Laser-induced cavitation bubble manometer system for measuring pressure of living cells

Abstract

A laser-ablation hardware is coupled to a compound light microscope to deliver a nanosecond pulse of energy into living cells. This process vaporizes the liquid inside the cell which forms a gas or vapor bubble. By observing the maximum bubble radius, the bubble shape, the energy required to generate a bubble, and the rate at which it collapses using a high-speed camera, the internal pressure of the cell can be determined.

Description

LASER-INDUCED CAVITATION BUBBLE MANOMETER SYSTEM FOR MEASURING PRESSURE OF LIVING CELLSCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present invention claims the benefit of U.S. Provisional Application Serial No. 63 / 569,898, the subject matter of which is herein incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 2333889 from the National Science Foundation. The U.S. Government has certain rights in the invention.FIELD OF THE INVENTION

[0003] The present invention relates generally to a laser-induced cavitation bubble manometer system for measuring pressure of living cells and a method of using the same.BACKGROUND OF THE INVENTION

[0004] Measuring the internal pressure maintained by living cells (including plant cells and animal cells) is extremely difficult to achieve due to the size and delicate nature of cell membranes and walls.

[0005] Various methods have been used to measure internal pressure (also known as turgor pressure) of plant cells including, for example, the use of pressure probes, atomic force microscopy (AFM), and indentation techniques.

[0006] The use of pressure probes is one of the more common method and typically requires the use of highly artificial environmental conditions as well as an invasive pressure probe that pierces the plant cell wall and membrane. It is unclear whether such measurements are entirely accurate not only because of the damage resulting from inserting the probe, but also because of the potential inadequate seal between the probe and cell wall and membrane at the entry point. Furthermore, many plant cells of interest are too small to use with the pressure probe.

[0007] In the pressure probe method, a glass microcapillary tube is heated and pulled to a tip of approximately <5 pm in diameter, polished, and then mounted in a micromanipulator andconnected to a pressure transducer. The tip of the pressure probe is positioned adjacent to the cell of interest, the pressure is recorded, and then the tip of the probe is advanced to impale the cell, at which point the change in pressure is measured and recorded by the transducer and associated equipment.

[0008] The yield for this type of measurement is often quite low, allowing for approximately 1 measurement per day if the equipment is working well, and only in cells large enough to allow probing without measurement error or significant damage to the cell wall and membrane.

[0009] Because of the small size of plant cells and that the cell and the pressure probe tip need to be viewed simultaneously for positioning, the process is very difficult. Furthermore, light transmitted through the leaf becomes scattered and illumination becomes a problem because of the reduced visibility. Some research groups have used reflected light (instead of transmitted light), which can resolve some imaging issues but produce other imaging issues. Finally, most of the pressure probe measurements to date in guard cells (i.e., the pair of cells that form a stomate on the leaf surface) are dissected samples, where the epidermis is peeled from the plant and then experimentally manipulated. This condition removes the plant cells from their natural state, and thus introduces a range of sampling artifacts.

[0010] AFM works by measuring the force needed to displace the surface of the sample by a given distance, allowing for the calculation of the apparent stiffness of the cell wall and estimation of turgor pressure. While this method is less invasive than the use of pressure probes and allows for repeated measurement and analysis of small cells, it requires specialized equipment and a high level of expertise. In addition, it is difficult to evaluate the cells in their natural state and / or under different environmental conditions.

[0011] Indentation methods include cellular force microscopy (CFM) and microcompression which are used to measure the force required to indent the cell wall, providing information about its mechanic properties and turgor pressures. While this method is also less invasive than pressure probes, it also requires specialized equipment and a high level of expertise. As with AFM, it is difficult to evaluate the cells under different environmental conditions.

[0012] Thus, there remains a need in the art for an improved method measuring the internal pressure of living cells, including plant cells and animal cells and that can be used to evaluate such cells in their natural state and / or under different environmental conditions in an efficient manner.SUMMARY OF THE INVENTION

[0013] It is an object of the present invention to provide a less invasive method and system for measuring the internal pressure of living cells.

[0014] It is another object of the present invention to provide a non-invasive method and system for measuring the internal pressure of living cells.

