EP2780695A1 - Vorrichtung und verfahren zur nicht-invasiven erfassung von wachstumsprozessen und simultanen messung von chemisch-physikalischen parametern - Google Patents
Vorrichtung und verfahren zur nicht-invasiven erfassung von wachstumsprozessen und simultanen messung von chemisch-physikalischen parameternInfo
- Publication number
- EP2780695A1 EP2780695A1 EP12798135.5A EP12798135A EP2780695A1 EP 2780695 A1 EP2780695 A1 EP 2780695A1 EP 12798135 A EP12798135 A EP 12798135A EP 2780695 A1 EP2780695 A1 EP 2780695A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- growth
- optode
- light
- unit
- chemical
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6408—Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/0098—Plants or trees
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N2021/6463—Optics
- G01N2021/6471—Special filters, filter wheel
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6497—Miscellaneous applications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
- G01N2021/7706—Reagent provision
- G01N2021/773—Porous polymer jacket; Polymer matrix with indicator
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7769—Measurement method of reaction-produced change in sensor
- G01N2021/7786—Fluorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
- G01N2021/8466—Investigation of vegetal material, e.g. leaves, plants, fruits
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/783—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour for analysing gases
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
- G01N21/80—Indicating pH value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/062—LED's
- G01N2201/0627—Use of several LED's for spectral resolution
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/06—Illumination; Optics
- G01N2201/064—Stray light conditioning
- G01N2201/0642—Light traps; baffles
Definitions
- the invention relates to an apparatus and a method for detecting growth processes and simultaneous measurement of chemical-physical parameters.
- WinRHIZO Software “WinRHIZO” (Regent Instruments, Inc.). This typically examines washed-out roots after the invasive process of digging or sampling with the aid of a sample cylinder driven into the ground The obtained photographic images can then be analyzed with the aid of the software, whereby parameters of the root morphology, such as root length, root diameter and more can be measured (morphometric analysis) To non-invasively record morphometric data using the WinRHIZO software by measuring photographic images obtained, for example, from rhizotron experiments without the detour of scanning out washed roots.
- WinRHIZO Registered Instruments, Inc.
- Root parameters include weighing fresh and dry matter. Once the roots have been dug up, many parameters of the root architecture are irretrievably lost. These include, for example, parameters such as the exact root length density, the total width of the root system, branch angles, and the like. In addition, it is not possible to carry out further measurements on the same test object. This can only be done via comparison populations, which significantly increases the number of plants to be grown compared to non-invasive measurements.
- invasive methods for determining the growth of aboveground plant organs are variously described in the literature. This includes, for example, the weighing of cut leaves or the scanning / photographing thereof, including morphometric, image-analytical methods.
- auxanometers or resistance transducers have been used for decades to detect the growth of aerial shoots or leaves under various environmental conditions.
- the spatial resolution is also limited and limited to the simple detection of the longitudinal growth.
- dicotyledonous leaf waxes In addition, these study methods have the disadvantage that no difference can be made between the lengthening of the petiole and leaf sheath (or even individual segments of the leaf sheath) (growth of dicotyledonous plants).
- transducers and auxanometers can not be used for technical reasons. Basic applications in hydroponics or aeroponics cultures appear to make little sense here.
- root growth and shoot growth of the same plant can only be studied to a limited extent, depending on each other. This applies in particular to growth analyzes which are intended to capture growth in high spatial and temporal resolution.
- This is one of the current limitations of the current state of the art, which can be solved for example by simultaneous, automated detection of root, shoot and / or leaf growth with high-resolution methods.
- all investigations of growth involve the disadvantage that a simultaneous detection of environmental influences in high spatial and temporal resolution is typically not possible.
- This disadvantage relates in particular to the simultaneous detection of chemical and / or physical properties of the rhizosphere in measurements of root growth in high spatial resolution.
- the prior art has not made it possible to simultaneously detect changes in the chemical-physical properties of the growth medium and the dynamics of growth parameters in high spatial and temporal resolution. In order to investigate the interplay between growth and growth medium, this information had to be recorded in separate measurements and as a rule invasive and therefore could not be performed on the same object in parallel and continuously. According to the prior art, it is only possible to carry out quantitative measurements of chemical-physical parameters of a growth environment of living beings by using miniaturized measuring probes, such as microelectrodes or optical measuring systems (optics), which enable simultaneous detection of environmental influences in high spatial environments and temporal resolution typically did not allow (Strömberg 2008, Pijnenborg et al 1990, Blossfeld & Gansert 2007). With indicators for pH changes such. B.
- Bromocresol purple which have already been used in agar systems for the study of rhizosphere effects, quantitative pH measurements are not possible for all growth media and only for limited ranges of the pH scale. Quantitative measurement can only be done within the envelope of the indicator (Jaillard et al 1996, Marschner et al., 1986). Outside the cargo handling area, only a qualitative measurement can be made. (Marschner 1986). Other indicators for detecting, for example, changes in the redox potential are known, for. B. methylene blue. Microelectrodes for the specific measurement of the concentration of ammonium, pH, calcium, chloride, sodium, oxygen, carbon dioxide and more are known (Microelectrodes Inc.).
- the concentration of the measurement parameter is changed or consumed as a result of the measuring principle, which can lead to problems, in particular in the case of measurements over a longer period of time, in particular if reciprocal interactions are the subject of the investigation.
- the spatial resolution is limited by using microelectrodes by their geometry.
- the dimensions of the microelectrodes limit the spatial resolution to be achieved, the reaction time the achievable temporal resolution. If a flat measurement in high spatial and temporal resolution, several microelectrodes must be used, which is associated with relatively high cost and high costs. If simultaneously several chemical-physical parameters are to be measured continuously, this is also at the expense of the spatial resolution.
- planar optodes also referred to below as optodes
- Optodes are known, for example, for the specific measurement of the concentration of ammonium, oxygen, carbon dioxide, pH and various ions and organic compounds. The measurement is based on the use of specific, on the measured parameters tuned fluorescent dyes that are excited in the short term with light of one or more specific wavelengths. The dye fluoresces depending on the quantity of the parameter to be measured and the intensity of the exciting light for a few milliseconds, or nanoseconds.
- fluorescence To be distinguished from the fluorescence is the phosphorescence, in which the light emission lasts much longer, u. U. up to several hours. Both terms (fluorescence and phosphorescence) are subordinated to the general concept of luminescence.
- the dye is fixed in or on a polymer matrix and applied in or onto the growth medium to be examined.
- the optical measurement takes place by means of a suitable optical structure (eg CCD camera) outside the growth medium.
- a suitable optical structure eg CCD camera
- the sensor eg the optode
- the detector eg the CCD chip
- Another possibility for optical detection of fluorescence is the application of a stepper motor-bound system (Blossfeld & Gansert 2007, Biossfeld et al., 2010). This is a camera-independent system in which both the excitation light and the fluorescence are conducted via a glass fiber from the light source to the sensor or from the sensor to the detector. The glass fiber is driven in a defined grid from the outside over the sensor.
- the fluorescent dye is excited with light of a specific excitation wavelength, and after the excitation has ceased, the cooldown of the
- Fluorescence measured Fluorescence lifetime
- Fluorescence lifetime Fluorescence measured (fluorescence lifetime), which is dependent on the analyte concentration.
- some fluorescent dyes have an extremely short fluorescence lifetime (a few nanoseconds), so that it may be necessary to integrate an analyte-sensitive reference dye into the optodes in addition to the analyte-sensitive fluorescent dye (Schröder 2006). Since this reference dye fluoresces independently of the analyte concentration in the millisecond range and is also excited by the excitation wavelength used, the two fluorescence signals (dual lifetime referencing) overlap. This mixed signal is therefore likewise dependent on the analyte concentration and thus allows the fluorescence lifetime measurement of fluorescence dyes with short-lived fluorescence lifetime.
