KR20090008689A - Method of fluorometric determination of photoautotrophic organisms photosynthesis parameters, device for its realization and measuring chamber - Google Patents

Abstract

A method of fluorometric determination of photoautotrophic organisms photosynthesis parameters, a device and a measuring chamber for embodying the method are provided to obtain data for short-term prediction of dynamic mechanics of phytoplankton in phytocoenoses. A method of fluorometric determination of photoautotrophic organisms photosynthesis parameters comprises a step of exposing a sample of analysis medium to excitation light, and a step of additionally evaluating the fluorescence intensity determining the photosynthesis parameter of an object. A device for embodying the method comprises a measuring chamber, one or more light sources which are optically connected with the measuring chamber, a control block which is connected with a computer, a current stabilizer whose output part is connected with the electric port of the light source and port is connected with the control block, and a natural irradiation sensor which is connected with the current stabilizer though the control block.

Description

METHODO OF FLUOROMETRIC DETERMINATION OF PHOTOAUTOTROPHIC ORGANISMS PHOTOSYNTHESIS PARAMETERS, DEVICE FOR ITS REALIZATION AND MEASURING CHAMBER}

TECHNICAL FIELD The present invention relates to biology, and can be used for environmental protection techniques, in particular for grammology and marine hydrology, and for assessing algal concentrations and aqueous environmental conditions for their photosynthesis, Continuous analysis of aqueous environments using fluorimeters can be used in any other field of science, engineering and environmental protection.

It is known that the concentration and photosynthetic activity of photosynthetic biological cells are greatly affected by various ecological factors and human pollution effects on land and aqueous environment. Their change also changes all other chains in the ecosystem. For this reason, photosynthetic apparatus (PSA) has been found to be the most important for measuring plant conditions. Thus, regulating the photosynthetic characteristics of phytoplankton is one way to comprehensively assess the aqueous environment.

Currently adopted models of primary photosynthetic reactions include photo systems PS1 and PS2. PS2 uses oxygen release and protons to oxidize water and regenerate primary and secondary quinone acceptors (Qa and Qb). FS1 transfers electrons from a pool of plastoquinones (PQs) to CO ₂ , the final acceptor of electrons.

The reaction center (RC) of PS2 consists of a special molecule of chlorophyll (P680), the primary electron donor of the quinone receptor (Qa) upon excitation. Photon energy absorbed in PS2 can be converted into energy of separation charge (P680 + Qa-), which is used for further photosynthesis reaction, lost by fluorescence quantum, or forming heat Is divergent. These are three steps characterized by the rate constants Kph, Kf and Kd respectively.

The initial state where P680 is regenerated and Qa is oxidized is called the open state. The state of forming itself after charge separation in the initial pair (P680 + Qa-) is called the closed state of RC. In this state, the new excitation can be used after returning to the initial open state due to the reduction of P680 + from the secondary donor and the oxidation of the initial receptor by the secondary electron acceptor.

In the open state of RC, the efficiency of the use of excitation energy in photosynthesis is high, the potential for energy loss is minimal, and the quantum yield of fluorescence equal to the Kf / (Kph + Kf + Kd) ratio is at least about 2%. . If excitation light acts continuously (in this case, the RC closed condition does not occur), the fluorescence intensity is the F0 value. The low yield of chlorophyll fluorescence by the use of light energy in the first photosynthetic reaction is explained by the photochemical quenching of chlorophyll. When RC is closed, photochemical charge separation becomes impossible and the fluorescence quantum yield increases to Kf / (Kf + Kd), which corresponds to the intensity value (Fm) and is about 5%. The difference between the maximum and minimum values of fluorescence intensity (Fv = Fm-F0 _ variable fluorescence) is proportional to the light energy used for the photochemical photosynthetic reaction when the reaction center PS2 is opened. The ratio of variable fluorescence intensity to maximum fluorescence intensity (Fv / Fm) (relatively variable fluorescence) is equal to the efficiency of RC light energy use which determines the light use efficiency in the path of photosynthesis. Similar to the coefficient of efficiency, the dimensionless characteristic of photosynthesis is universal and does not depend on the specific characteristics of a particular species of organism.

