Direct measurements of cell turgor and cell membrane water permeability with intact plant cells - the cell pressure probe technique

Qing Ye, Whitney Leigh Kress & Maciej Zwieniecki

Method Overview
Step 1 - pulling micro-capillary	(A) a glass capillary is secured in a pipette puller. (B) heat softens glass to be drawn out and (C) produces two micro-capillaries with the tip size in the order less than a micron in radius.
Step 2 - grinding the tip	A pipette is ground down to open its tip to 5-10 micromiter in size at an angle of 45 degrees. Each pipette tip is visually inspected under the microscope and tested for blockeage by pushing air through it.
Step 3 - filling pipette with oil	A pipette is fixed in a plastic screw. A syringe is used to fill it with silicon oil, and no air bubbles are left in the pipette.
Step 4 - attaching pipette to pressure probe	The plastic screw is tightened into a holder that connects to the pressure probe.
Look at the pressure probe	Pressure probe is constructed out of plexi-glass. It allows for attachment of pressure transducer (best with small internal volume measuring pressures up top 3.0 MPa) and a port for movable rod allowing for changes in internal volume of the pressure probe via use of the micro-positioning screw.
Step 5 - probing the pressure of leaf epidermal cell	The pipette is maneuvered into place above the leaf, and now is ready to be used to enter into a cell.

Step 5 - establishing the meniscus in teh capillary tip	Once the pipette has entered the cell, a cell sap/oil meniscus is formed in the tip of the pipette which can be seen under the microscope. This serves as a point of reference during the measurements in the experiments. With the aid of the pressure probe, the position of the meniscus can be changed forward or backward and kept stable after the change. This results in pressure relaxations.
Step 8 - measurments	The cell turgor pressure measured by the probe is recorded and graphed by a computer. Four hydrostatic pressure relaxation curves are shown here that can be used to determine the cell membrane water permeability (hydraulic conductivity, Lp).

Setaup overview A: micro-manipulator B: cell pressure probe C: microscope D: illuminator (fiber optics) E: glass micro-capillary and its holder