[0015] It is another object of the present invention to provide a non-invasive method and system for measuring the internal pressure of living plant cells.

[0016] It is still another object of the present invention to provide a non-invasive method and system for measuring the internal pressure of living plant cells in their natural condition.

[0017] It is still another object of the present invention to provide a non-invasive method and system of measuring the internal pressure of living plant cells in which different environmental conditions can be evaluated.

[0018] It is still another object of the present invention to provide a non-invasive method and system for measuring the internal pressure of living animal cells.

[0019] To that end, in one embodiment, the present invention relates generally to a method of determining an internal pressure of a living cell, the method comprising the steps of: a) arranging a sample of living cells in a sample chamber or on a sample platform, wherein the living cells contain water; b) delivering a focused pulse of energy from a dye laser system into the living cell arranged in the sample chamber or on the sample platform to nucleate a vapor bubble within the living cell, wherein at least a portion of the water within the living cell vaporizes to form a gas bubble; c) measuring and recording a maximum diameter of the gas bubble and a dynamic growth and collapse of the gas bubble; and d) calculating the internal pressure of the living cell based on the maximum bubble diameter, the dynamic growth and collapse of the gas bubble, and the energy required to nucleate the gas bubble.

[0020] In one embodiment, the present invention also relates generally to a laser-induced cavitation bubble manometer system for measuring pressure of living cells comprising:a. a dye laser system, wherein the dye laser system comprises a laser coupled to a compound light microscope, wherein the dye laser system is configured to provide a focused pulse of energy, the dye laser system comprising: i. a laser that is capable of producing a high intensity, rapidly pulsed light source at a desired output wavelength, preferably wherein the desired output wavelength is in the UV range, ii. a compound light microscope, preferably a compound fluorescent light microscope coupled to the laser, and iii. a series of mirrors, lenses, and optical components arranged between the laser and the compound light microscope to control the intensity of the pulsed light source and tightly focus the pulsed light source to a small region, preferably wherein the pulsed light source is capable of being focused to a living cell arranged in a sample chamber, preferably wherein the pulsed light source is capable of being focused to a portion of the living cell; b. a sample chamber or sample platform arranged proximate to the dye laser system, wherein a sample of living cells is positionable within the sample chamber or on the sample platform, whereby when the dye laser system delivers the focused pulse of energy to a living cell within the sample of living cells, a gas bubble within the living cell nucleates and causes at least a portion of the water within the living cell to vaporize to form a gas bubble; and c. a high speed camera for imaging and recording the dynamic growth and collapse of the gas bubble.BRIEF DESCRIPTION OF THE FIGURES

[0021] The accompanying figures illustrate several aspects of the disclosure, and together with the description, illustrate various aspects of the present invention.

[0022] Features and aspects of embodiments are described below with reference to the figures, in which elements are not necessarily depicted to scale, and in certain views, parts may have been exaggerated or removed for purposes of clarity.

[0023] The figures are included to illustrate certain aspects of the present disclosure and should not be viewed as exclusive embodiments. The subject matter disclosed is capable of considerable modifications, alterations, combinations, and equivalents in form and function, as will occur to one having ordinary skill in the art and the benefit of the disclosure.

[0024] Fig. 1 depicts a laser-induced cavitation bubble manometer system in accordance with one aspect of the present invention.

[0025] Fig. 2 depicts a representative image sequence of the formation of a bubble inside a stomatai guard cell.

[0026] Fig. 3 depicts a representative dataset for three different fem species in accordance with one aspect of the present invention.

[0027] Fig. 4 depicts a micro-fluidic testing apparatus in accordance with one aspect of the present invention.DETAILED DESCRIPTION OF THE PRFERRED EMBODIMENTS

[0028] As described in further detail herein, the goal of the present invention was to develop a non-invasive (or less invasive), method of measuring the internal pressure of cells, in order to observe their physiological state under natural conditions in intact living tissues, including plant tissues. In one embodiment, the living cells are plant cells and the physiological state of the living can be observed under natural conditions, which natural conditions can be varied. In another embodiment, the living cells are animal cells.