If the excitation light affects plants adapted to darkness, a change in the chlorophyll fluorescence intensity over time has been detected (characteristic induction curve of fluorescence). First, the fluorescence intensity increases from the open RC to the level corresponding to the fluorescence quantum yield (F0). Next, when the light activity becomes sufficiently strong, the fluorescence yield reaches Fm. An additional step of chlorophyll fluorescence induction eventually reduces fluorescence yield. This leads to an increase in photochemical fluorescence disappearance in the acceleration of electron movement, a corresponding reduction in the reduction of quinone receptor (PS2), and also development of a non-photochemical extinction step associated with the formation of gradients of protons on the photosynthetic membrane. Is explained by.

In this way, the measurement of different chlorophyll fluorescence parameters offers the possibility of obtaining information about the photosynthesis device.

So, the following fluorescence parameter measurement

Chlorophyll fluorescence intensity values under conditions of poor illumination after long adaptation of the assay to excitation by test light, without closing Fo-RC and changing the photosynthetic device state.

Fm-Chlorophyll fluorescence intensity value under conditions of low illumination after long adaptation of the analyte to the excitation by the test light, which does not close the RC forming the stationary level.

Ft-Chlorophyll fluorescence intensity value in long time continuous lighting conditions.

F'm-Chlorophyll fluorescence intensity value at conditions of its excitation by continuous illumination and light, which completely closes the RC of the photosynthetic device.

These parameters of photosynthetic organisms,

1.Maximum quantum yield of charge separation in PS2 as ratio Fv / Fm = (Fm-Fo) / Fm (this parameter is proportional to the share of active RCPS2 in the dark and corresponds to the potential efficiency of the photosynthetic process),

2. background light-photochemical disappearance at qP = (F'm-Ft) / (F'm-Fo '),

3. background light-non-photochemical disappearance at NPQ = (Fm / F'm) -1,

4. Quantum yield of photochemical conversion of light energy absorbed in PS2 as ratio Y = (Fm'-Ft) / Fm 'reflecting the parameters of acyclic electron transfer during photosynthesis,

5. The absolute value of Fo as the index of abundance of phytoplankton in water,

6. The size of the pool of quinine and light antenna, which can be calculated by the kinetics of chlorophyll fluorescence intensity growth from Qa to Qb, and from Fo to Fm.

Let the calculation

The fluorescence method of the physiological state of plant organisms allows real-time information on the most objective, non-destructive, and PSA objects in the invasive habitat.

Single beam fluorescence using object illumination by constant light through a short wavelength (typically blue) filter with the additional registration of fluorescence intensity induced changes through a corssed red filter that protects the fluorescence detector from excitation light. Registration methods exist (Matorin DN, Venedityov PS microalgal cultures and chlorophyll fluorescence in natural populations of phytoplankton // M .: Bnjgi nauki tekhniki, ARISTI. Series Biophysics. 1990. V. 40. P. 49-100). Since the fluorescence yield is not large, electron multiplication phototubes are used as detectors. Using one and the same ray to initiate fluorescence excitation and photosynthesis process in which the quantum yield of fluorescence and the state of the photosynthetic device do not change does not allow clear interpretation of these measurement results.

Dual fluorescence registration methods exist (Lyadskiy VV, Gorbunov MA, Venediktov PS Pulse Fluorometer to study the primary photochemical process of green plants // High School Scientific Report. Biology. 1987. V.11.P. 31 -36.) In this method, a change in the PSA state is made by excitation of constant light flux and fluorescence to perform FSA change observation with weakly controlled light and to evaluate the FSA state by the change in quantum fluorescence yield.

In order to register the regulated fluorescence signal in this method, a resonance amplifier is used that does not pass a constant fluorescence signal excited by the acting light.

This method has a rather narrow dynamic range of measurement and intensity of working light. Thus, the increase in saturated light requires a somewhat intense sounding light to make the variable fluorescence signal larger than the noise of the constant fluorescence signal induced by the constant working light. However, in this case, the sounding light can cause significant electron flux, which will result in a mistake in the F0 measurement. In addition, well-known methods presuppose the use of inhibitor diurone, which makes the measurement difficult and practically impractical under conditions of immersion and water.

There is a method of fluorescence registration by control of the detection light (Ounis A., Evain S., Flexas J., Tosti S., Moya I. Adaptation of PAM-fluorometer for remote sensing of chlorophyll fluorescence // Photosynth.Res 2001. V.68.No.2.P. 113-120), in this method, the sensing light source is a very short (650 nm) light of red (650 nm) light having a high on-off time (the interval between impulses is about 1000 mcsec). 1mcsec) LED to generate an impulse. The impulse fluorescence signal is detected by the LED and augmented by a pulse amplifier with a synchronous detector.

