Abstract:
A hand-held, battery operated, single photon sensitivity phytoluminometer is claimed for rapid, simple nondestructive, noninvasive evaluation of photosynthetic topography in green leaves. All the steps for generating phytoluminescence are performed within a single, light tight enclosure. The sensitivity of the instrument is such that stress induced changes in photosynthesis can be detected seconds after occurrence. Thus phytoluminography is made an easily applied technique for practical use by all those concerned with plant life.

Description:
This is a continuation of application Ser. No. 07/889,177, filed on May 27, 1992, which is a continuation of application Ser. No. 07/700,556, filed on May 15, 1991, now abandoned, which was a continuation of prior application Ser. No. 421,835, filed on Oct. 16, 1989, now abandoned. 
    
    
     BACKGROUND OF THE INVENTION 
     A bluish-green &#34;Phosphorlicht&#34; of living leaves was first observed by Carl von Szutz, an apothecary of Hungary, on the leaves of a garden plant, Phytolacca decanda  (Linn), between 9 and 10 p.m. one evening in September, 1800. Szutz picked the leaves and the light continued to shine, sometimes yellowish-green, sometimes bluish, sometimes stronger, sometimes weaker, depending on whether the &#34;Zug der Lade&#34; was stronger or weaker. The phosphorescence lasted until after midnight and then disappeared. The light may have been an electrical phenomenon, although luminous fungi growing on living leaves is a second possibility (As quoted by E. Newton Harvey, in A History of Luminescence, The American Philosophical Society, Philadelphia, pp486, 1957). 
     Phytoluminescence was first described by Strehler and Arnold in 1951 (Strehler B. L. and Arnold W. 1951, Light production by green plants--J Gen Physiol 34:809-820). This extremely short lived light emission by living green plants is not visible to the unaided eye and not at all related to the observations of von Szutz. This emission of light is part of a photosynthetic reaction by excited states of chlorophyll and is dependent upon photosynthetic electron transport. A reversible injury causes an increase in light output probably by a decrease in photosynthesis diversion of energy to phytoluminescence. Irreversible injury damages the entire photosynthetic mechanism abolishes phytoluminescence. Phytoluminography is two dimensional imaging of phytoluminescence using high sensitivity light amplifiers. The technique was first described by Sundbom and Bjorn in 1977. Its use in early detection of plant stress has been frequently described in Soviet literature. A few selected references are listed: 
     1) E. Sundbom &amp; L. O. Bjorn, Physiol Plant 40:39-41, 1977 
     2) L. O. Bjorn &amp; A. S. Forsberg, IBID 47:215-222, 1979 
     3) A. A. Krasnovskii, Jr., Yu. V. Kovalev, et al., Biofizika 25(5), 1980 
     4) J. L. Ellenson &amp; R. G. Amundson, Science 215:1104-1106, 1982 
     5) R. Blaich, O. Bachmann, &amp; I. Baumberger, Z Naturforsch Sect C Biosci 37 (5-6):452-457, 1982 
     P. F. Daley, K. Raschke, J. T. Ball, &amp; J. A. Berry, Plant Physiol 90: 1233-1238, 1989 
     Phytoluminescence has been arbitrarily divided into chlorophyll fluorescence and delayed light emission by all the authors. We attribute this division to insensitive imaging techniques and can demonstrate these to be parts of a negative exponential luminescence decay. The prior art involves large, expensive apparatuses using conventional air coupled lenses, mechanical shutters, long optical paths and light sources that do not resemble sunlight. Since the apparatuses are not hand-held or battery operated, they are inconvenient for use outside the laboratory. This limits the use of this valuable technique and puts it beyond the reach of common farmers and horticulturists. 
     Schematics of typical apparatuses using the prior art are reproduced in FIGS. 1, 2, and 3 as published in references 1, and respectively. 
     SUMMARY OF THE INVENTION 
     The above and other disadvantages of prior art phytoluminography apparatuses are overcome by the present invention which provides a hand-held, battery operated, single photon sensitivity, large format device for quantitative imaging of phytoluminescence, referred to hereinafter as a &#34;Phytoluminometer&#34;. 