[0029] It is known that laser ablation systems can be used to destroy individual cells for various research purposes. In contrast, in the invention described and claimed herein, a laser pulse is used to nucleate a bubble inside a living plant cell, and a high speed camera is used to record the bubble growth and collapse dynamics. Thereafter, the pressure of the plant cell can be estimated using bubble growth and collapse dynamics. While similar techniques have been used by physicists to generate bubbles and observe the collapse dynamics using high-speed cameras, this technique has not previously been used for living tissue, including both plant tissue and animal tissue, and has not be used to observe and study living cells under various environmental conditions.

[0030] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which thisdisclosure belongs. It will be further understood that terms used herein should be interpreted as having a meaning that is consistent with their meaning in the context of this specification and the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0031] As used herein, “a,” “an,” and “the” refer to both singular and plural referents unless the context clearly dictates otherwise.

[0032] As used herein, the terms “comprises,” “comprising,” “includes” and / or “including” specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0033] As used herein, the term “about” refers to a measurable value such as a parameter, an amount, a temporal duration, and the like and is meant to include variations of + / -15% or less, preferably variations of + / -10% or less, more preferably variations of +7-5% or less, even more preferably variations of + / -!% or less, and still more preferably variations of + / -0.1% or less of and from the particularly recited value, in so far as such variations are appropriate to perform herein. Furthermore, it is also to be understood that the value to which the modifier “about” refers is itself specifically disclosed herein.

[0034] Relative terms such as “below” or “above” or “upper” or “lower” or “horizontal” or “vertical” may be used herein to describe a relationship of one element, layer, or region to another element, layer, or region as illustrated in the Figures. It will be understood that these terms and those discussed above are intended to encompass different orientations of the device in addition to the orientation depicted in the Figures.

[0035] It will be understood that when an element such as a layer, region, or substrate is referred to as being “on” or extending “onto” another element, it can be directly on or extend directly onto the other element or intervening elements may also be present. In contrast, when an element is referred to as being “directly on” or extending “directly onto” another element, there are no intervening elements present. Likewise, it will be understood that when an element such as a layer, region, or substrate is referred to as being “over” or extending “over” another element, it can be directly over or extend directly over the other element or intervening elements may also be present. In contrast, when an element is referred to as being “directly over” or extending “directly over” another element, there are no intervening elements present. It will also beunderstood that when an element is referred to as being “connected” or “coupled” to another element, it can be directly connected or coupled to the other element or intervening elements may be present. In contrast, when an element is referred to as being “directly connected” or “directly coupled” to another element, there are no intervening elements present.

[0036] As used herein the term “substantially-free” or “essentially-free” if not otherwise defined herein for a particular element or compound means that a given element or compound is not detectable by ordinary analytical means that are well known to those skilled in the art of metal plating for bath analysis. Such methods typically include atomic absorption spectrometry, titration, UV-Vis analysis, secondary ion mass spectrometry, and other commonly available analytically methods.

[0037] In one embodiment, the present invention relates generally to a method of determining an internal pressure of a living cell, the method comprising the steps of: a) arranging a sample of living cells in a sample chamber or on a sample platform, wherein the living cells contain water; b) delivering a focused pulse of energy from a dye laser system into the living cell arranged in the sample chamber or on the sample platform to nucleate a vapor bubble within the living cell, wherein at least a portion of the water within the living cell vaporizes to form a gas bubble; c) measuring and recording a maximum diameter of the gas bubble and a dynamic growth and collapse of the gas bubble; and d) calculating the internal pressure of the living cell based on the maximum bubble diameter, the dynamic growth and collapse of the gas bubble, and the energy required to nucleate the gas bubble.

[0038] The focused burst of energy is delivered at an intensity and for a duration that is sufficient to nucleate a vapor bubble within the cell so that at least a portion of the water within the cell vaporizes to form a gas bubble. The focused pulse of energy from the laser typically has a duration of about 0.5 to about 5 nanoseconds, more preferably about 1 to about 3 nanoseconds, most preferably about 1 nanosecond. The energy required to nucleate the gas bubble is typically within the range of about 15 to about 1000 pj. The pulse of energy vaporizes a smally volume of the plant cell contents (mostly water and dissolved solutes) and creates a visible bubble with amaximum diameter of less than 10 pm or less than 8 pm, or less than 6 pm, or even less than 5 pm.