This method increases the ratio of working to sensed light up to 106 and allows to register the level of fluorescence F0 over a wide fluorescence intensity range of working light. At the same time, this method proposes the use of constant light as an activator to induce RC multiple turnover. This makes the interpretation of the data difficult at rectifying conditions because the fluorescence yield is affected by many factors (which make it difficult to define each contribution, resulting in the possibility of multiple valuation of the results).

Photoautotrophic organisms for chlorophyll fluorescence measurements based on a pump-and-probe method that illuminates a sample with continuous flash ("light sensing-powerful saturation-light sensing") There is a fluorescence definition of the photosynthetic parameter of US Pat. No. 4,942,303, MPK G 01 N 21/64, 1990. Powerful flash after pumping generates a single turnover of photosynthetic RC, and photosensing flashes supplied at different times after pumping are used to determine the kinetics of chlorophyll quantum fluorescence yield while RC is moved from closed to open. .

Measuring fluorescence yield before and after saturated flash in the presence of continuously acting light of known intensity offers the possibility of measuring photosynthetic rates. The pump and probe method allows the range expansion of the PSA parameters to be limited.

At the same time, the absorption cross-section and the velocity measurement of the electron flow from PSA2 to PSA1 are repeated 30 times (the sequence of flash is “sense-pump-sensing”), and the intensity of the flash after pumping is saturated at zero. The saturating level changes, or the delay time between pumping and the second sense flash varies from 80 mcsec to 300 mcsec. The realization of this experimental protocol requires 5 to 10 minutes of fluorometer operation to perform the measurement.

This is, for example, the application of this method in obtaining a phytoplankton fluorescence vertical profile in the ocean where this protocol has to be implemented on each meter of water polls and the indicated time becomes unacceptably large. Limit the scope. The need to keep the intensity of the sensing flash below 1% of the PS2 saturation level results in a low ratio of signal / noise, especially when the concentration of chlorophyll is low, which makes it impossible to obtain the required measurement sensitivity.

Known pump and probe fluorometers implement the use of two separate excitation channels (two flashes), which make fabrication difficult and increase the cost of the fluorometer. In particular, in marine phytoplankton studies, the full implementation of the experimental protocol according to the method described above requires a significant amount of electrical energy. This requirement limits the possibility of long independent (above rich buoy) measurements using electric batteries as the power source for the fluorometer.

The work defines a complex of variable parameters of the photosynthetic device and uses this or other methods of excitation and registration of fluorescence.

One of the results of the known technical solutions for the described invention is the method of fluorescence determination of photosynthetic parameters of photoindependent nutritional organisms (US Pat. No. 5,426,306, MKP G 01 N 21/64, 1995). And the additional measurement of the fluorescence intensity to determine the light effect on the assay medium sample and the photosynthesis parameter of the test specimen by an excitation light impulse having sufficient energy to stimulate chlorophyll excitation in the sample.

In this method, the fast repetition rate (FRR) flashes at a controlled energy rate, and high frequency is used as the excitation light impulse to saturate the PS2 constantly with plankton. This method makes it possible to obtain data on the RC absorption cross section, data on the function of phototransmitter energy transfer between PS2, photochemical and non-photochemical fluorescence extinction, and on the kinetics of electron transfer in the acceptor plane of PS2. However, the possibility of the above-described method of fluorescence determination of the activity and state of PSA photosynthetic organisms does not presuppose multiple measurements of fluorescence intensity (F 0) for a single sample resulting in serious error in the measurement of key parameters. In this method, individual samples are used to determine each parameter of the PSA status, which can also cause large mistakes. This method does not determine the different types of extinction, and then does not calculate the light curve of electron transfer and the confinement of the optimal photosynthetic region. In this way it is not possible to define the stability of the antioxidant system.

In addition, this measurement method does not determine the contribution of individual species in the production characteristics of the phytoplankton family. In addition, it is impossible to apply mathematical modeling techniques to this method because each impulse must be modeled separately, and this becomes very difficult if the series contains 100-200 impulses. For this reason, using mathematical techniques in this method to determine (calculate) the rate constant of the electron transfer reaction in the photosynthesis process provides only approximate values or is not at all possible.