     The present invention uses easily available electronic flashguns with xenon flashlamps to excite luminescence in leaves. Green leaves exposed to other types of light such as sunlight, fluorescent light, incandescent light will also exhibit this phenomenon. However, these sources of illumination are non-uniform in spatial intensity distribution, spectral emission pattern, non-reproducible and not suitable for quantitative work. The preferred configuration of the electronic flashgun would be in the form of a ring light for ease of incorporation into the phytoluminometer and uniform distribution of light when used with a diffuser. Other configurations would work for less critical applications. The light source can be used in a white light (polychromatic) mode. It can also be filtered with glass or gelatin filters for monochromatic mode. Xenon flashlamp sources may be operated at varying repetition rates for luminescence lifetime studies. Planar electroluminescent lamps are suitable for less intense excitation. Solid state laser diode sources can be used after beam expansion and can be pulsed. Two dimensional arrays of light emitting diodes (LEDs) can be used but provide nonuniform illumination. 
     The leaf surface is viewed by the light amplifier assembly without delay after luminous excitation with the source of excitation switched off. The leaf surface is placed or held directly against the planar input surface of a fused, coherent, rigid, fiberoptic light pipe, taper or flexible fiberoptic bundle. Gradient index (GRIN) self-focussing fibers may be used in the fiberoptics. A fiberoptic taper allows minification or magnification depending upon the application. Conventional air coupled lenses may be used if provided with plano concave fiberoptic faceplates to optimize coupling between the leaf and the light amplifier. Projection of the image of the leaf using standard focal length lenses is not acceptable because of severe transmission losses. When air coupled, multi-element, glass lenses are used the surface coupled to the light amplifier should be of a plano type again to reduce coupling losses. 
     Reflection losses caused within the coupling of polished planar surfaces are reduced by the use of optical greases such as Dow Corning Silicon Grease #Q2-3067 optimized for refractive indices of the glasses. 
     The light amplification can be performed with first, second, or third generation image intensifiers of the types commonly used in night vision devices. Second generation image intensifiers are preferred because of optimal spectral match between the phytoluminescent emission and the photocathode spectral response. Proximity focused, electrostatic inverter, gated or nongated image intensifiers with one or more microchannel plates may be used. Multistage, low noise amplification with luminous gains greater than 10 8  is necessary for imaging the entire luminescence decay. This necessitates cascading at least two second generation image intensifiers. Hybrid types with second generation intensifier at the input end and first generation at the output end can be used but are currently twice as expensive. All of the above image intensifiers can be procured with self contained battery operated power supplies and automatic bright source protection and can be gated. All of them are available with plano fiberoptic input and output faceplates. This facilitates ease of coupling to imaging optics and to each other. The entire assembly is encased in a light tight aluminum or plastic housing. The material of the enclosure is not critical as long as phosphorescent or fluorescent materials are excluded. The batteries for operating the flashgun, image intensifiers as well as the variable gain controls, switches are external to the light tight enclosure. The intensified image may be viewed in subdued light, recorded with conventional 35 mm, Polaroid, still or movie film, video, analog or digital cameras. Analog images may be digitized for image storage, processing, analyses, transmission by use of commercially available software and hardware. The intensified image may be quantified in its entirety or fraction thereof using optical to electrical probes in conjunction with oscilloscopes or photometers. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1. is a schematic of one type of phytoluminography apparatus using prior art. 
     FIG. 2. is a schematic of a second type of phytoluminography apparatus using prior art. 
     FIG. 3. is a schematic of a third type of phytoluminography apparatus using prior art. 
     FIG. 4. demonstrates a schematic of the phytoluminometer using the current invention. 
     FIG. 5. represents a &#34;second generation&#34; image intensifier comprising multi-alkali photocathode, an electrostatic focussing system, a microchannel plate (MCP) as electron multiplier and a luminescent screen positioned immediately behind the MCP. A high voltage power supply operating from low voltage batteries is included in the encapsulation. 
     FIG. 6. represents a proximity focussed gated image intensifier with MCP and a gating power supply. This enables the tube to be switched within 5 nanoseconds for rapid electronic shuttering during luminescence lifetime studies. 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The present invention is a process and apparatus for obtaining quantitative images of luminescence arising from plant leaves and other surfaces excited by an external source of light. The luminescent light distribution represents active sites of photosynthesis. Perturbation of photosynthesis caused by darkness, high concentrations or lack of CO 2 , lack of O 2 , extremes of temperatures, noxious gases, chemicals, bacterial, viral, and fungal infections, can be rapidly and easily demonstrated using this invention. The perturbed area will manifest itself as brighter than non-perturbed area if recoverable, and darker if irreversibly damaged. These changes are visible with the instrument described herein seconds after the damage and days or weeks before the damage is visible to the unaided eye. Consequently, this invention helps in very early detection of plant stress and prediction of recovery. Since the apparatus is small, portable, low cost, sensitive, easy to use, it is useful to farmers, plant nurseries, plant breeders and others involved in plant research. Herbicide and pesticide research as well as ecological and pollution research would also benefit from use of the instrument. Further scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. 