[0039] In one embodiment, the maximum bubble diameter is proportional to the pressure. Therefore, depending on the pressure of the cell, the bubble diameter will vary. In addition it is also believed that other systems (including i.e., animal cells) may have larger bubbles.

[0040] A high-speed camera records the maximum bubble radius and the rate at which the bubble collapses over time, which typically occurs in less than one second. The time to bubble collapse is less than about 1 second, preferably in the range of about 1 to about 1,000 ms, or in the range of about 5 to about 500 ms, or within the range of about 10 to about 100 ms.

[0041] The high speed camera records the maximum diameter of the gas bubble and the rate at which the vapor bubble collapses over time. In one embodiment, the high speed camera records at least 100 frames per second or at least 200 frames per second or at least 300 frames per second or at least 400 frames per second or at least 500 frames per second or at least 600 frames per second, or at least 700 frames per second or at least 800 frames per second, or at least 1,000 frames per second. After collapse of the gas bubble, gas dissolves back into the living cell.

[0042] Using this bubble collapse information, the rate of collapse in a range of plant species and cell types and over a range of physiological conditions can be compared to identify differences in internal cell pressure. The high speed camera is coupled to a controller that includes a computer to record the high speed images of the camera. Then, established theory and equations from the physics literature can be used to approximate the absolute internal cell pressure based on the energy required to nucleate a bubble, the maximum bubble radius, and the rate of collapse.

[0043] The focused pulse of energy is delivered from a laser that is coupled to a compound fluorescence light microscope. The laser produces a high intensity, rapidly pulsed light source at a desired output wavelength. Preferably, the desired output wavelength is in the UV range. In one embodiment, the laser is a nitrogen laser.

[0044] The light source is directed to a dye cell containing a solvent and a dye and the selection of the solvent and the dye depend on the desired output wavelength of the laser. The laser is coupled to a compound light microscope, preferably a compound fluorescent light microscope, and a series of mirrors, lenses, and optical components arranged between the nitrogen laser and the compound light microscope to control the intensity of the pulsed light source and tightly focus the pulsed light source to a small region. Preferably, the pulsed light source is focused tothe living cell arranged in the sample chamber or on a sample platform, more preferably the pulsed light source is focused to a portion of the living cell. In one embodiment, the pulse of light is focused into a single plant cell on the surface of leaves. An example of a commercially available dye laser system is a MicroPoint laser system available from Andor-Oxford Systems.

[0045] In one embodiment, the sample is arranged on a sample platform. In another embodiment, the sample chamber is arranged in a sample chamber.

[0046] In one embodiment, the sample chamber is connected to a gas exchange chamber. The gas exchanger is capable of conditioning the air to provide different environmental conditions, including, for example, environmental conditions that are manipulated to stimulate stomatai opening or stomatai closing. In one embodiment, the gas exchange chamber monitors photosynthetic rate and stomatai conductance of the plant cells.

[0047] In one embodiment, the different environmental conditions are achieved by manipulating a physiological response of living cells in the sample of living cells in response to varying levels of illumination from a light source. The intensity of the light source may be modified over a prescribed time period from substantially zero, where the living cell is in the dark, and is increased over time to provide different levels of illumination. The changes in the maximum diameter of the gas bubble and a dynamic growth and collapse of the gas bubble are observed in response to varying levels of illumination and differences in the internal pressure of the living cell can be calculated over a range of light intensities.

[0048] The step of measuring the dynamic growth and collapse of the gas bubble comprises measuring and recording (i) a maximum diameter of the gas bubble; (ii) a shape of the gas bubble; and (iii) a rate at which the gas bubble collapses.

[0049] In one embodiment, the present invention also relates generally to a laser-induced cavitation bubble manometer system 10 for measuring pressure of living cells.