Of all the known devices for determining photosynthetic biological states, the closest to the invention described is a device for realizing the principle according to the invention described above, which is a measuring chamber, a measurement designed to have the possibility of inducing fluorescence of a sample. It consists of a light source, an apparatus for measuring sample fluorescence, a control panel connected to a computer, a measuring light source, and a sample fluorescence measuring apparatus.

Known fluorometer designs allow the continuous measurement of photosynthetic parameters and the continuous measurement of photosynthetic rate in the dark as well as in daylight. However, the functional possibilities of this device are limited, and the necessity of defining the PSA of the organism necessary to calculate and form the light curve of electron transfer or to create a PSA mathematical model that can determine the signals and parameters of the PSA that cannot be measured directly. Do not allow a number of parameters to be measured simultaneously. Since small fluorescent signals in known devices are measured against a background of strong blocking, it is impossible to measure in an open chamber of a water surface layer with strong light.

In addition, the measurement chambers of all devices known for fluorescence measurements do not provide signal suppression from the photoreceiver due to the fact that a significant share of scattered light falls into the excitation light which hits the walls of the measurement chamber and other components. . At the same time, the device must be very sensitive if phytoplankton fluorescence parameters characterized by low content of phytoplankton cells are measured in the natural environment.

All of the above factors limit the applicability of both known methods and devices.

In this way, the technical results obtained from the realization of the described invention allow for the study of the production characteristics of phytoplankton and the abundance of phytoplankton and the determination of the contribution of individual species in their functional state at the same time to a single sample. Fluorescence evaluation of the activity of photosynthetic devices of photoindependent organisms at the expense of defining multiple test conduction with high temporary resolution for, and also defining its production characteristics in real time in the natural environment. It is aimed at increasing objectivity and accuracy in fluorometric assessment.

The technical results also include the expansion of the scope of application at the expense of the realization of the possibility that electron transfer calculates light curves, applies mathematical modeling methods and defines additional parameters that define the photosynthetic process in this way.

The technical consequences of realizing the described invention also include the expansion of the functionality and operational possibilities of the device for determining the state of photosynthetic organisms.

The technical results described above include the steps of exposing a sample of the analytical medium to light by an impulse of excitation light having sufficient energy to excite chlorophyll fluorescence and further measuring the fluorescence intensity to define the photosynthetic parameters of the object under study In the method for fluorescence determination of photosynthetic parameters of photoindependent nutritional organisms, the fluorescence measurement is performed on the same sample of the analytical medium, and the impulse of the excitation light has the same amplitude, the length of which is an individual chain of the electron and mobile chain of photosynthesis. The chlorophyll photosynthesis is characterized by a constant background illumination, which changes continuously according to the electron's travel time in daylight, adapts the sample to daylight and darkness, and then matches the state of the photosynthetic device by a range of total fluorescence intensity values. This is due to the fact that it defines the parameters of.

In addition, in order to define the chlorophyll fluorescence intensity determination in which the impulse of the excitation light does not affect the state of the photosynthesis apparatus, the impulse time is selected from 1 to 5 mcsec, with a pulse interval of 5 to 100 ms.

Samples were fed with an optical impulse with a time of 100 to 200 mcsec to measure the relative dimensions of the light-harvesting complex of the pigment at the center of the photosynthetic reaction, and start the optical impulse by further calculation of the chlorophyll fluorescence intensity during the impulse action time. From chlorophyll fluorescence intensity measurements of less than 10 mcsec each.

Receptors of PS2 in three consecutive groups of optical impulses with the same amplitude and in each group of time 1-5 mcsec, 100-200 ms and 200-1000 mcsec (average density over 3000 Jm ² s ⁻¹ ), respectively It is suitable to influence the object in order to evaluate the regeneration rate of the component in the part, and to measure the change in fluorescence intensity individually under the influence of each of these impulses, and the fluorescence intensity at an impulse time of 1 to 5 mcsec. The impulse of the excitation light corresponds to the chlorophyll fluorescence intensity which does not affect the state of the photosynthetic apparatus, and the inclination angle of the induction curve of the fluorescence intensity change in response to the impulse of the excitation light having a time of 100 to 200 mcsec, indicates that The relative dimension is determined and defines the relative dimension of the quinone dimension of the induction curve of the fluorescence intensity change in response to the impulse of the excitation light having a time of 200 to 1000 ms.

The maximum level of chlorophyll fluorescence can be determined by supplying light scintillation to a sample at a time of 200 to 500 ms and an irradiation capability density of 3000 wt / m ² .