- rhizosphere parameters are, for example, oxygen or carbon dioxide concentration ([0 2 ], [C0 2 ]) and pH (pH), but fluorescent dyes are also available for measuring the concentration of a large number of other parameters (eg. Metal ions, ammonium nitrogen).
- Table 1 Examples of fluorescent dyes and the corresponding possible parameters that can be analyzed
- pH-sensitive fluorescent dye for the pH range 7.3 - 9.3
- HPTS 8 - hydroxypyrene - 1, 3,6 - trisulfonic acid as trisodium
- pH-sensitive fluorescent dye for the pH range 5.5 - 8.6 (Zhu et al., 2005) or C0 2- sensitive fluorescent dye 0 - 20%
- PtOEP platinum (II) 2,3,7,8,12,13,17,18-octaethyl-21H, 23H-porphyrin
- Pt-PFP platinum (II) mesotetra (pentafluorophenyl) porphyrin
- Ruthenium (II) diimine complex ruthenium (II) tris-4,7-diphenyl-l, 10-phenanthroline, with trimethylsilylpropanesulfonate as counterion
- the spatial resolution is generally dependent on the pixel resolution of the camera used and the camera distance (eg total area 101 cm 2 with a pixel size of 277 ⁇ m x 277 ⁇ m, Hakonen et al., 2010). In the case of the stepper motor-bound system, the spatial resolution depends on the step size of the motors and the diameter of the light cone (eg total area 80 cm 2 at a step size of 1.5 to 3.0 mm per step, Biossfeld & Gansert 2007).
- the temporal resolution between two image acquisitions is the use of camera-based systems in the range of seconds or milliseconds and depends on the camera characteristics and the associated data processing routine (eg ring buffer, real-time processing, etc.). Long-term measurements of several days to weeks are possible, but may require a large data memory of several MB to GB with sufficiently fast access times, data transfer rates and temporary storage (Hakonen et al., 2010).
- the temporal resolution between the individual steps is in the range of seconds (2-3 seconds), the temporal resolution between two complete raster cycles depends on the number of steps and is usually in the minutes range (Blossfeld & Gansert 2007). Long-term measurements of several days to weeks are also possible and require a comparatively small data memory of a few MB.
- optodes When using optodes in the context of z. B. soil or convincedwurzeluntersu- ments they can be optically read through a transparent glass pane from the outside. An opening of the system is thus not necessary for the measurement, and said changes of the system, for. B. with regard to a disturbance of the gas or moisture balance are excluded.
- the use of optodes for the general detection of chemical-physical parameters, such as the pH or the determination of salt concentrations is known.
- a measurement of chemical-physical parameters simultaneously with the imaging acquisition of growth processes in high spatial or temporal resolution using optodes is not yet possible.
- Imaging techniques such as DISP root, GROWMAP and GROWSCREEN root (Walter et al 2009, Nagel et al 2009) are not compatible with measurement via optodes for various reasons.
- the hitherto known optodes have in their physical properties not the required optical characteristics, which are necessary for such a detection. It is essential, first of all, that planar optodes have a suitable trans- missivity, and the highest possible resolving power not impaired by (coherent and incoherent) scattering effects.
- the growth object to be examined must be continuously identifiable for imaging growth analyzes as clearly as possible, rich in contrast and sharp through an optode.
- abiotic growth processes for example, physical growth processes, such as the growth of soil physical parameters, e.g., soil cracks or soil pores, and their interaction with the other named parameters are of interest and can be analyzed using this apparatus and method of operation.
- “biotic growth processes” also referred to as differentiation and development processes, also include those processes which take place in cultures / tissue cultures of animal, plant or fungal organisms / cells or populations of microorganisms Collected and analyzed together in one study or separately in different studies.
- the invention also allows, among other things, observation of the growth and development of fungal hyphae or living organisms of the marine or terrestrial soil fauna, generally with simultaneous detection of the physicochemical properties and alteration of the surrounding medium in high spatial and temporal resolution.
- growth processes is understood to mean both biotic and abiotic growth processes.
- Non-invasive imaging of growth parameters such.
- the detection of biotic differentiation, development and growth processes of organisms, organs, tissues or cells, which are of particular interest in the context of this invention include, for example, the spatial and temporal resolution of growth.
- the term "spatial and temporal resolution of growth” is understood to mean, for example, tropisms, growth rates, relative growth rates, spatial distribution of growth rates and the temporal dynamics of changes in growth rates of plant roots and rhizomes, as well as their dynamic alteration of the root architecture it, imaging these growth parameters by means of an imaging method within the device according to the invention with an optical detection to automatically detect and analyze a camera system and to analyze it with image-analytical methods.
- the term "detection” is understood to mean the collection and evaluation of the data which has been determined / provided by the optical detection unit / camera system. be used to evaluate the growth behavior as a function of the physicochemical parameters and to be able to make statements about the dependence of the growth of, for example, pH, ammonium concentration and light intensity.
- the invention enables detection and analysis of the biotic / abiotic growth processes as well as analysis and detection of fluorescence-spectrometrically detectable, dynamically changing physicochemical parameters and processes within the growth medium or, with the aid of the planar, transparent optodes.
- the continuous simultaneous detection of growth processes and chemical-physical environmental parameters according to the invention is automated and non-invasive, with both a qualitative, as well as a quantitative evaluation of growth processes and chemical-physical parameters or their dynamic change over time is possible.
- automation with regard to the acquisition of growth parameters and chemical / physical parameters, both a mechano-automated positioning of the camera system in front of the examination object and vice versa a mechanically-automated positioning of the examination subject in front of the camera is possible.
- the collection of the measured parameters should take place in sufficiently short time intervals, so that the relevant growth dynamics, ie the change over time, z. As growth rates, can be recorded and described so that a causal analytical interpretation is made possible.
- a high temporal resolution in the measurement of chemical-physical quantities is achieved in that an elevation of the measured parameters takes place in sufficiently short time intervals, so that the relevant change in the parameters, eg. For example, concentrations of an analyte over time may be causally related to the measurement of biotic or abiotic growth processes.
- the time resolution for measuring chemical-physical quantities should normally be 1 image / hour (1 image / h), as chemical processes in the rhizosphere may also change during the day.
- a survey of the measured parameters should be carried out in sufficiently high spatial resolution, which in the sense of the invention is primarily two-dimensional, but under certain circumstances can also be understood three-dimensionally
- the maximum resolution may allow detection of the structures to be measured, for example using the DISP method for spatial distributions of relative growth rates within 125 px (pixels) / mm resolution should be measured by growing root tips over time, but a resolution of approximately 5 - 10 px / mm is usually sufficient for taking root-growing roots in rhizotron systems.
- the survey of the measured parameters should be carried out in sufficiently high spatial resolution, which is primarily two-dimensional in the context of the invention, but under certain circumstances may be understood in three dimensions, so that the change in question the parameter, eg Concentrations of an analyte over time can be brought into a causal analytical relationship with the measurement of biotic or abiotic growth processes.
- the maximum resolution must enable detection of the structures to be measured.
- the spatial resolution for measuring chemical-physical quantities should normally be at least 1 px (pixels) / mm. At any time of measurement, it may be a two-dimensional map of, for example, the concentration of the chemical analyte or the physical parameter
- the reaction of the fluorophores on the surface of the optode will vary depending on the concentration of the analyte. This allows mapping of the respective parameters.