[0050] As shown in Fig. 1, a dye laser system comprising a laser 50 is coupled to a compound light microscope 60 and is configured to provide a focused pulse of energy. In one embodiment, the dye laser system comprises a nitrogen laser 50 that is capable of producing a high intensity, rapidly pulsed light source 90 at a desired output wavelength. In one embodiment, the desired output wavelength is in the UV range. In addition, the dye laser system comprises a compound light microscope 60 coupled to the nitrogen laser 50. In one embodiment, the compound light microscope 60 comprises a compound fluorescent light microscope.

[0051] In one embodiment, the dye laser system comprises a dye laser assembly as described, for example in U.S. Pat. No. 5,933,274 to DeSimone, the subject matter of which is herein incorporated by reference in its entirety. In one embodiment, the nitrogen laser 50 produces a nanosecond pulse of light in the ultraviolet (UV) range (e.g., 365 nm). That light is directed to a ‘dye cell’ containing a solvent (usually methanol or ethanol) and a dye, which may be a powdered dye. The dye and solvent can be optimized to achieve the desired output wavelength. In one embodiment, the dye laser system of the invention uses Coumarin as the dye in a methanol solvent to achieve an output wavelength of approximately 465 nm (blue).

[0052] As discussed above, instead of using the light energy generated by the nitrogen laser 50 to ablate cells, the nitrogen laser 50 is instead used to generated bubbles in living cells and, in combination with a high-speed camera 20, observe the dynamics of bubble growth and collapse that are generated by the laser pulse.

[0053] The dye laser system also comprises a series of mirrors, lenses, and optical components 65 arranged between the nitrogen laser 50 and the compound light microscope 60 to control the intensity of the pulsed light source 90 and tightly focus the pulsed light source 90 to a small region of a sample of living cells 80 arranged in a sample chamber 70. In one embodiment, the pulsed light source 90 is capable of being focused to a living cell of the sample of living cells 80 arranged in the sample chamber 70. In one embodiment, the pulsed light source is capable of being focused to a portion of the living cell of the sample of living cells 80 arranged in the sample chamber 70.

[0054] In one embodiment, the series of mirrors, lenses, and optical components 65 arranged between the nitrogen laser 50 and the compound light microscope 60 comprises a MicroPoint system that includes a series of mirrors and lenses that allow the user to focus the energy pulse to a very small region, just a few micrometers in diameter and in the same focal plane as the microscope objective. The MicroPoint system is coupled to the epifluorescence port of a standard fluorescence microscope. The intensity of the pulse delivered to the sample can be controlled by using a neutral density gradient filter, or by placing other filters in the optical path. Other systems of mirrors, lenses, and optical components that allow the user to focus the energy pulse to a specific region and that can be coupled to the compound light microscope 60 would also be usable in the practice of the instant invention. In one embodiment, the energy required to nucleate the gas bubble is in the range of about 15 to about 1000 pj.

[0055] In one embodiment, the sample of living cells 80 is arranged on a sample platform and the dye laser system delivers the focused pulse of energy 90 to a living cell within the sample of living cells 80 arranged on the sample platform so that the dye laser system delivers the focused pulse of energy 90 to a living cell within the sample of living cells 80 to nucleate a gas bubble within the living cell and cause at least a portion of the water within the living cell to vaporize to form a gas bubble.

[0056] In another embodiment, the sample of living cells 80 is housed in a sample chamber 70 arranged proximate to the dye laser system. The sample of living cells 80 is positioned within the sample chamber 70 so that the dye laser system delivers the focused pulse of energy 90 to a living cell within the sample of living cells 80 to nucleate a gas bubble within the living cell and cause at least a portion of the water within the living cell to vaporize to form a gas bubble.

[0057] In one embodiment, a gas exchange chamber 30 is coupled to the sample chamber 70. The gas exchange chamber 30 is capable of conditioning the air in the sample chamber 70 to vary environmental conditions within the sample chamber 70.

[0058] High speed camera 20 is used to image and record the dynamic growth and collapse of the gas bubble and is coupled to controller 100 that includes a computer to record images from the high speed camera 20.

[0059] In one embodiment, the sample of living cells 80 comprises plant cells and the plant cells comprise stomata or other plant cells. In one embodiment, the sample of living cells 80 is a leaf and the stomata are on the surface of the leaf.