It is suitable to measure the kinetics (induction curve) of the change in fluorescence by affecting the probe sample by light flashes of time 300 to 1000 v ms, or to measure the chlorophyll fluorescence intensity during its action of less than 1 ms.

Based on the results of fluorescence kinetics measurements, a mathematical model of the photosynthetic device can be made, and through this mathematical model, constant values and quality parameters of electron transfer reactions not defined in the experiment can be determined.

The fluorescence value change is carried out on the same probe sample of the analysis medium through the continuous conversion of the excitation impulse regime after the measurement of each parameter, and the irradiation time in each successive regime is fixed to a larger value than before.

It is suitable to use the method described above to determine photosynthetic characteristics of phytoplankton.

In the production characteristics of phytoplankton, and also in the heterogeneity of individual cell populations, it is desirable to simultaneously use selected media to define the contribution of each type of individual algae.

In order to define the contribution of each type of individual algae to the production characteristics of phytoplankton and also to the heterogeneity of the individual cell populations of the primarily selected sample, for example, by filtration of water through a nuclear filter. The secondary sample of the more concentrated assay medium is extracted and then placed in one layer of cell, for example in a Najotte chamber, and then visually defined to define the composition of the cells of the phytoplankton organism.

In addition to evaluating the type of population, it is desirable to measure each cell parameter of fluorescence and determine its heterogeneity according to the methods described above, wherein the distribution of cells in the population is dependent upon the efficiency of the photosynthetic process and the relative content of pigments in the cells. The impulse of the excitation light is defined by the value of chlorophyll fluorescence intensity that does not affect the state of the photosynthetic device in the absence of constant illumination after the sample is continuously adapted in the dark.

A photoindependent nutrient organism comprising a measurement chamber, a light source, a sample fluorescence measurement module, and a control block connected to a computer, which are optically connected to the measurement chamber and designed with the potential to induce sample fluorescence. An apparatus for fluorescence determination of photosynthetic parameters comprises at least one light source such that the number of light sources optically connected to the measurement chamber is equal, the output being connected to an electrical port of the light source, the port being connected to a control block. Due to the fact that it includes a sensor of natural irradiation which is connected to the current stabilizer of the light source via a current stabilizer and a control block, technical results are obtained.

The light sources are produced identically and each of them is used as a measuring and / or saturation and / or working light source.

The sample fluorescence measurement module is a photo-amplifier connected through a fluorescence detector connected to an independent high power supply, for example a signal processing block and a control block with a registration device (e.g. a personal computer). Can be prepared.

The signal processing block may include at least one intensifier connected to analog and digital converters having an output connected to the control block through a synchronous detector.

It is appropriate to connect the signal processing block of four consecutively connected operational multipliers, each of which is connected to an analog and digital converter, with a control block.

In a preferred variant, the apparatus for fluorescence determination of photoindependent nutritional bio photosynthesis parameters comprises a pump, a collector having a first output connected to a measurement chamber and a second output connected to a concentration system of a secondary sample of the medium. collector, an additional measurement chamber for the determination of the fluorescence parameters of individual cells (suitable to use the Najotte chamber as the second measurement chamber), a microfluorescence desorption device consisting of a luminescent microscope with a fluorometric attachment and a current It may include an LED light source connected to the control block through the ballast.

The fluorometer attachment can be designed as a sample fluorescence measurement module.

The technical results indicate that the inlets and outlets are designed to have the possibility of feeding and removing the light source, the fluorescence detector and the irradiated sample into the chamber, respectively, from the skeleton, the window of the skeleton, The measurement chamber comprising an outlet) is due to the fact that it has the possibility of absorbing light from the light source located opposite and comprises at least one additional light source located opposite the first light source.

The measuring chamber comprises two or more even light sources located in pairs opposite each other on a plane perpendicular to the axis of the framework, each light source being designed to have the possibility of absorbing light from the light sources located opposite.

The fluorescence detector can be designed as an optical amplifier, and the axis of the optical system coincides with the framework axis.

In order to solve the above problems, the constituent means constituting the method for measuring the fluorescence of the photoindependently affected bio photosynthesis parameter of the present invention is a method for measuring fluorescence of the photosynthetic parameter of a photoautotrophic organism. Exposing a sample of the analytical medium to light by excitation light having sufficient energy to excite the chlorophyll fluorescence of and further measuring the fluorescence intensity to determine the photosynthetic parameter of the object under study, The impulse of the light has the same amplitude and variable time, and at the same time, the chlorophyll fluorescence characteristic is measured under constant background illumination, similar to the object irradiation intensity during irradiation and after adapting the sample to the dark, and over a full range of fluorescence intensity values. By defining the parameters of the photosynthetic device state .