- the invention relates to a device and a method by means of which measurements of a) optically detectable growth processes and b) fluorescence-spectrometrically detectable, dynamically changing chemical-physical parameters and processes within a growth medium or substrate, and their Interaction with the detected growth processes, imaging in high temporal and spatial resolution, can be simultaneously recorded and analyzed by image analysis. Furthermore, it is also possible to determine parallel or alternative chemical-physical parameters outside the growth medium / substrate, such as a gas analysis of the shoot in a gas-tight space in addition to the analysis of the growth processes of the roots and the chemical-physical parameters in the growth medium.
- the measurement with optodes is based on the use of specific fluorescent dyes (fluorophores) which are tuned to the parameters to be measured and which are excited in the short term by light of one or more specific wavelengths.
- the dye is in or on a polymer matrix, for. B. polymer film fixed and / in / on the medium to be examined / is in contact with the object or medium to be examined in chemical and / or physical contact.
- the optical measurement is carried out by means of a suitable optical structure (eg CCD camera) outside the medium to be examined.
- the simultaneously detectable and coregistratable chemical-physical parameters in the context of this invention can be of different nature. Can be measured For example, temperature, pH values, specific substance concentrations (for example of ammonium ions, oxygen, C0 2 ). Further parameters and their respective influence on determinable growth parameters are listed in Table 2.
- the selection of the parameters to be observed is very versatile, since in principle a large number of fluorophores and coated optodes can be used or described. It is also possible to measure several of these parameters simultaneously.
- the system can be flexibly adapted in its spatial and temporal "magnification" or resolution to the respective requirements of the experimental setup
- magnification e.g. lens change, working distance, etc.
- the structure can be used within an automated system. For example, this flexibility allows high throughput screening of plants with a high number of replicates that can be studied simultaneously.
- Table 2 Examples of chemical-physical parameters and growth parameters that can be measured (independently of each other) in the context of this invention.
- pH value of substrate structure e.g. Belly
- growth media is to be understood in general as meaning the space which surrounds the object to be observed, such as, for example, an organism / a living being or the organism, and which permits its growth and thus represents the living environment.
- the growth medium can be of a varied nature in the method of the method used, which can be both a natural substrate (for example soil, soil, sand) and "semi" synthetic substrates (for example agar, hydroponics, air cultures) from other growth media, such as fully synthetic, swellable substitution media is possible.
- the growth medium is referred to as the rhizosphere.
- the Rizosphere can be defined as the soil volume surrounding living plant roots, which is affected by root activity. Hinsinger et al. (2005) New Phytologist, 168: 293-303.
- the term organism / living being may, for example, be a plant root system, which is imaged together with the surrounding growth medium.
- the term organism may generally be understood as meaning plant or animal organisms or else bacteria, fungi.
- the device according to the invention comprises a system which has one or more cameras, an optical configuration, a light source for illuminating the examination subject, e.g. As the rhizosphere, a special planar and thus sufficiently transparent optode, so that growth measurements are ensured (the optode has at least one immobilized fluorophore) and a digital computer unit (or an embedded computer system) uses. With the aid of this arrangement, it is possible to take non-invasive and non-destructive photographic recordings or time-lapse recordings of a growing organism, organ or tissue.
- the biological object and the growth medium are within a transparent growth container, which is at least partially transparent.
- a transparent growth container which is at least partially transparent.
- a second group of created images and image series allows the simultaneous detection of one or more chemical and / or physical properties within the growth medium, wherein the recordings of all parameters can be made in high temporal and spatial resolution.
- planar, transparent optode used according to the invention has advantageous optical properties in comparison to known, commercially available planar optodes.
- the planar, transparent optode according to the invention makes it possible for the first time to simultaneously look through and photograph through an optode.
- the optode according to the invention is thus transparent.
- optical properties is understood to mean the parameters resolving power, contrast and transmissivity.
- the advantageous optical properties in particular the transparency, simultaneous other measurements in the form of image analysis and imaging are also possible for the first time.
- the advantageous optical properties of the optode which are desired in the context of the invention, and which can be used in photography or looking through the optode, the following properties are to be understood, which together ensure a high image quality and object-faithful image: a) achievable transmissivity (ie low absorption of light different
- the achievable gray-scale contrast photometric contrast, differences in the brightness between light and dark areas in the recorded object are retained when the gray-scale contrast is maintained
- the advantageous properties of the optode are particularly important because, according to the invention, photographic images of the objects or organisms to be examined are to be made through the optode, and the objects are behind the planar optode.
- the aforementioned advantageous optical properties mean that only minimal changes are caused with regard to the imaging quality or the image quality of the object / organism photographed through the optode. The change is so small that objects / organisms located behind the optode can be recognized by the optode through object-true, and in photography through the optode through object-faithfully and significantly equal to the reference can be reproduced.
- the aforementioned advantageous properties of the planar, transparent optode used according to the invention have certain correspondences with regard to the image quality, which are mentioned below: a) The desired high transmissivity of the optode is limited by a small amount
- the light intensity here as a signal to be measured of importance
- the light intensity is the signal
- the point response (lintel, Green's function, transmission function) says like a sharp point of light
- these optical properties are considered to be advantageous when it is intended to look through an object, such as in the case of viewing windows (window glass screens). ben).
- the absorption must be as low as possible, the gray-scale contrast as high as possible, and the resolution as high as possible.
- the device according to the invention for non-invasive detection of growth processes and simultaneous measurement of chemical and / or physical parameters consists of the following components:
- the light source should emit light having a wavelength spectrum suitable for both illuminating / exciting the optodes used with the particular fluorophore (s) and emitting the wavelength spectrum (s) for the imaging photographic measurement Growth processes is needed. However, it is also possible for one light source to illuminate / excite the optode and another light source to emit the wavelength spectrum / wavelength needed to detect and analyze the growth processes.
- the required wavelengths are to be selected in each case as a function of the fluorophore immobilized on the planar optode and its excitation maxima. In the case of HPTS, for example, these are the wavelengths 405 and 450 nm.
- HPTS high-temperature temperature
- the light source (s) should therefore emit light of a wavelength in the range of, for example, 200 to 2500 nm. The number of light sources needed depends on the particular wavelength spectrum that the light source can emit and the wavelengths it needs.
- the chosen wavelength should be both the biological object, and the
- roots show no influence from near-infrared (NIR) and short-wavelength infrared (SWIR) spectra. iii.
- the wavelength must not overlap with the excitation and emission ranges of the flurophore used.
- One possible light source may be a (monochromatic) infrared illumination which emits in the wavelength range between 700 nm and 2500 nm.
- the wavelength range of the light source is limited to the range up to about 1100 nm, since the CCD camera sensors there reach their upper wavelength sensitivity limit.
- the light source may preferably provide additional monochromatic wavelengths in the region of the excitation maximum or of the excitation maxima of the immobilized fluorophore (s) used on the optode. In this regard, the wavelength of the excitation maximum may vary depending on the fluorophore used.
- the selection of the desired / required fluorescence measurement also determines the number of monochromatic wavelengths (eg dual excitation - single emission or dual lifetime referencing, see paragraph on ratiometric fluorescence measurement) in the case of HPTS and the dual excitation - single emission Fluorescence measurement methods are the required excitation wavelengths, for example, 405 nm and 450 nm, with 405 nm corresponding to the unprotonated form and 450 nm to the protonated form of HPTS.
- the light sources can be one or more monochromatic LEDs or an LED cluster in which the emission of the LEDs for excitations of the individual excitation wavelengths of the fluorophores has a non-overlapping spectrum.
- the "Fill Half Width" of the LEDs must ideally be as narrow as possible.