[0060] Stomata are pores that form across the epidermal cell layer of plant leaves and stems. They connect the inner air space of these organs with the atmosphere, serving as the major route for gaseous exchange, stomatai guard cells are the premier plant cell model for ion transport and cell signaling, and are key target for biological engineering to improve crop production and reproduce water demand. Stomata respond to environmental and endogenous signals, opening and closing the pore in order to satisfy the needs of the mesophyll cells for carbon dioxide in photosynthesis while limiting water loss via transpiration to the atmosphere.

[0061] Fig. 2 depicts a representative image sequence of the formation of a bubble inside a stomatai guard cell. Stomatai guard cells are often used as a plant cell model for membrane transport, signaling, and homeostasis. Stomatai guard cells surround pores in the epidermis of plant leaves, controlling the aperture of the pore to balance carbon dioxide entry into the leaf forphotosynthesis with water loss via transpiration. Therefore, the position of the stomatai guard cells in the epidermis makes them suitable for study using the method and system described herein.

[0062] The arrow in Fig. 2 at t = 0 points to the bubble generated by firing the laser at the first frame in the image sequence where the bubble is visible. The next three frames are each 10 milliseconds apart. In the second and third frames the bubble can be seen collapsing. After 40 ms, and as depicted in the fourth image, the bubble has completely collapsed and the gas has dissolved back into solution.

[0063] By measuring the maximum bubble radius, the time it takes for the bubbles to collapse, and the energy required to nucleate a bubble, which is typically between about 15 and about 1000 nJ, the pressure can be determined based on an understanding of cavitation bubble collapse dynamics using the theory of Epstein and Plesset (P. S. Epstein and M. S. Plesset, “On the Stability of Gas Bubbles in Liquid Gas Solutions,” The Journal of Chemical Physics, Vol. 18, No. 11, November 1950).

[0064] Thus, it can be seen that an application of existing technology and an understanding of the physics of bubble collapse are applied in a new way. For the first time, it is now possible to measure the pressure of individual cells in plants while they are in a natural, unperturbed state and to manipulate environmental conditions within the system to stimulate cells opening and closing. In addition, while the process described herein is described in reference to plat cells, it is also believed that this system could also be used to measure pressure in non-plant cells (e.g., animal cells).

[0065] As described herein, in one embodiment, the sample of living cells 80 is mounted in the sample chamber 70 that can be observed with a microscope. In one embodiment, the sample chamber 70 may be 3D printed to provide a chamber that is customized to observe leaf surfaces at high magnification (e.g., 200x) while conducting photosynthesis measurements simultaneously. The leaf surface is illuminated with a standard light source coupled to a bifurcated optical goose neck fiber. The light intensity may then be increased or decreased using the light source to induce stomatai opening or closing.

[0066] In one embodiment, the sample chamber 70 may be connected to a portable gas exchange chamber 30 (a commercial example of which is available from LiCor Environmental under the Tradename LI-6800) that can be used to monitor the photosynthetic rate and stomataiconductance of the leaf (i.e., sample of living cells 80), and to condition the air to provide a range of environmental conditions.

[0067] By imaging the leaf surface with the microscope system while the leaf is in the sample chamber 70, the stomata opening or closing can be observed in response to light intensity. In addition, the stomatai closure can be observed if the leaf petiole is cut so that it is disconnected from the plant. Similarly, the petiole can be reconnected to a water source (i.e.,. a short length of plastic tubing filled with water) to manipulate the water status of the leaf and then the stomatai aperture. When stomata are closed, the internal pressure is believed (and measured) to be low. As stomata open, the pressure inside the cells increases. This is accomplished by the movement of solutes across the cell wall and membrane which induces a solute gradient, leading to the movement of water along that gradient as well. Water floods into the guard cells and increases their internal pressure. The pressurization forces the guard cells to bend, and that bending leads to an open pore though which air and water vapor can travel through. When leaves are in the dark, stomata tend to close. When leaves are illuminated the guard cells open to allow CO2 to diffuse into the leaf to sustain synthesis.