In addition, in order to define the chlorophyll fluorescence intensity determination that the impulse of the excitation light does not affect the state of the photosynthesis device, the time of the impulse is selected from 5 to 100 ms between pulses and 1 to 5 mcsec. .

In addition, the sample was supplied with an optical impulse with a time of 100 to 200 mcsec to measure the relative dimensions of the light-harvesting complex of the pigment at the center of the photosynthetic reaction, and further calculation of the chlorophyll fluorescence intensity growth during the working time of the impulse. To measure the chlorophyll fluorescence intensity of less than 10 mcsec each from the start of the optical impulse.

In addition, in each group of three consecutive optical impulses with the same amplitude and having a time of 1 to 5 mcsec, 100 to 200 ms and 200 to 1000 mcsec (average density of 3000 Jm ² s ⁻¹ or more), respectively. The fluorescence intensity affecting the object to assess the rate of regeneration of the components in the receptor portion of PS2, measuring the change in fluorescence intensity individually under the influence of each of these impulses, and reacting at an impulse time of 1-5 mcsec, The impulse of the excitation light corresponds to the chlorophyll fluorescence intensity which does not affect the state of the photosynthesis device, and the inclination angle of the principal curve of the fluorescence intensity change in response to the impulse of the excitation light having a time of 100 to 200 mcsec indicates that the light harvesting complex Determine the relative dimension and define the relative dimension of the quinone pool dimension by the inclination angle of the induction curve of the fluorescence intensity change in response to the impulse of the excitation light having a time of 200 to 1000 ms. And that is characterized.

In addition, the maximum level of chlorophyll fluorescence is characterized in that it is determined by supplying light flash to the sample at a time of 200 to 500 ms, irradiation ability density 3000 wt / m ² .

In addition, in order to determine a constant value of the electron transfer rate in the chain of photosynthetic electron transfer, chlorophyll fluorescence intensity during the action of less than 1 ms or by affecting the probe sample by light scintillation of 300-1000 v ms. Is performed, and the reaction kinetics (induction curve) of the fluorescence change are obtained according to the measurement results.

In addition, the fluorescence kinetics measurement results, to create a mathematical model of the photosynthesis device, and through this mathematical model, characterized in that the constant value and quality parameter (quality parameter) of the electron transfer reaction not defined in the experiment.

In addition, the measurement of the fluorescence parameters is carried out on the same probe sample of the analytical medium through the continuous conversion of the excitation impulse regime after the measurement of each parameter, in which the irradiation time is fixed to a larger value than before. It is characterized by.

It is also characterized by being used to determine photosynthetic characteristics of phytoplankton.

In addition, the selected medium is characterized in that it is used simultaneously to define the contribution of each type of individual algae in the production characteristics of phytoplankton and also in the heterogeneity of the individual cell populations.

In addition, in order to define the contribution of each type of individual algae in the production characteristics of phytoplankton and also in the heterogeneity of the individual cell populations of the primarily selected sample, for example, in the filtration of water through a nuclear filter. Extract the secondary sample of the more concentrated assay medium, and then, for example, put the concentrate in one cell layer in a Najotte chamber, and then define the composition of the cells of the phytoplankton organism by visual assessment. It is done.

In addition to assessing the type of population, the fluorescence parameters were measured, the distribution of cells in the population was determined by the efficiency of the photosynthetic process and the relative content of pigments in the cells, and the samples were continuously adapted in the dark. The impulse of the excitation light in the absence of constant illumination is determined by the value of the chlorophyll fluorescence intensity which does not affect the state of the photosynthesis device.

On the other hand, the constituent means constituting the device for fluorescence determination of photosynthesis parameters of the photoindependently affecting organisms of the present invention is a device for fluorescence determination of photosynthesis parameters of the photoindependent nutritional organisms, optically measured with a measurement chamber, Apparatus for fluorescence determination of photoindependent trophic biophotosynthesis parameters, consisting of a light source, a sample fluorescence measurement module, and a control block connected to a computer, designed with the potential to induce sample fluorescence, At least one light source such that the number of light sources optically connected to the measurement chamber is even, and an output is connected to an electrical port of the light source, the port being a current stabilizer and a control block of the light source connected to the control block. Sensor of natural irradi connected to the current stabilizer of the light source ation).