- the light source may further consist of one or more layers of transparent, self-illuminating monochromatic OLEDs for each of the wavelengths required for the fluorophores.
- the emission of the OLEDs for excitations of the individual excitation wavelengths of the fluorophores should also have a non-overlapping spectrum. Ideally, the "Fill Half Width" of the OLEDs must be chosen as narrow as possible.
- a filter wheel or a filter changer can be arranged, which allows a change between at least two filters.
- a filter for example, a "narrow band" filter having a small “small half width" of, for example, 15 nm for the excitation wavelength or wavelengths resulting from the selection of the immobilized fluorophores and the respective fluorescence measurement method can be used.
- the dual excitation / single emission method for example, requires the use of two filters in the range of 405 nm and 450 nm, respectively.
- an infrared transmission filter which transmits only infrared light above 700 nm but no light from the range of the visible and UV light ranges.
- the optical detection unit serves, on the one hand, to acquire the image data / image signals which are emitted by the organism / growth system to be examined and which are required for detecting the growth process and, on the other hand, to detect the fluorescence light emitted by the fluorophores.
- a CCD camera can be used as a camera.
- the camera may be provided with a filter / notch filter which allows only the passage of light of a certain wavelength. This may, for example, be the light of the wavelength corresponding to the excitation wavelength of the fluorophore on the optode or, in the case of acquisition of image data of the growth process, the light of the wavelength required to acquire and analyze the growth of the organism to be examined. For the detection of growth processes, for example, light in the near infrared light spectrum is suitable.
- the filter can be arranged in front of the lens of the camera. It can alternatively be arranged behind the lens with an optional tube extension in front of the sensor of the camera.
- the optical detection unit can also have at least two cameras.
- the first camera then serves to detect and record the chemical-physical parameters (eg pH value) in which the emission of the fluorescent light is measured.
- This camera should have a narrow-band filter with a small fill half width (width of the total emitted wavelength spectrum at half height of the emission maximum).
- the second camera is then used to capture the growth data.
- an infrared pass-through filter can be arranged on the camera, which allows only infrared light to pass through the filter. It is thus from this camera detects only the infrared light that was used to capture the object to be examined for illuminating the same.
- At least one growth container having at least partially a transparent wall surface may also have two or more transparent wall surfaces.
- the transparent wall surface should be chosen so large that the optode sufficiently excited with light and the signal of the optode can be evaluated and the organism to be examined, also sufficiently exposed by the optical detection unit and the image data can be detected.
- the transparent wall surface should be transmissive at least to light of the wavelength emitted by the illumination unit. This can be, for example, light in the infrared light spectrum and visible light spectrum.
- the wall surface which is located behind the transparent wall surface, covered with a dark, non-reflective and highly absorbent material. This can be, for example, black velvet or a similar material.
- the growth container should have a three-dimensional design and may for example have the shape of a cube, cuboid or a cylinder. Petri dishes are also suitable as growth containers.
- the growth container is filled with growth medium.
- growth medium is to be understood in general as the space surrounding the organism or organ to be observed, which allows its growth and thus represents the living environment.
- the growth medium may be of a variety of nature, which may be both a natural substrate (e.g., soil, soil, sand) and "semi" synthetic substrates (eg, agar, hydroponics, air cultures), as well as the use of other growth media.
- the growth medium is typically referred to as the rhizosphere, in which case the growth container is referred to as rhizotron, for example, the growth medium should be agar with the appropriate organism
- a Hoagland solution in various modifications or any other nutrient solution suitable for plant cultivation can be used. which can then be automatically exchanged and measured, for example.
- a Petri dish wheel can be measured in which the Petri dishes are always exchanged by a controlled motor and brought into the required direction / position for the lighting unit and optical detection unit (see also FIG. 1).
- the illumination unit can be applied directly to the front of the transparent growth container.
- the lighting unit can be mounted both on the camera side and on the opposite side.
- the lighting unit is to be positioned in this embodiment such that the transparent side of the container is illuminated as homogeneously as possible.
- positioning it is important to avoid total reflections and other lighting effects as possible.
- This can be done, for example, by arranging the illumination unit at an angle of less than 90 ° (angle between camera and beam path of the illumination unit, either the camera or the illumination unit being oriented orthogonally to the surface of the transparent growth container and the surface of the transparent optode).
- the light source can be mounted at a 30 ° angle. Deviating angles in a range of 0 to 90 ° degrees are also possible when measuring if required.
- the growth container can also be arranged at an angle of less than 90 ° horizontally inclined to the illumination unit and the optical detection unit, so that on the one hand, total reflections are avoided and on the other hand, the growth in the direction of the optode takes place, so that the transparent planar optode contact the growth medium can. d) a planar, transparent optode
- a planar, transparent optode suitable for the purposes of the invention for measuring the chemical and / or physical parameters, for example the pH value, which, when wetted with a film of liquid water, is distinguished by a transparency which, for example, all in Walter et al. (2009) for measuring growth processes of roots (such as DISP and GROWSCREEN root), and immobilizing the fluorescent dye, can be prepared as follows: A cellulose acetate film (eg from Clarifoil®) is coated with an ethylcellulose polymer into which the fluorescent dye HPTS (8-hydroxy-1,3,6-trisulfonic acid) is immobilized. To prepare the solution with which the film is coated, two solutions must first be provided.
- Clarifoil® eg from Clarifoil®
- HPTS 8-hydroxy-1,3,6-trisulfonic acid
- solution 1 referred to as solution 1 and solution 2, which are then mixed.
- solution 3 z. B. be applied by dip coating process on the carrier film.
- the reagents for preparing solution 1 may be: a.) Tetraoctylammonium hydroxide solution (TOA +; 10% solution in methanol)
- the reagents for preparing the solution 2 may be:
- Ethyl cellulose (ethoxyl content 48%) is added to and dissolved in this total solution from d) and e), the proportion of ethyl cellulose then being 0.59% by weight of the total solution of d) and e).
- solutions 1 and 2 are mixed in a ratio of 1 to 2.125.
- the optode is dried, rinsed in Milli-Q water and can be kept dried.
- all conditions known in the art may be selected.
- a particularly suitable solution is immersion of the carrier material in the solution containing fluorescent dye 1 to 3 times for about 10 seconds, with the carrier material subsequently being withdrawn from the solution at a rate of about 1 cm / second.
- the optodes used should have the visible range of the spectrum as well as the near-infrared range of the spectrum 1.) maximum transmittance, 2. maximum resolution and 3.) maximum gray-scale contrast in order to obtain the measurement as far as possible from growth parameters (see Walter et al., 2009).
- polymer layer can be kept under running water without peeling
- HPTS is particularly well-researched compared to other fluorophores (Zhu et al.
- ionophores can also be incorporated or applied in or on the optode.
- the optode is arranged so that the side coated with the fluorophore contacts the growth medium.
- a control unit This can be used to control the device components such as the illumination unit and the optical detection unit.
- An electronic data acquisition and evaluation unit (or embedded computer).
- the determined data / image data can be stored and evaluated by means of special evaluation methods and evaluation programs.
- the device in an advantageous embodiment, the device
- the optical detection unit such as, for example, B of the camera, the optics, the growth container, the lighting unit such.
- the optodes and filters from external influences such as external light, temperature fluctuations.
- An optional access door can facilitate optional maintenance.
- the invention further relates to a method for non-invasive detection of growth processes and simultaneous measurement of chemical and / or physical parameters, comprising the following method steps:
- the invention offers novel solutions and applications for several economic and scientific purposes.
- crop science should be mentioned, which open up the methods and methods of the invention, the possibility to perform screenings in the fields of plant science and crop science.