[0068] As discussed above, when using the sample chamber 70 in combination with the gas exchange chamber 30, environmental conditions can be manipulated to stimulate stomatai opening or closing, and during transitions or under steady state conditions, and then the nitrogen laser can be triggered to nucleate a bubble inside the leaf cells under these different environmental conditions. For example, the leaf inside the sample chamber 70 may be exposed to a series of step changes in light intensity. After the leaf stabilizes in response to the each step change in light intensity, several guard cells can be sampled. This process can then be repeated by moving to the next light intensity.

[0069] For example starting from a leaf that has been in the dark, the light intensity can be slowly increase to sample guard cells that should start at low pressure and then increase in pressure as stomata continue to open over time and in response to increasing light intensity. In this way, it is possible to observe the bubbles in leaf cells at different physiological states over the full range of internal pressures that are generated by the plant.

[0070] Fig. 3 depicts a representative dataset for three different fern species. In this set of experiments the light intensity supplied to the plant was manipulated to induce opening or closing of the stomata. When stomatai conductance is high (where stomata are open and thepressure should be high) the bubble collapse time is fast. As stomatai conductance decreases, the bubble collapse time increases. Thus, there is a proportionality between how open the stomata are (which determines stomatai conductance) and the bubble collapse time. Because bubble collapse time is a function of pressure, where higher pressure will cause bubbles to collapse faster, we can determine the range of both collapse times in an intact, actively transpiring plant, but we can also approximate the pressure because of the well-described physical processes that govern bubble collapse.

[0071] In addition, a micro-fluidic testing apparatus was constructed to evaluate whether the phenomenon could be replicated in a synthetic system as depicted in Fig. 4. Here, the microscope objective (e.g., 20X) focuses the light on a micro-fluidic test cell. The test cell used was a microfluidic flow cell available from Darwin Microfluidics and comprised a clear slab of plastic with cylindrical microchannels embedded in the plastic and inlet and outlet ports. The microchannel is 100pm in diameter). The channel was flooded with pure water and small particles, and then the cell was connected to a pressure transducer (PT) and a pressure chamber (PC). Using the pressure chamber, it was possible to increase the pressure applied to the microfluidic cell and generate bubbles and observe them with the high speed camera as described above. In this way it was possible to mimic a range of pressures that might exist in living plants. In this instance, the absolute pressure of the system was known and it was possible to generate bubbles and observe their collapse dynamics. Based thereon, it was determined that bubble collapse time is faster when pressure is high.

Claims

What is claimed is:

1. A method of determining an internal pressure of a living cell, the method comprising the steps of: a) arranging a sample of living cells in a sample chamber or on a sample platform, wherein the living cells contain water; b) delivering a focused pulse of energy from a dye laser system into the living cell arranged in the sample chamber to nucleate a vapor bubble within the living cell, wherein at least a portion of the water within the living cell vaporizes to form a gas bubble; c) measuring and recording a maximum diameter of the gas bubble and a dynamic growth and collapse of the gas bubble; and d) calculating the internal pressure of the living cell based on the maximum bubble diameter, the dynamic growth and collapse of the gas bubble, and the energy required to nucleate the gas bubble.

2. The method according to claim 1, wherein the living cells comprise plant cells or animal cells.

3. The method according to claim 2, wherein the living cells are intact plant cells, wherein a physiological state of the living cells is observed under natural conditions.

4. The method according to claim 2, wherein the plant cells are stomata or other plant cells.

5. The method according to claim 4, wherein the sample of living cells is a leaf and the stomata are on a surface of the leaf.

6. The method according to claim 1, wherein a high speed camera records the maximum diameter of the gas bubble and the rate at which the vapor bubble collapses over time, optionally wherein the high speed camera records at least 100 frames per second or at least 200 frames per second or at least 300 frames per second or at least four hundred frames per second or at least 500 frames per second or at least 600 frames per second, or at least 700 frames per second or at least 800 frames per second, or at least 1,000 frames per second.

7. The method according to claim 1, wherein the energy required to nucleate the gas bubble is in the range of about 15 to about 100 pj.

