Chemistry: molecular biology and microbiology – Plant cell or cell line – per se ; composition thereof;...
Reexamination Certificate
1999-03-23
2001-08-21
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Plant cell or cell line, per se ; composition thereof;...
C435S001100, C435S420000
Reexamination Certificate
active
06277637
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods and apparatus for measuring turgor pressure of a cell or cells by determining the area of contact between a probe and a specimen, and more particularly to an instrument including a transparent mechanical probe and its use to view the area of its contact with a specimen.
BACKGROUND OF THE INVENTION
A characteristic of a deformable specimen that can be related to area of contact between a mechanical probe and the specimen is the turgor pressure of a cell. Growing plants are hydrostatic structures. Plant form is maintained by turgor pressure. In most of the biomechanics of plant growth, an understanding requires some knowledge of turgor pressure changes to determine the physical properties of the plant, such as yield threshold and wall modulus. However, turgor pressure is not readily measured in a nondestructive, noninvasive way.
Traditional approaches for determining turgor pressure in plant cells were conducted using either an incipient plasmolysis method, a pressure bomb method, or a micropipette-pressure-probe or “micropressure probe” method (see Park S. Nobel, Physicochemical and Environmental Plant Physiology, 103, 176-180, Academic Press Inc., New York, 1991). These traditional methods are laborious and subject to artefactual error. For example, the incipient plasmolysis method is highly subjective, and it radically alters the environment of the cells being measured. The “micropressure probe” method, in contrast, is potentially precise and accurate, but inherently difficult to perform. The micropressure method necessarily destroys the cells whose turgor is being measured. Finally, other techniques, such as the pressure-bomb method, are only suitable for whole organs and are generally characterized as single use, one-shot methods. Thus, the traditional ways of determining turgor pressure are invasive or disruptive to the cellular specimen, thereby interfering with the normal dynamics of the cell, including cellular behavior.
In contrast, the present invention relates to a method and an apparatus for measuring the contact area or contact patch between a specimen and a mechanical probe, and this can be used to determine, virtually instantaneously and repeatably, the turgor pressure in a cellular specimen. The method and apparatus can be non-invasive and non-destructive to the specimen. In cellular specimens the present invention's method can be repeated from point to point, for example, along a growing axis.
SUMMARY OF THE INVENTION
In accordance with this invention a method and an apparatus for determining the area of contact between a convex probe surface and a cellular specimen uses a transparent probe and an optical viewing path through the probe to the area of contact. The area of contact can be described as the contact area or contact patch between the objective and the specimen. In one embodiment, the specimen is located at the working distance of a microscope objective, and the probe is introduced between the objective and the specimen. A known force is applied by the probe to a deformable specimen. The amount of deformation of the specimen will depend on the force. If the probe's contact surface is of known geometry, for example spherical, and of known dimensions, the contact area between the probe's contact surface and the cellular specimen will be a function of the turgor pressure.
Contact area image information, i.e. the optical image or data descriptive of the optical image, is conveyed to an image analysis system. This calculates the contact area and consequently, permits calculation of the specimen characteristic affecting contact area.
By applying a series of known forces via the probe and measuring respective contact areas it is possible to derive data representing a plot of area versus force. This enables extrapolation of the specimen characteristic at zero force. As discussed in more detail below, when method and apparatus of the invention is used to determine turgor pressure in a cell, an extrapolation of this kind permits determination of the turgor pressure when no force is applied by the probe.
The turgor pressure of a cell is the hydrostatic pressure contained in a constraining membrane of each individual cell. Given a constant force and a spherical probe contact surface, the greater the internal pressure of the cell, the smaller will be the contact area or patch between probe and cell. In measuring this pressure in a cellular specimen, composed of one or more cells, the method and apparatus of the invention have the capability of making such measurement without invasion or destruction of the cellular specimen or any cell of the cellular specimen.
The method of nondestructively and noninvasively calculating the turgor pressure in the cellular specimen uses an appropriate proportionality relationship between the turgor pressure in a supported cell that has a substantially smooth upper surface, and the contact area between the transparent mechanical probe, having a known geometry. The contact area is viewed by a microscope, which will have a suitable support for the specimen and may have associated with it an appropriate means for illuminating the contact area, either by a substage light source and condenser, by a fiber optic light guides brought in at substantially the level of the microscope stage and providing oblique illumination at approximately ninety degrees to the optical axis of the microscope, or by epi-illumination through the objective lens itself The light source is manipulated until a clear image of the outline bordering the contact area is observed.
The term view or observe as used here is meant to include both observation by an individual using the method and apparatus of the invention and retrieving of image information optically, electrically or otherwise. For example, the apparatus for determining the contact area may include an image capturing system using a CCD camera to which the image is exported. A video frame grabber and image analysis station can be used to arrive at the actual contact area.
A force controllable mechanical probe support provides an accurately determined contact force between the probe and the specimen. The probe is an optically neutral element. In a preferred embodiment the probe's contact surface was spherical, formed by a sphere of glass, diamond, or quartz and affixed to a strip of cover glass by a drop of ultraviolet cured optical adhesive. This arrangement avoids distortion at the spherical ball surface remote from the specimen. Essentially, the contact area is being viewed through a flat window to the far surface of the ball. The force controllable mechanical probe may employ a jewel bearing system for reducing friction, e.g. one employing a sapphire or like-bearing material.
A field instrument used to measure the turgor pressure of leaves of crop plants is one application of the turgor pressure measuring embodiment of the invention. Such a device can be employed to quantify water stress on plants quickly in the field to serve as a “go-no go” gauge for irrigation, that is to say, to indicate whether or not irrigation is required.
REFERENCES:
Derwent WPI Abstract of SU 1766363.
Lintilhac Philip M.
Outwater John O.
Baker & Botts L.L.P.
Lankford , Jr. Leon B.
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