- these screenings may be used to study the potential of various plant types (e.g., species, biotypes, ecotypes, mutants, species, transgenes, hybrids, and the like) to interact with their environment in the desired manner in a chemical-physical manner.
- applications are found in the combined, simultaneous measurement of chemical-chemical interactions which emanate from living beings, tissues or cell colonies on the growth substrate or growth medium, or conversely from the growth substrate or growth medium to living beings, tissues or cell colonies, as well as the spatial and temporal effects and distributions of pollutants, xenobiotics, nutrients or other biological, chemical and physical stimuli and simultaneous imaging and image analysis of growth parameters, with the aim of: As a variety / ecotype screening, metabolic monitoring, tolerance and / or resistance screening and / or monitoring, in high spatial and temporal resolution.
- An application may, for example, also consist in the investigation of the nutrient uptake capacity of a plant, or in the investigation of a herbicidal effect on metabolic processes of the rhizosphere, or tolerance of plants to environmental toxins. From an economic point of view, the latter plants are of particular interest, in particular crops and ruderal plants / weeds.
- Another exemplary field of application thus also exists in environmental sciences and environmental toxicology.
- a conceivable application could z. This includes, for example, identifying and quantifying the influence of environmental toxins (eg heavy metals) on root-soil interactions in the rhizosphere.
- environmental toxins eg heavy metals
- the deposition and concentration of heavy metals in ecosystems is currently increasing worldwide and has been a major focus of environmental toxicological research for years.
- FIG. 1 Schematic overview of the device:
- FIG. 2 Growth container in the form of a Petri dish:
- FIG. 3 growth container in the form of a rhizotron
- FIG. 4 Growth container in the form of a hydroponic culture:
- Figure 5 Arrangement of the device with a schematic representation of
- FIG. 6 Relative transmissivity (%) of an optode according to the prior art (a) in FIG.
- FIG. 1 shows, by way of example, a schematic overall overview of the device with: a: root of a plant sprout,
- growth containers eg rhizotron, petri dish
- f light source, e.g. To excite the fluorophores,
- n filter or filter wheel
- FIG. 2 shows the side view of a cross section through a possible embodiment of the growth container as a petri dish system
- FIG. 3 shows a side view of a cross section through a possible embodiment of the growth container as a rhizotron system a: soil / substrate surface,
- FIG. 4 shows the side view of a cross section through a possible embodiment of the growth container as a hydroponic system
- FIG. 5 schematically shows the connection of the individual device components with the aid of which growth processes and dynamically changing chemical-physical properties of the growth medium can be recorded simultaneously.
- the following device components are shown:
- Control unit which controls the alternating illumination of the examination subject by individual monochromatic light sources at intervals, as well as the triggering of the camera.
- the interval between the respective single image recordings, or individual triggered light flashes, is determined by an existing predefined configuration file and by the runtime program of the control unit.
- LED 2 Light source for recording growth processes (eg 880 or 920 nm).
- LED 3 light source which corresponds to the second absorption maximum of the absorption spectrum of the fluorophore applied to the optode.
- the light source may be, for example, an LED with a narrowband Trading maximum emission. (In the case of HPTS this is for example the wavelength 450 nm).
- the light source which corresponds to the first absorption maximum of the absorption spectrum of the applied on the opto-fluorophore.
- the light source may be, for example, an LED with a narrowband emission maximum (narrow band). (In the case of HPTS this is for example the wavelength 405 nm).
- the control unit activates LED2 as a prerequisite for recordings corresponding to 25 (growth recordings in a spectral range which is not affected by the emission of the fluorophore).
- Monochromatic light of the wavelength of LED4 which impinges on the optode which corresponds to the wavelength of the first absorption maximum of the absorption spectrum of the fluorophore deposited on the optode.
- narrow-band stop filter which only reads through the light of a wavelength which corresponds to the emission maximum of the fluorophore used and applied on the optode (in HPTS for example 515 nm, when excited with light of a wavelength of the first absorption maximum).
- Narrow-band stop filter which only reads through the light of a wavelength which corresponds to the emission maximum of the fluorophore used and applied to the optode (in the case of HPTS, for example, 515 nm, when excited with light of a wavelength of the second absorption maximum).
- Broadband blocking filter for growth measurements, for example in the infrared spectrum.
- the selected spectral range of the filter can be chosen as wide as desired, but ideally includes a range in which desired structure discriminations are ensured optimally and without influencing the structures to be investigated (eg root growth).
- Sequential number n of the recording made in 25. within a sequence of images created as a file.
- Multi-TIFF file for storing
- Image evaluation software eg DISP
- a control unit (1) controls the activities of various light sources (2, 3, 4) and an optical detection unit / camera (12, 17).
- the light sources LED 3, 4 serve to excite a planar, transparent optode (6) and LED 2 serves to detect growth processes.
- LED 4 is activated while LEDs 2 and 3 remain inactive (7d & 8g).
- the monochromatic light from LED 4 (step 9) excites the planar optode (6) and the fluorescence light of the optode emitted thereby (step 14 m) passes through a narrowband notch filter (13j) and impinges on the optical detection unit / camera (12, 17).
- a two-dimen- sional recording of the fluorescence corresponding to the first absorption maximum of the fluorophore used is made (18).
- a total of 10 such recordings are created one after the other (step 38) and then provided with a sequential number (19) as an averaged image using conventional methods, and a time stamp (29) for each individual image is stored in a log file (30) ,
- the subsequent fluorescence uptake is used in accordance with the second absorption maximum Fluorophore created.
- LED 3 is activated (8h), while LEDs 4 and 2 remain inactive (7e & 5b).
- the monochromatic light from LED 3 (10) excites the planar optode (6) and the fluorescent light emitted thereby (step 14n) passes through a narrow-band notch filter (13k) and strikes the optical detection unit / camera (12, 17).
- a two-dimensional recording of the fluorescence corresponding to the second absorption maximum of the fluorophore used is produced (22).
- For denoising (38) a total of 10 such recordings are created one after the other (38) and then provided with a sequential number (23) as an averaged image by conventional methods, and a time stamp (29) for each individual image is stored in a log file (30) ,
- the subsequent recording of the growth processes and structures is created.
- the LED 2 is activated (7f), while LEDs 3 & 4 remain inactive (8i & 5c).
- the monochromatic light from LED 2 passes through the transparent planar optode (6) and strikes the sample behind it.
- the light (14o) subsequently reflected by the sample passes through a broadband blocking filter (131) and strikes the optical detection unit / camera (12, 17).
- a two-dimensional recording of the growth processes and structures is made (25).
- For denoising (38) a total of ten such recordings are created one after the other (38) and then provided with a sequential number (26) as an averaged image by conventional methods, and a time stamp (29) for each individual image is stored in a log file (30).
- the image processing of the fluorescence images (40) begins with the formation of a ratiometric image from 18. and 22. (41). This image is then offset with a dark image (39) to filter out the black noise.
- the resulting corrected image (43) is then subjected to pixel-by-pixel correction according to relevant literature (44).
- the resulting completely corrected image (45) is stored in a so-called multi-TIFF format (33q) and can be read from there and further processed.
- the growth or structural images (25) are stored directly in a so-called multi-TIFF format (33p). This image is then further processed during the further image analysis (36) with relevant software (37).
- FIG. 6 and some exemplary measured values in Table 3 show the relative transmissivity in% of an optode according to the prior art (a) in comparison with the new optode (b) according to the invention as a function of the wavelength.
- a maximum possible light transmittance and thus transparency in the entire spectrum would be given at a 100% transmission of light (transmissivity) at all wavelengths.
