Abstract:
Turgor pressure is measured in a cellular sample by contacting the sample with a convex probe, measuring the area of contact and using a formula to convert the area into turgor pressure.

Description:
This is a continuation of copending PCT application No. PCT/US971/17442 filed Sep. 30, 1997 (now PCT Publication WO 98/14754), which claims priority from U.S. application Ser. No. 60/027,392, filed Sep. 30, 1996. 
    
    
     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&#39;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&#39;s contact surface is of known geometry, for example spherical, and of known dimensions, the contact area between the probe&#39;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&#39;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. 
    
    
     DESCRIPTION OF THE DRAWINGS 
     The invention will be more readily understood from the description of a preferred embodiment that follows and from the diagrammatic figures of the drawings. 
     In the drawings: 
     FIG. 1 is a cross-sectional view of a spherical force controllable transparent mechanical probe contacting a cellular specimen; 
     FIG. 2 is a schematic view partially in section and partially in block diagram form and illustrates a transparent mechanical probe in a system to determine the contact area between the probe and a specimen; 
     FIG. 3 is a schematic view like that of FIG.  2  and diagrammatically illustrates an alternative means of illumination of the probe-specimen contact area; and 
     FIG. 4 is a graphical illustration plotting a relationship between contact area derived turgor pressure (Bars) and medium osmotic pressure (Bars). 
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     I. Theoretical Background for a Proportionality Relationship between the Turgor Pressure and the Contact Area of a Spherical Force Controllable Transparent Mechanical Probe Contacting a Cellular Specimen 
     In a preferred embodiment of this invention a cellular specimen  10 , shown as an individual cell, is contacted by a spherical surface of a transparent optical probe  20 , as shown in FIG.  1 . The individual cell is treated as a thin-walled pressure vessel to which an external load W is applied for a theoretical understanding of the invention. The cell has a substantially smooth upper surface  12 . By “substantially smooth upper surface” is meant a surface not having features, such as epidermal hairs, that would interfere with a force controllable transparent optical probe&#39;s contacting that surface of the specimen. 
     For the general case of a thin-walled pressure vessel to which an external load is applied by means of a rigid probe, the area of the contact patch is related to the internal pressure, turgor pressure, of the individual cell by: 
     
       
           W=Ap;   (1) 
       
     
     wherein: W is the force applied to the cell through the force controllable transparent mechanical probe (measured in Newtons or other units of force); A is a contact area (measured in square meters or other units of area); and p is a turgor pressure (measured in Bars, Pascals or other units of pressure). 
     Referring to FIG. 1, where the contact surface of the probe  20  is spherical, the probe causes an indentation in the surface  12  of the pressure vessel or cell  10 . There is an additional supporting force which results from the stress in the skin acting to lift the load, as shown in FIG.  1 . Thus, the force acting on an indentor of this nature will be balanced by the internal pressure, turgor pressure, of the individual cell according to the relationship: 
     
       
           W=pπr   2 +2 πrσt  sin(θ);  (2) 
       
     
     wherein: W is the force applied to the cell through the force controllable transparent mechanical probe causing the indentation (measured in Newtons or other units of force); t is the cell wall thickness (measured in meters or other units of length); p is a turgor pressure (measured in Bars, Pascals or other units of pressure); σ is a stress in the cell wall (measured in Bars, Pascals or other units of pressure); R is a radius of the spherical, force-controllable transparent, mechanical probe (measured in meters or other units of length); r is a radius of the contact patch (measured in meters or other units of length); and θ is a contact angle with respect to the center of the sphere and the outline of the contact area (measured in degrees). 
     If one keeps the indentation of the force controllable transparent mechanical probe into the individual cell small, sin(θ) is approximated by r/R; such that equation (2) reduces to: 
     
       
           W=pπr   2 +2 πr   2   σt/R.   (3) 
       
     
     Moreover, the wall stress, σ, is related to internal pressure according to the following relationship: 
     
       
         σ= pD/ 4 t;   ( 4 ) 
       
     
     where D is the approximate cell size (measured in meters or other units of length). Substitution of equation (4) into equation (3) produces the following relationship: 
     
       
           W=pπr   2 (1+½ D/R )= pA (1+½ D/R ).  (5) 
       
     
     Because D generally has a dimension near 50 μm and R in this case is 1000 μm, equation (5) can be reduced for these specific dimensions to the following relationship: 
     
       
           W=pA ×1.05.  (6) 
       