     Referring to FIGS. 1, 2, and 3, it will be apparent to those skilled in the art that most of the luminescent photon flux generated within the leaf surface is lost during transmission to the front surface of the air-coupled, multi-element imaging lenses. This reduces the sensitivity of the apparatus, creates multiple optical interfaces which are subject to moisture condensation, contamination, misalignement by shocks or vibration and need to be focussed. The mechanical shutters are too slow to respond to rapid luminescent changes. The illuminating systems have similar drawbacks of long distances, use of reflecting mirrors, and lamps not resembling solar spectral output. The bulky nature of the apparatus is immediately apparent. The use of only one stage of light amplification further contributes to the decreased sensitivity of the technique. Thus the use of the apparatus is limited to the laboratory. 
     FIG. 4 refers to the schematic of the present invention. The excitation source A for illuminating the leaf is shown as an electronic flashgun with the xenon filled tube in a ring form. The light from the ring flash is diffused by a Lucite disc B within the ring. The Lucite disc B also serves as a support for the leaf D to be studied. Narrow or wide band, glass or gelatin filter C may be interposed between the Lucite support and the leaf D for monochromatic excitation. The power source E for the ring flash A is battery operated and external to the assembly X. The mechanical or electronic switch F allows either the excitation source A or the light amplifier G and H to be operational thus preventing damage to the light amplifiers by inadvertent exposure to high intensity light. A micro-switch I in the front removable cap J housing the ring flash A, diffusing disc B, leaf D switches off the light intensifiers when the cap J is removed thus protecting the amplifiers from ambient light. The imaging optic K is a fiberoptic taper with planar large and small surfaces. In operation leaf D on support B is directly in contact with the front surface L of the taper K. The rear surface M of the taper is coupled to the front fiberoptic faceplate N of the first stage image intensifier O. The rear fiberoptic output phosphor P of the first stage image intensifier is coupled directly to the front fiberoptic faceplate Q of the second stage image intensifier R. The output phosphor S of the second stage intensifier R is not covered by the enclosure X and thus is visible to the eye. The output phosphor S can also be coupled to optical cameras. The power source for the image intensifiers is provided by the batteries T providing power to E as well. 
     All fiberoptic coupling surfaces should use a thin layer of Dow Corning Q2-3067 optical couplant taking great care to exclude air bubbles. This reduces reflection losses, maintains tight but removable contact between surfaces and is immune to humidity, temperature changes. 
     The image intensifier tubes used in this apparatus should have the highest available photocathode sensitivity, in the spectral emission band of phytoluminescence, best signal to noise ratio, lowest equivalent background illumination (EBI), minimum number of blemishes, hot spots. Standard military grade image intensifiers used in night vision devices are not preferred. Astronomical grade tubes should be specified. Second generation tubes with high output type (HOT) MCPs are preferred. When research applications with luminescence lifetime detection are involved fast output phosphors such as P-11 should be specified for the image intensifiers. Multi-colored current sensitive phosphors such as PT-452 will provide good contrast discrimination without computer enhancement. 
     Proximity focus, gated wafer tubes may be used in the first or second stage for rapid electronic switching of light amplification and for protection during strobing. It is preferable to operate photocathode and output phosphor surfaces close to ground potential to eliminate arcing, ion flashes, leakages between opposing coupled surfaces. A thin conductive coating with optical transparency such as tin oxide (NESA) should be applied to output fiberoptics for grounding it. 
     The entire assembly is encased in a light tight plastic or metal enclosure with care taken to exclude all fluorescent or phosphorescent materials such as paints, adhesives, detergents, optical brighteners. The low voltage battery power supply and adjustable gain controls for the image intensifiers are located outside the light enclosure but are connected to it by a cable or mounted on it. The switching functions and gain control can be controlled by a computer for nonportable applications. The manual gain controls should preferably be ten turn, wire wound potentiometers with turns indicator dial for precise setting and resetting of gain. 
     If an analog video camera is used for recording, then the video signal may be digitized for image acquisition, storage, analyses, quantitation, and enhancement. The hardware and software for accomplishing these functions are widely available commercially and need not be described here.