- the optode according to the invention has a transmission of more than 90% from a wavelength of about 280 nm, while the optode of the prior art at 280 nm only has a relative transmission of about 14.66% up to a maximum of 62% at 852 nm.
- FIG. 7 shows relative absorption spectra in% and, for example, a relative emission spectrum in% of the optode according to the invention, which contains the fluorophore HPTS, at different pH values.
- Ordinate X wavelength (nm); Abscissa Y: Relative absorption or emission in% based on the amount of light irradiated.
- the optode has two absorption maxima in the range of 1.) 405 nm and a second absorption maximum at 450 nm. Furthermore, it has an emission maximum at 515 nm. This shows that the optode according to the invention is suitable for ratiometric fluorescence measurements, as already described above. Two excitation wavelengths of 405 nm and 450 nm can be used and an emission wavelength of 515 nm.
- Table 4 Relative gray value contrast (% of gray values) in% relative to the reference (without optode) in black and white photographic photographs of Times New Roman point dots with a dot size of 128 exposed to 880 nm wavelength light ,
- Table 4 shows the relative gray value contrast (gray value difference) with respect to the reference (without optode) in photographic black-and-white photographs of Times New Roman point dots with a dot size of 128 (See also Table 8) with one exposure Light of the wavelength of 880 nm. Location in the middle of the rightmost black dot (font size 128) in the respective photographic image using a narrow-band filter, which transmits only light with the wavelength 880 nm. A small value shows a small gray value contrast between white and black areas in the photograph. Desirable is a value as close as possible to the value without optode (100%).
- the optode according to the invention clearly has the desired improved property compared with the optical device of the prior art.
- Table 5 Relative gray level contrast (% of gray values) in% relative to the reference (without optode) in black and white photographic photographs of Times New Roman point dots with a 128 spot size exposed to visible light
- Table 5 shows the relative gray value contrast (gray value difference) with respect to the reference (without optode) in photographic black-and-white photographs of points of the Times New Roman type with a dot size of 128. (See also Table 8) with exposure to visible light. Place of measurement: in the middle of the rightmost black dot (font size 128) as well as in the dot between the two dots with the font size 128 and 64 in the respective photographic shot without the use of a filter. A small value shows a small gray value contrast between white and black areas in the photograph. Desirable is a value as close as possible to the value without optode (100%).
- the optode according to the invention clearly has the desired improved property compared to the optode of the prior art.
- Table 6 shows the relative diameter in percent of the rightmost point in the photographic image (font size 128) when exposed to light of wavelength 880 nm as a measure of the resolution. A value close to 100% indicates a slight blurring and thus high selectivity. Desirable is a value as close as possible to the value without optode.
- the optode according to the invention clearly has the desired improved property over the prior art optode.
- Table 7 Relative diameter as a percentage of the rightmost point in the photographic image (font size 128) when exposed to visible light as a measure of resolving power
- Table 7 shows the Relative Diameter as a percentage of the rightmost point in the photographic image (font size 128) when exposed to visible light as a measure of resolving power. A value close to 100% indicates a slight indistinctness and thus high selectivity. Desirable is a value as close as possible to the value without optode.
- Plant seeds of the species to be tested are externally sterilized within a sterile bank by first placing the seeds in 70% ethanol, in aqueous solution, for three minutes and then in 0.5% sodium hypochlorite (including one drop of Tween to 10 ml ), in aqueous solution, for ten minutes and then rinsed thoroughly with autoclave water.
- the medium is poured into Petri dishes. After curing of the agarose, the optodes are placed on the agarose gel so that the side coated with the fluorophore rests on the agarose as far as possible without air inclusions. Petri dishes are sealed with micropore tape and parafilm.
- the sterilized seeds are sown within a sterile bench in perforations on the narrow side of the Petri dish and sealed the Petri dish with parafilm.
- the plants are grown in suitable climatic conditions, the Petri dishes with the side which rests the optode to z. B. 45 ° is tilted inwards (see Fig. 2).
- the optode used in this example is comparable in terms of transparency with glass or plastic vision, and allows an evaluation, for example with the software GROWSCREEN-Root or the software GROW Map-Root. Other evaluation methods are also possible.
- the rhizotrons are z. B. filled with conventional Anzuchterde. Then the lens is removed and cleaned from adhering soil. On the bottom surface exposed in the rhizotron, the planar optode is placed on the bottom with the side coated with the fluorophore. Then the lens is placed on the optode and re-attached. Alternatively, the planar optode can also be attached to the appropriate location of the lens with, for example, silicone grease before the soil is poured into the rhizotron. Thus, it would be avoided that when opening an already filled rhizotrons the soil structure is changed.
- the rhizotrons are at an angle of z. B. stored for 45 ° in the direction of the lens permanently.
- the seeds of the plants to be examined are sown in the rhizotron and cultured under suitable, species-specific culturing conditions (see FIG.
- An optical detection unit such as a CCD camera or the like is locked to the viewing window of the rhizotron at a defined distance and angle to create reproducible recordings.
- the illumination unit usually monochromatic LEDs, are also locked analogously to the optical detection unit.
- Illumination and detection units are controlled by a PC in order to record at defined intervals recordings of the root system and the rhizosphere pH dynamics.
- the detection of the growth of the root system for example, using the software ROOTFIND and is usually based on images of the optical detection unit in bitmap format.
- An upstream conversion of the images in black / white format etc. is possible, if the downstream evaluation software requires it.
- the length and the number of visible roots are automatically recorded and stored in separate data files.
- the calibration of the images is done by comparing the counted root pixels with the number of pixels of objects whose length and thickness are known, and which are also captured in the image (eg capillaries, etc.).
- the recording of the rhizosphere pH dynamics is achieved by comparing the generated images (eg TIFF or RAW format) with previously created calibration data.
- Conventional and commercially available evaluation software can be provided by the "Image processing toolbox" from Matlab or Photoshop The error correction of the generated images can be done analogously to the description by Strömberg & Hulth (2005):
- the black noise is subtracted from the images of each wavelength.
- the pH-Optode can e.g. calibrated as follows (modifications are also possible):
- ⁇ 1 and ⁇ 2 describe the asymptotic Minimum and maximum of the sigmoid function
- ⁇ 3 is the apparent pKa4 of HPTS (ie, the inflection point of the sigmoid function)
- ⁇ 4 is a constant describing the slope of the function between ⁇ 1 and ⁇ 2.
- “dual excitation” (ex) and “dual emis- sion” (em) properties of HPTS are used (eg, Fl, ex / em: 405 / 440nm and F2, ex / em: 465/510 um ).
- the so-called “time correlated pixel-by-pixel” calibration can be applied (also described by Strömberg and Hulth 2005).
- the optode to be calibrated is immersed in a vessel containing the respective buffer solution and the ratiometric response is detected through a viewing window or through the glass of the beaker, etc. with the detection unit.
- the result of the calibration can be registered for each pixel individually or as a sum response and processed further later.
- the root images and pH images generated during the experiment can then be overlaid with appropriate image manipulation programs (e.g., Corel Draw, Photoshop, ImageJ, etc.) to elucidate correlations between pH and root architecture.
- image manipulation programs e.g., Corel Draw, Photoshop, ImageJ, etc.
- Transmissivity of the optode input intensity of the light - absorption
- the optodes were placed in a Petri dish (120 * 120 * 17 mm, Greiner Petri dishes with cams) filled with 20 ml deionized water (Milli-Q, Millipore Corporation). square, Greiner Bio-One item No .: 688102), and the bottom of the Petri dish turned upside down so that the optodes were planar fixed.
- the lid of the Petri dish was then set to a standard consisting of a high resolution print on printer paper, in which different symbols (dots) of the font "Times New Roman" of the software Microsoft Word in the font sizes 1, 2, 4, 8, 16 , 32, 64 and 128.
