and larvae. No study comparing the accuracy of pneumatic and hydraulic injectors under the same conditions was found.
Sources: specifications: Eppendorf 2017 p.11, p.12, Drummond Nanoject III · the knob and the time to leave the tip: this page’s computation
Questions
Frequently asked questions
What does a microinjector do?
It is a device that pushes and pulls the liquid in a glass needle to put nL to fL of solution, or cells, into cells or embryos. It is used with a manipulator, which moves the needle. There are pneumatic injectors, which push with air, and hydraulic injectors, which push with oil.
How do pneumatic and hydraulic microinjectors differ?
A pneumatic injector pushes the sample by applying a regulated air pressure for a set time. The volume delivered changes with the tip size, the viscosity and the pressure on the receiving side. In a hydraulic injector, a piston or plunger pushes oil, which moves the sample at the tip by the volume pushed.
Why apply a compensation pressure?
It is a low positive pressure kept on between injections. It stops surface tension at the air–sample boundary in the needle from drawing medium into the needle (WPI μPUMP manual). The pressure needed is set by the inner diameter where the boundary sits, not by the tip: about 80 psi at 0.5 µm and 0.08 psi at 0.5 mm.
How is the microinjection volume calibrated?
Shoot a droplet of the sample onto mineral oil, measure its diameter d and get the volume from V = πd³/6. A diameter of 124 µm is 1 nL (WPI μPUMP manual). With a pneumatic injector, this droplet calibration resets the injection time or pressure every time the needle changes.
Does the pressure inside a cell change the injected volume?
Yes, when the injection pressure is low. The inner pressure of two cultured cell lines (RT4, T24) averaged 341 Pa and 405 Pa. Injecting at 1 kPa delivered 20–35% less than calibrated in open medium (Wang X et al. 2018). At an injection pressure of 100 kPa (about 15 psi) this share is 0.3–0.4% (this page’s computation).
How does a positive displacement injector like the Nanoject work?
A motor pushes a wire plunger a set distance into an oil-filled glass needle, which sends that much sample out of the tip. The Nanoject III injects 0.6–999.9 nL in steps of 0.1 nL (Drummond). Because the plunger sets the volume, there is no need to measure a droplet for each needle.
Why are oil injectors used for ICSI?
Because a single sperm or nucleus has to be drawn into the needle, held still and pushed out. Injection and enucleation use oil injectors; the holding side may use air (Ogura 2017). For the same hand movement, an oil injector changes the pressure 16–160 times as much as an air injector (this page’s computation).
How do the FemtoJet 4i and 4x differ?
Both are electronic pneumatic injectors that set the injection pressure, the compensation pressure and the injection time. The 4i has a built-in compressor and is recommended for series of injections of fL to 100 pL. The 4x uses an external pressure supply and is recommended for volumes up to 1 µL and long series of injections (C. elegans, early fish embryos, frog oocytes) (Eppendorf 2017 p.12).
Further reading
- Eppendorf, Smooth Operator: micromanipulator and injector brochure (2017)
- WPI, µPUMP manual (2024)
- Drummond, Nanoject III manual (2023)
- Forman et al., Openspritzer: an open hardware pressure ejection system (Scientific Reports, 2017)
- Wang X et al., Effect of Cell Inner Pressure on Deposition Volume in Microinjection (Langmuir, 2018)
- Wang W et al., A Fully Automated Robotic System for Microinjection of Zebrafish Embryos (PLoS ONE, 2007)
- Yuan, Sun, Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos (JoVE, 2009)
- Ding et al., Automated microinjection for zebrafish xenograft models (bioRxiv, 2024)
- Liu et al., Pronuclear Microinjection and Oviduct Transfer Procedures for Transgenic Mouse Production (2013)
- Ogura, Cloning Mice (Cold Spring Harbor Protocols, 2017)
- Guo et al., Design of an automated robotic microinjection system for batch injection of zebrafish embryos and larvae (2024)
- Gholizadeh et al., Fluid Bulk Modulus: A Literature Survey (International Journal of Fluid Power, 2011)
- Casey, In Search of the Perfect Hydraulic Fluid – Part II (Machinery Lubrication)
- Computation: a lumped model (Python) with the compressibility of air, oil and tubing and the resistance of the tip was used to check this page’s numbers and Try it. The tube’s elasticity and the needle’s cone angle are assumptions.