Page 4 - Microinjection

First, let's consider volumes.

• A milliliter (mL) is one thousandth the volume of a liter (L) or 10^-3L
• A microliter (µL) is one thousandth the volume of a mL (10^-6L)
• A nanoliter (nL) is one thousandth the volume of a µL (10^-9L)
• A picoliter (pL) is one thousandth the volume of a nL (10^-12L)

This is graphically represented at the right. Notice that the mL is one trillion times larger than the picoliter. The table (right) shows that the side of a cube with a volume of 1mL is 1cm long. Likewise, the side of a cube with a volume of 1pL is 10µm long. Just for comparison, it shows that the diameter of a sphere with a volume of 1mL is 1.24cm, and the volume of a sphere with a volume of 1pL is 12.4µm.

With these volume comparisons in mind, lets consider some of the available options for microinjection pumps.

World Precision Instruments' PUL-1000 is a microprocessor controlled, four-stage, horizontal puller for making glass micropipettes or microelectrodes used in intracellular recording, microperfusion and microinjection. It offers programmable sequences of up to four steps with complete control over the heating, force, movement and cooling time. This allows graduated cycles for a variety of applications. PUL-1000 can produce pipettes with tip diameters from less than 0.1µm to 10+ µm.

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Micro4™ from World Precision Instruments is an easy-to-use but very capable controller for WPI's Nanoliter Injector and UMP3 UltraMicropPump.

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WPI's Nanoliter 2010 Microinjection Pump is ideal for many applications, including zebrafish, xenopus oocytes and drosophila. MICRO4, an optional microprocessor-based controller, can provide an "intelligent" and easy-to-use interface to up to four Nanoliter Injectors. Operating parameters are set with the membrane key-pad and LCD display.

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Watch how researchers from the University of Chicago inject adult zebrafish using a 10μl NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III (UMP3-1 includes one UMP3 pump and a Micro4 controller). 

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References

Warmerdam, T., Schröder, F., Wit, H., & Albers, F. (n.d.). Perilymphatic and endolymphatic pressures during endolymphatic hydrops. European Archives of Oto-Rhino-Laryngology, 260(1), 9–11. http://doi.org/10.1007/s00405-002-0518-2 

Wei, J., Song, J., Jiang, S., Zhang, G., Wheeler, D., Zhang, J., … Liu, R. (2017). Role of intratubular pressure during the ischemic phase in acute kidney injury. American Journal of Physiology - Renal Physiology, 312(6), F1158–F1165. http://doi.org/10.1152/ajprenal.00527.2016 
Petrie, R. J., Koo, H., & Yamada, K. M. (2014). Generation of compartmentalized pressure by a nuclear piston governs cell motility in a 3D matrix. Science, 345(6200), 1062–1065. http://doi.org/10.1126/science.1256965 
Petrie,...more

Chiara Cianciolo Cosentino, at the University of Pittsburgh, describes how she uses intravenous microinjections of zebrafish larvae to study acute kidney injury in this JoVE video. You can also watch this video on JoVE. WPI equipment shown in this video includes:

• MMP Manual Microsyringe Pump
• PV820 Pneumatic PicoPump
• M3301 Manual Micromanipulator
• M10 Magnetic Stand
• 5052 Steel Base Plate

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Researchers at the University of Michigan are using WPI's PV820 for injecting a morpholino solution into the lumen of the otic vesicle of 1-day old zebrafish embryos.  Then, they use electroporation to introduce mif and mif-like morpholinos into the developing inner ear tissues.

Check out the JOVE video to see:

• How to make electrodes for electroporation
• How to setup the electroporation
• How to inject the morpholinos
• Examine some of the results

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References

JOVE video: Holmes, K. E., Wyatt, M. J., Shen, Y., Thompson, D. A., Barald, K. F. "Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development". J. Vis. Exp. (47), e2466, doi:10.3791/2466 (2011).

Warmerdam, T., Schröder, F., Wit, H., & Albers, F. (n.d.). Perilymphatic and endolymphatic pressures during endolymphatic hydrops. European Archives of Oto-Rhino-Laryngology, 260(1), 9–11. http://doi.org/10.1007/s00405-002-0518-2 

Wei,...more

In this video, Mike Pizza demonstrates how to front fill a Nanofil syringe using MicroFil.

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The Nanoliter injector is a micro-processor controlled injection system that uses direct piston displacement. The collet assembly (exploded view) is shown in the image below. The glass pipette fits into the white, plastic sleeve (spacer). When the collet is screwed in place securely, the black rubber washers are compressed and hold the glass firmly in place.

Nanoliter 2000

A side view of the assembled Nanoliter injector is shown below.

Nanoliter 2010 

As the plunger is depressed, it pushes into the glass to expel the liquid in the pipette. Eventually, the diameter of the plunger matches that of the glass, and the plunger cannot expel any more liquid. If the plunger continues to press on the tip of the pipette, the pipette could crack or become dislodged from the plastic sleeve (spacer) inside the collet assembly. If that happens, the Nanoliter will begin to leak.

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When it comes to setting up microinjection systems, the options appear endless. The pictures below give some broad suggestions on how you might set up your own system. Keep in mind that many parts are interchangeable depending on your needs or preferences.

In general, you will need a stereo microscope on a stand, a light source, one or two micromanipulators with stands, and one or two injection systems. The following images show various setups for microinjection, and all the WPI part numbers are included for easy reference.

Remember, when you set up your own system, choose the parts that fit your needs. For example:

• M10 or the M9 magnetic base could be used.
• PZMIV stereo microscope could be used instead of the PZMIII stereomicroscope.
• M3301 or the KITE micromanipulators can be used, and these micromanipulators can be placed on either side. (Keep in mind, though, if you wanted to use a KITE micromanipulator on the right side of the setup below, you would order a KITE-R (right hand), or...more

Eliminate the cost and trouble of making your own micropipettes — WPI can quickly supply your need for consistently sized pre-pulled glass micropipettes for injection of dyes or proteins into cells, oocytes and for many other biomedical laboratory applications. Tip diameters (ID) range from 0.1 to 10 micrometers. Micropipettes are available as plain shank or with luer fittings.

• Schott Duran borosilicate glass
• 0.5 micrometer and smaller ID micropipettes include an internal glass fiber for easy filling
• Tip inner diameter tolerance ±20%
• Short taper yields high strength
• Nominal length ≈ 50 mm
• OD:ID = 1.33:1
• Standard capillary outer diameters are 1.0 mm (thin-wall) or 1.14 mm
• Every pipette individually tested and inspected
• Vacuum packed

Silanized Tips (Luer Shank)

Silanization waterproofs the glass to retard water when inserting into cell. This will not let the outside fluid run down the pipette and get inside so easily.