- a standard objective lens 25 mm, Cosmicar / Pentax, The Imaging Source, Bremen, Germany
- a filter or an infrared pass-through filter 880 nm, Edmund Optics, Düsseldorf, Germany.
- a constant illumination was carried out either by means of a halogen lamp (for measuring the optical properties of the optodes in visible light at about 400-800 nm) or by means of infrared LEDs (880 nm, Conrad Electronics, Hirschau, Germany).
- Photographs were taken using the root leaf aquisition software (Schmundt et al., 1998) and stored in multi-TIFF format, after which the photographs were taken using the IrfanView software (http://www.irfanview.com /) and measured with the tools of the IrfanView software.
- the measuring tool "Measure tool” of the IrfanView software was used to measure the resolving power.
- the start and end points of the measurement of the diameters of the point of the font size 128 were determined by the respective change of the gray value of more than 3 gray scale levels compared to the background Diameter was measured.
- the gray value was measured with the IrfanView software, whereby the measurement points were selected either in the middle of the dot of the font size 128 or in the middle between the dots of the font size of 128 and 64. Then the difference of the gray values was calculated.
- one strip of the optode was planarly placed in a crystal glass cuvette and the cuvette filled with 1 ml of deionized water (Milli-Q, Millipore Corporation).
- a glass cuvette filled with 1 ml of deionized water (Milli-Q, Millipore Corporation) was used.
- the measurement was carried out using a two-beam spectrophotometer (UVIKON xl, Bio-Tek Instruments).
- a photometric reference measurement can be set 100%.
- the curve corresponding to this measurement would have a value of 100% over the entire range of the spectrum in the representation chosen in FIG. 6 a) / b) (relative transmission relative to the reference).
- Table 8 shows the photographic images determined in the previously described measuring methods. The two optodes to be compared and the blank measurement with the same measurement setup can be compared to obtain a first impression.
- Table 8 Photographic black-and-white photographs of Times New Roman dots at various dot sizes from dot size 1 to dot size 128 a) the blank reading (reference; without optode) with exposure to visible
- the optode according to the invention achieves transmissivity values which lie above 90% in the spectrum (relevant for the purposes of the invention) between 300 and 900 nm. In the near-infrared region of the spectrum (700 to 900 nm), the new optode achieves a transmissivity of 100%. The area of near-infrared light is of particular importance for the purposes of the invention.
- transmissivity for the purposes of the invention is not only relevant, but also the parameters resolving power and gray value contrast are important for achieving a maximum image quality, these parameters were determined for a commercially available pH optode and the new optode.
- the obtained selectivity (resolving power) of the new optode was little and not significantly worse using both visible light and near-infrared light (880 nm) than the resolution achieved when no optode was placed in the beam path (Fig. Petri dish) (4% degradation on exposure to light in the visible region of the spectrum; 1% degradation on exposure to light of wavelength 880 nm).
- the commercially available optode deteriorated resolving power as compared with the reference (measurement without optode in the visual axis) by using light of the visible spectrum by 27%, and by exposure to near-infrared light of wavelength 880 nm by 24%.
- the deterioration of the gray scale contrast compared to the achieved gray value contrast without optode in the beam path is 26% when using visible light in the new opto and 6% when using near-infrared light (880 nm).
- the gray level contrast degradation is 93% using visible light, and 89% using near infrared light (880 nm).
- the new optode in the context of the invention has significantly and significantly better optical properties than the commercially available optode.
- the new optode is proven to be suitable for use in imaging and image analysis techniques such as DISP, WinRHIZO and GROWSCREEN root.
- Image analysis is the systematic analysis of the image content by means of visual image interpretation.
- Imaging method An imaging method generates an image from measured variables of a real object, wherein the measured variable or information derived therefrom is visualized spatially resolved and coded via brightness values or colors. (Www.wikipedia.org).
- Image processing refers to all processes by which images are processed and altered, i. the preparation and analysis of image or raster data. Designates a set of digital methods used to create, analyze, enhance, interpret, or display image data or raster data.
- Grayscale is the number associated with a pixel, e.g. in raster data or in digital image processing. Depending on the number of bits which a gray value can take, a distinction is made between binary image (1 bit, states 0 and 1), gray value image (8 bits, states between 0 and 255) and color image (3 * 8 bits each with states between 0 and 255). These values are associated with entries of a color table (LUT) for display on a screen, i. the values are interpreted as indices of a color table. (Geoinformatik-Service of the University of Rostock, 2008, Chair of Geodesy and Geoinformatics (GG) AT Vietnamese Rostock, www.geoinformatik.uni-rostock.de).
- the photometric contrast characterizes the brightness difference between two luminous surfaces: (Mütze et al., 1972).
- Monomers are low molecular weight, reactive molecules that can combine to molecular chains or networks, to unbranched or branched polymers (wikipedia). l l. optode:
- angular second Measurable in arc seconds (synonyms angular second, unit of measurement of the angle). 60 arc-seconds equals one minute of arc, 60 arc-minutes equals one degree. The resolution of 1 '(an angular minute) corresponds to a spatial resolution of about 1.5 mm at 5 m distance. The smaller the angular acuity, the better the visual acuity (Mütze et al., 1972).
- Plant Soil 126 155-160
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Botany (AREA)
- Plasma & Fusion (AREA)
- Optics & Photonics (AREA)
- Wood Science & Technology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Ecology (AREA)
- Forests & Forestry (AREA)
- Environmental Sciences (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102011118619A DE102011118619A1 (de) | 2011-11-16 | 2011-11-16 | Vorrichtung und Verfahren zur Erfassung von Wachstumsprozessen und simultanen Messung von chemisch-physikalischen Parametern |
PCT/DE2012/001023 WO2013071902A1 (de) | 2011-11-16 | 2012-10-24 | Vorrichtung und verfahren zur nicht-invasiven erfassung von wachstumsprozessen und simultanen messung von chemisch-physikalischen parametern |
Publications (1)
Publication Number | Publication Date |
---|---|
EP2780695A1 true EP2780695A1 (de) | 2014-09-24 |
Family
ID=47323794
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP12798135.5A Withdrawn EP2780695A1 (de) | 2011-11-16 | 2012-10-24 | Vorrichtung und verfahren zur nicht-invasiven erfassung von wachstumsprozessen und simultanen messung von chemisch-physikalischen parametern |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP2780695A1 (de) |
DE (1) | DE102011118619A1 (de) |
WO (1) | WO2013071902A1 (de) |