     
     If a spherical probe is applied to a surface of a cellular specimen that comprises a multicellular tissue, and the probe contact area spans more that one cell, the additional support offered by the anticlinal walls may be considered. The internal support of the anticlinal walls could cause a decrease in the contact area and an apparent increase in the measured turgor pressures. However, if the additional support of the anticlinal walls within the contact area is negligible with respect to the turgor pressure, then the contact area is related to the average turgor pressure of the cells in contact with the probe. Considering the delicate nature of the cell walls and the fact that the experimentally measured pressures were consistently lower than would otherwise be predicted it is presently believed that the anticlinal walls may at this time be safely ignored. 
     Additionally, there is the possibility of compartmentalization in a cellular specimen composed of a multicellular tissue resulting in a lack of fluid mobility. under the probe. This lack of fluid mobility could result in higher pressures at the center of the probe producing an apparent increase in the cell pressure. 
     The thin-walled model discussed above for the embodiment described here does not incorporate any correction for either subsurface support or fluid compartmentalization, but corrections for these effects may be included where necessary. 
     II. Method and Apparatus for the Measurement of Turgor Pressure 
     To determine specimen turgor pressure, an accurate measurement is made of the contact area between the cell  10  and the force-controllable, transparent mechanical probe  20 , of known geometry. Such probe may have, but is not limited to, a contacting surface that is spherical, hemispherical, or cylindrical. A calibrated load is applied to the specimen via the probe by a suitable accurate force producing mechanism. The specimen  10  is supported from below by support  11 . The transparent probe  20  may be made of any light transparent material, such as, but not limited to, glass, diamond, and quartz. The cellular specimen may be composed of a plurality of eukaryotic, either plant or animal, cells; a plurality of procaryotic cells; a plurality of organic micelles; a plurality of inorganic ricelles; or a single cell or micelle, provided that the cellular specimen includes a constraining membrane  14 . 
     The probe  20  is small enough to be inserted directly beneath an objective lens  32  of a standard compound microscope  30  as shown in FIG.  2 . The particular probe of this embodiment includes a strip  22  of No.  2  cover glass, acting as a support beam, and a 1 millimeter diameter ball lens  24  cemented to the strip  22  with a drop of ultraviolet cured optical adhesive  26 . The probe is thus inserted into the optical path of the microscope  30 . There it is manipulated into the working distance of the objective  32 , and carefully lowered onto the cellular specimen  10 , supported by the microscope stage  11 . The adhesive prevents distortion at the spherical surface remote from the specimen. 
     The ball lens  24  serves as a spherical mechanical indentor, while at the same time it provides an optically neutral, flat window at the upper surface through which the contact area can be observed directly. Because the image formed by the microscope is of the tissue in contact with the lower surface of ball lens itself, the optical properties of the ball lens do not contribute to total magnification of the system. This results in a reasonably clear observable image of the cell or cells of specimen  10  on which the ball lens is resting. 
     In an actual embodiment, the total mass of the ball lens  24  and its support  22  was 150 milligrams. The actual controlled force applied to the cellular specimen  10  was 45 milligrams times gravity. For the purpose of applying the controlled force, any accurate force producing mechanism  35  may be coupled to the probe. 
     As shown in part in FIG. 2, the cellular specimen  10  and the contact patch formed with the probe  20  are transilluminated by a standard substage condenser  41 , and light source  40 . Alternatively, as illustrated in FIG. 3, illumination may be by fiber-optic light guides  42  brought in at the substantially the level of the microscope stage, providing oblique illumination at approximately ninety degrees to the optical axis of the microscope. In still another alternative, illumination may be by epiillumination through the objective lens itself These means for illumination are manipulated to provide sufficient contrast to reveal the contact area. 
     In the preferred embodiment, as shown in FIG. 2, the image of the contact area is exported to an image capturing system, via, for example, a CCD camera  44 , thence to a video frame grabber  46  and finally to an image analysis station  48  where the contact area is determined directly. The image analysis station is suitably a computer running OPTIMAS™ or another commercially available image analysis program. The area may also be determined directly by the use of an eye piece incorporating a measuring reticle. 
     Measurements of neighboring cells in the cellular specimen  10  can be rapidly assessed using a translation device  50  as shown in FIG.  2 . The translation device is movable in either one, two, or three dimensions. The translation device allows the probe  10  to slide over the surface of the specimen  20 . Multiple measurements can be taken as fast as the probe can be moved to another cell and the image captured using the image analysis system. The capturing of the image is generally accomplished by clicking the “Freeze” button on the image analysis system. 
     The turgor pressure is then directly calculated from the observed and measured contact area using the relationships described in Equations (1) through (6). By repeating the measurement of the contact area at a variety of different forces of indention, data representing a plot of turgor pressure versus force are developed. The turgor pressure at zero force thus may be extrapolated. 
     The method and apparatus described above was used to determine if the measured areas and the calculated turgor pressures varied linearly with cellular osmotic pressure. In this test, peeled patches of onion leaf-base adaxial epidermis were incubated in mannitol solutions of varying osmolality, where one osmole equals one mole of nonpermeating molecules plus ions per liter. These solutions corresponded to 1 Bar increments in osmotic pressure from distilled water to −6 Bars. A slight meniscus of liquid around the contact patch facilitated observation of the contact patch outline. 
     FIG. 4 shows a plot of the turgor pressure, calculated by the contact area method, against the ambient osmotic pressure of the incubating medium. This plot shows a basically linear relationship between the calculated turgor pressure of the target cells, and the osmolarity (water potential) of the incubating medium. Sources of scatter in the graph may include: inaccuracies in the method employed, real differences in turgor pressure from cell to cell in the cellular specimen, and the presence of the contact meniscus which inflates the area and consequently lowers the apparent turgor pressure. 
     It should be noted that several factors can contribute to real differences in turgor pressure from cell to cell in cellular specimen. These factors include: deformations of the cellular specimen resulting from constraining a spherical layer of cells onto a flat microscope slide which can result in local strains that could either act to raise or lower the turgor pressure in the cells, and the geometry of individual cells comprising the cellular specimen can also give rise to different measured turgor pressures. 
     A hand held version of the turgor pressure measuring apparatus may be fabricated. This would include the same elements as the described embodiment. The addition of a portable power supply to power the means for illumination is envisioned. In certain applications natural light may suffice to illuminate the contact patch. 
     Whereas a specific preferred embodiment of this invention has been described it will be understood that variations and modifications may be made without departure from the spirit and scope of the invention as set forth in the appended claims.