8. The method according to claim 1, where the laser dye system comprises: a laser that produces a high intensity, rapidly pulsed light source at a desired output wavelength, preferably wherein the desired output wavelength is in the UV range, wherein the light source is directed to a dye cell containing a solvent and a dye, wherein the selection of the solvent and the dye depend on the desired output wavelength of the laser, wherein the laser is coupled to a compound light microscope, preferably a compound fluorescent light microscope, and a series of mirrors, lenses, and optical components arranged between the laser and the compound light microscope to control the intensity of the pulsed light source and tightly focus the pulsed light source to a small region, preferably wherein the pulsed light source is focused to the living cell arranged in the sample chamber, preferably wherein the pulsed light source is focused to a portion of the living cell.

9. The method according to claim 1, wherein the sample of living cells is arranged in the sample chamber and the sample chamber is connected to a gas exchange chamber, wherein the gas exchange chamber conditions the air to provide different environmental conditions, optionally wherein the environmental conditions are manipulated to stimulate stomatai opening or stomatai closing.

10. The method of according to claim 9, wherein the gas exchange chamber monitors photosynthetic rate and stomatai conductance of the living cell.

11. The method according to claim 9, comprising the step of manipulating a physiological response of living cells in the sample of living cells in response to varying levels of illumination from a light source, optionally wherein the intensity of the light source is modified over a prescribed time period from substantially zero, wherein the living cell is in the dark, and is increased over time to provide different levels of illumination, wherein changes in the maximum diameter of the gas bubble and a dynamic growth and collapse of the gas bubble are observed in response to varying levels of illumination and differences in the internal pressure of the living cell can be calculated over a range of light intensities.

12. The method of claim 1, wherein the step of measuring the dynamic growth and collapse of the gas bubble comprises measuring and recording (i) a maximum diameter of the gas bubble; (ii) a shape of the gas bubble; and (iii) a rate at which the gas bubble collapses.

13. The method according to claim 1, wherein the focused pulse of energy from the laser has a duration of about 0.5 to about 5 nanoseconds, more preferably about 1 to about 3 nanoseconds, most preferably about 1 nanosecond.

14. The method according to claim 1, wherein the time to bubble collapse is less than about 1 second, preferably in the range of about 1 to about 1,000 ms, or in the range of about 5 to about 500 ms, or within the range of about 10 to about 100 ms.

15. The method according to claim 1, wherein after collapse of the gas bubble, the gas dissolves back into the living cell.

16. A laser-induced cavitation bubble manometer system for measuring pressure of living cells comprising: a. a dye laser system, wherein the dye laser system comprises a nitrogen laser coupled to a compound light microscope, wherein the dye laser system is configured to provide a focused pulse of energy, the dye laser system comprising: i. a nitrogen laser that is capable of producing a high intensity, rapidly pulsed light source at a desired output wavelength, preferably wherein the desired output wavelength is in the UV range, ii. a compound light microscope, preferably a compound fluorescent light microscope coupled to the nitrogen laser, and iii. a series of mirrors, lenses, and optical components arranged between the nitrogen laser and the compound light microscope to control the intensity of the pulsed light source and tightly focus the pulsed light source to a small region, preferably wherein the pulsed light source is capable of being focused to a living cell arranged in a sample chamber, preferably wherein the pulsed light source is capable of being focused to a portion of the living cell b. a sample chamber or sample platform arranged proximate to the dye laser system, wherein a sample of living cells is positionable within the sample chamber or on the sample platform, whereby when the dye laser system delivers the focusedpulse of energy to a living cell within the sample of living cells, a gas bubble within the living cell nucleates and causes at least a portion of the water within the living cell to vaporize to form a gas bubble; and c. a high speed camera for imaging and recording the dynamic growth and collapse of the gas bubble.

17. The laser-induced cavitation bubble manometer system according to claim 16, comprising the sample platform, wherein the sample of living cells is positionable on the sample platform.

18. The laser-induced cavitation bubble manometer system according to claim 16, comprising the sample chamber, wherein the sample of living cells is positionable within the sample chamber.

19. The laser-induced cavitation bubble manometer system according to claim 18, wherein the sample chamber is connected to a gas exchange chamber, wherein the gas exchange chamber is capable of conditioning the air in the sample chamber to vary environmental conditions within the sample chamber.

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