Families Citing this family (19)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE102012105291A1 (de) * | 2012-06-18 | 2013-12-19 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Verfahren, Vorrichtung und tragbares Messgerät zur Detektion biologischer Moleküle in Schichten eines Schichtsystems |
DE102013022032A1 (de) * | 2013-12-19 | 2015-06-25 | Technische Universität Ilmenau | Verfahren zum Nachweis von Fremdstoffen oder Degradationsprodukten in verkapselten Systemen sowie dessen Verwendung |
DE102014000816A1 (de) * | 2014-01-22 | 2015-07-23 | Technische Universität Bergakademie Freiberg | Kammersystem für die Analyse von Gasflüssen von Ökosystemen |
DE102014212657B4 (de) * | 2014-06-30 | 2016-03-10 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | System und Verfahren zur bedarfsgerechten Zuführung von Beleuchtungsenergie an Pflanzen |
DE102016204541B4 (de) | 2016-03-18 | 2019-11-14 | Technische Universität Dresden | Verfahren und Vorrichtung zur zeitlichen und lokal aufgelösten Detektion von Stoffkonzentration in Fluiden |
DE102017109968A1 (de) | 2016-05-10 | 2017-11-16 | Gesellschaft zur Förderung von Medizin-, Bio- und Umwelttechnologien e.V. | Gerät zur Kultivierung von phototrophen Organismen |
CN109005964B (zh) * | 2018-05-21 | 2020-06-12 | 华南农业大学 | 一种用于研究植物根系与环境相互作用的无菌滤纸栽培方法 |
CN109429768B (zh) * | 2018-12-21 | 2024-05-10 | 福建农林大学 | 一种具有特色光照装置的小型植物根系培养及探测装置 |
DE102019208833A1 (de) * | 2019-06-18 | 2020-12-24 | Robert Bosch Gmbh | Verfahren zum Kennzeichnen von Pflanzenbildinformationen, insbesondere für landwirtschaftliche Zwecke |
DE102019129924A1 (de) * | 2019-08-21 | 2021-02-25 | Endress+Hauser Conducta Gmbh+Co. Kg | Optochemischer Sensor, Sensorkappe, Verwendung des optochemischen Sensors und Verfahren zur Herstellung einer analyt-sensitiven Schicht des optochemischen Sensors |
DE102021102505A1 (de) | 2020-12-21 | 2022-06-23 | Endress+Hauser Conducta Gmbh+Co. Kg | Optochemischer Sensor sowie Verfahren zum Messen von lumineszierenden Analyten in einem Messmedium |
RU2765842C1 (ru) * | 2021-06-07 | 2022-02-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Петрозаводский государственный университет" | Приспособление для комплексного отслеживания сезонных изменений растений |
RU206755U1 (ru) * | 2021-06-07 | 2021-09-27 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Петрозаводский государственный университет" | Устройство для фиксации сезонного роста растений |
RU2765841C1 (ru) * | 2021-06-07 | 2022-02-03 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Петрозаводский государственный университет" | Способ комплексного отслеживания сезонных изменений растений |
RU206519U1 (ru) * | 2021-06-07 | 2021-09-14 | Федеральное государственное бюджетное образовательное учреждение высшего образования "Петрозаводский государственный университет" | Устройство для измерений сезонного роста растений |
BE1030100B1 (nl) * | 2021-12-24 | 2023-07-26 | Smo Bvba | Rhizotron |
CN114486824B (zh) * | 2021-12-27 | 2024-04-09 | 南京大学 | 一种模拟pH变化的面向土壤系统的高分辨技术耦合的重金属原位表征系统 |
DE102022107746A1 (de) | 2022-03-31 | 2023-10-05 | Lytegate GmbH | Verfahren und Messanordnung zur Untersuchung organischen Materials |
CN115436367B (zh) * | 2022-09-09 | 2024-05-03 | 中国农业大学 | 一种基于外置可变光源的根系土壤原位成像装置及方法 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3213183A1 (de) * | 1982-04-08 | 1983-10-20 | Max Planck Gesellschaft zur Förderung der Wissenschaften e.V., 3400 Göttingen | Anordnung zur optischen messung physikalischer groessen |
US5372784A (en) * | 1988-08-31 | 1994-12-13 | Baxter Diagnostics Inc. | Measurement of bacterial CO2 production in an isolated fluorophore by monitoring an absorbance regulated change of fluorescence |
AT391371B (de) * | 1989-05-12 | 1990-09-25 | Avl Ag | Verfahren und vorrichtung zur feststellung biologischer aktivitaeten in einer probe |
US5175016A (en) * | 1990-03-20 | 1992-12-29 | Minnesota Mining And Manufacturing Company | Method for making gas sensing element |
AT402452B (de) * | 1994-09-14 | 1997-05-26 | Avl Verbrennungskraft Messtech | Planarer sensor zum erfassen eines chemischen parameters einer probe |
US20060105174A1 (en) * | 2004-10-25 | 2006-05-18 | The Research Foundation Of State University Of New York | Optical pH sensor |
-
2011
- 2011-11-16 DE DE102011118619A patent/DE102011118619A1/de not_active Withdrawn
-
2012
- 2012-10-24 WO PCT/DE2012/001023 patent/WO2013071902A1/de active Application Filing
- 2012-10-24 EP EP12798135.5A patent/EP2780695A1/de not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO2013071902A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2013071902A1 (de) | 2013-05-23 |
DE102011118619A1 (de) | 2013-05-16 |
DE102011118619A8 (de) | 2013-10-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP2780695A1 (de) | Vorrichtung und verfahren zur nicht-invasiven erfassung von wachstumsprozessen und simultanen messung von chemisch-physikalischen parametern | |
DE19903506C2 (de) | Verfahren, Gefäß und Vorrichtung zur Überwachung der Stoffwechselaktivität von Zellkulturen in flüssigen Medien | |
Tambutté et al. | Morphological plasticity of the coral skeleton under CO2-driven seawater acidification | |
Kühl et al. | Functional and structural imaging of phototrophic microbial communities and symbioses | |
Hoefer et al. | Integrating chemical imaging of cationic trace metal solutes and pH into a single hydrogel layer | |
WO2003064990A2 (de) | Deckelelement | |
EP0515623A1 (de) | Verfahren und vorrichtung zur kontinuierlichen und reversiblen messung der konzentration einer chemischen spezies | |
EP2594601B1 (de) | Optode | |
EP1397672A1 (de) | Sauerstoffsensoren auf mikrotiterplatte | |
Han et al. | High-resolution imaging of pH in alkaline sediments and water based on a new rapid response fluorescent planar optode | |
JP4699214B2 (ja) | 有害物質の評価方法、及び有害物質の評価用キット | |
DE112011103545B4 (de) | Verfahren und Vorrichtung zur Diagnose von Pflanzenwachstumsbedingungen | |
DE102020109901A1 (de) | Optochemischer Sensor und Verfahren zur Messwertkorrektur | |
EP2805151A1 (de) | Optode zur bestimmung von chemischen parametern | |
WO2003098174A1 (de) | Verfahren und vorrichtung zur spektral differenzierenden, bildgebenden messung von fluoreszenzlicht | |
DE102011080696B4 (de) | Schnelldrehender Klinostat sowie Verfahren zur Untersuchung von Organismen | |
EP0091046B1 (de) | Anordnung zur Messung physikalischer Grössen | |
DE102013021097A1 (de) | Kalibriernormal für eine Vorrichtung zur bildlichen Darstellung biologischen Materials | |
DE102009036562B4 (de) | Verfahren zur Bestimmung der Wasserqualität eines Gewässers | |
WO2016066156A2 (de) | Mobile photometrische messvorrichtung und verfahren zur mobilen photometrischen messung an mikrotitierplatten | |
DE102016208967B4 (de) | Photometer mit quantitativer Volumenerfassung | |
DE102008026803A1 (de) | Analytisches Vorrichtungssystem und Verfahren zur Bestimmung von Substanzen in Flüssigkeiten | |
DE202008007512U1 (de) | Analytisches Vorrichtungssystem zur Bestimmung von Substanzen in Flüssigkeiten | |
Kvaternyuk | Multispectral control of pesticide concentrations in aquatic environments using bioindication on phytoplankton | |
Sanders et al. | An Introduction to Algae Measurements Using in vivo Fluorescence |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20140320 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: PFEIFER, JOHANNES Inventor name: NAGEL, KERSTIN Inventor name: SCHARR, HANNO Inventor name: WALTER, ACHIM Inventor name: BLOSSFELD, STEPHAN Inventor name: SCHURR, ULRICH Inventor name: MIELEWCZIK, MICHAEL |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN |
|
18W | Application withdrawn |
Effective date: 20141217 |