Tech.SightCell Biology

Gentle Slam

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Science  18 Dec 1998:
Vol. 282, Issue 5397, pp. 2213-2214
DOI: 10.1126/science.282.5397.2213b

Cell biologists are often interested in manipulating the environment of a single cell. When they want to affect the internal environment of a cell, they often use a technique called single-cell microinjection. Often referred to as “stab” microinjection, the approach consists of rapid insertion of the tip of a glass micropipette into the cell's cytosol under high pressures (100 to 200 mbar) to introduce sufficient material from the pipette. The injection pressure must be carefully controlled: too high a pressure will burst the cell, whereas too low a pressure results in delivery of too little material. The technique works best in large cells (because there is less chance of damaging intracellular organelles such as the nucleus) and in cells that are adherent, because they do not move away from the tip as they are being punctured. Stab injection is used to deliver synthetic molecules, nucleic acids, peptides, and proteins into the cytoplasm of these cells.

In small cells (2 to 15 μm in diameter), however, the approach becomes problematic because the nucleus-to-cytoplasm ratio of these cells increases, leading to a greater likelihood of damaging the nucleus during the stab. A report in last month's issue of the Biophysical Journal describes an interesting modification of the stab approach for injection of solutes into smaller and more mobile cells (1). The researchers refer to their technique as “slam” (simple lipid-assisted microinjection). This gentler approach consists of coating the tip of the micropipette with a lipid layer and letting this layer fuse with the plasma membrane of the cell to form a lipid bridge with an aqueous channel between the pipette and the cytosol. Through this channel, the solute gently diffuses inside the cell.

The glass micropipette is filled with injection medium, and the tip of the pipette is dipped in a 1 mM solution of phosphatidylcholine-oleyl-palmitoyl dissolved in chloroform and kept on ice. Next, the chloroform is allowed to evaporate, so that the synthetic lipid ends up coating the pipette. The pipette is then connected to a pressure control device, and the tip is soaked in aqueous medium to allow the dried lipid to swell and form a bilayer. The pressure inside the pipette is subsequently increased to 10 mbar. Absence of leakage of lucifer yellow, a fluorescent dye added to the injection medium, indicates that an effective lipid seal has been formed.

The authors tested their method on neutrophils, because these cells have features that are challenging obstacles to any microinjection strategy: small size, large nucleus-to-cytoplasm ratio, and loose adherence. The neutrophils were allowed to sediment onto a glass cover slip, and then the micropipette was brought to the surface of single cells with a motorized, microprocessor-controlled micromanipulator. Gentle contact with the cell surface led to transfer of the lipid and aqueous contents of the pipette to the cell. The pressure in the pipette was held constant at 10 mbar to ensure diffusion of its contents inside the cell. To monitor the transfer of the aqueous content, the motion of the lucifer yellow dye was monitored by fluorescence microscopy. The dye could clearly be seen to diffuse inside the neutrophil in a matter of seconds. To monitor the fate of the lipid bilayer, the authors used the fluorochrome 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbo-cyanine perchlorate; this dye is weakly fluorescent in water, but strongly fluorescent in lipid bilayers. Upon touching the cell, this dye was transferred from the micropipette to the cell membrane, where the fluorescence became uniform over time. The authors suggest that the lipid bilayer merges with the cell membrane, while the solutes are transferred to the cytosol. After several seconds, the pipette was retracted and the cells left in place for further observation.

The authors monitored cell damage using the trypan blue exclusion technique. Less than 5% of neutrophils survive the stab injection technique intact, whereas almost all survive the slam approach when the injection is at a pressure of 10 mbar. Higher injection pressures leads to cell damage or even complete cell rupture.

The method described appears simple and could be useful when cells need to be handled very gently. It appears not to damage cells and delivers a reasonable amount of material (about 1% of the cell volume). In addition, the approach could potentially be used to deliver material to the cell membrane by exploiting its fusion with the lipid bilayer. For example, the approach could be used to deliver membrane-soluble receptors to specific cell types. Of course, the method will need to be reproduced in other laboratories and with other cell types before the stab technique can be retired.


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