In addition, the light sources are identically produced and each of said light sources is used as a measuring and / or saturation and / or working light source.

In addition, the fluorescence measurement module includes a photo-amplifier connected through a fluorescence detector, eg, a signal processing block, connected to an independent high power source, and a control block with a registration device (eg, a personal computer). Characterized in that can be prepared).

In addition, the signal processing block may include at least one intensifier connected to an analog and digital converter connected to the control block through an synchronous detector.

In addition, the signal processing blocks of four consecutively connected operational multipliers, each of which is connected to analog and digital converters, are connected to a control block.

On the other hand, the present invention is an apparatus for fluorescence determination of photoindependent nutritional bio photosynthesis parameters, the pump, the first output is connected to the measurement chamber and the second output is connected to the concentration system of the secondary sample of the medium Microfluorescence consisting of a collector, an additional measuring chamber for the determination of the fluorescence parameters of individual cells (suitable for using Najotte chamber as the second measuring chamber), and a luminescent microscope with fluorometric attachment. Desorption device, characterized in that it can include an LED light source connected to the control block through the current stabilizer.

In addition, the fluorometer attachment is characterized in that it can be produced by a fluorescence measurement module.

It is also characterized by the use of the Najotte chamber as an additional measuring chamber.

On the other hand, the constituent means of the measuring chamber according to another embodiment of the present invention is a measuring chamber, which supplies a light source, a fluorescence detector, and a sample to be irradiated, which are installed in a carcass, a window of the skeletal structure, into the chamber, respectively. The measuring chamber, which includes an inlet and an outlet designed to have the possibility of removing out, comprises at least one additional light source positioned opposite the first light source with the possibility of absorbing light from the light source located opposite it. It is characterized by.

The measuring chamber also includes two or more even light sources located in pairs opposite each other on a plane perpendicular to the axis of the skeletal structure, each light source being designed to have the possibility of absorbing light from the opposite light sources. It is characterized by that.

In addition, the fluorescence detector is an optical amplifier, the axis of the optical system is characterized in that the axis of the skeleton structure.

Application of the microfluorescence method in this way allows us to evaluate the functional state of the photosynthetic device of microalgae cells and obtain data for short-term prediction of the kinetics of individual phytoplankton species in this phytocenosis. The high sensitivity and performance of these methods allow accurate measurements of plants in their natural environment, especially in the study of natural phytoplankton in the least productive marine regions.

The present invention is not limited to applications that measure fluorescence using photosynthetic organisms, and can be used in any case where detailed investigation of the process can be performed as a chemical or biological assay based on fluorescence change by a series of flashes. .

When a continuous light impulse is applied to a sample, photosynthetic reactions with different rates of action and different products of this reaction begin continuously. Chlorophyll fluorescence occurs and chlorophyll fluorescence is controlled by this reaction.

Each time interval exposes the sample to impulses at different times that correspond to the electron's transport time at the specific stages of the electron and transport chain of the photosynthesis, resulting in reactive fluorescence with parameters characterizing the reactions that develop in the photosynthetic stage. have. Thus, the normal time of one of the first reactions (reduction of Qa) takes about 100 mcsec, and in this way, if the impulse is shorter than 100 mcsec, the initiation of the photosynthetic reaction does not occur and 1-5 mcsec of light When impulse is supplied, the fluorescence level corresponds to the initial level of chlorophyll fluorescence when all RCs are in the open state. If the impulse time is 100-200 mcsec, the regeneration process of Qa starts. Qa is not oxidized because the further process of electron transfer along the electrons and transport chain proceeds at a slower rate. This makes it possible to measure the relative dimensions of the light collection complex by the inclination angle of the first part of the fluorescence intensity change. The larger the inclination angle, the larger the relative dimension of the light collection complex.

Exposure to excitation light with a time of 200-1000 ms results in complete regeneration of Qa and Qb and an additional pool of quinones. In further reactions, the oxidation of the quinone pool is slower. Under these conditions, the relative value of the quinone pool can be measured by the inclination angle of the fluorescence intensity change curve.

In this way, exposure of a sample to optical impulses that excite chlorophyll fluorescence of different amplitudes but with the same time has the potential to measure many parameters of the functional state of the object's photosynthesis device in one sample.

If the photosynthetic reaction is initiated and has the same characteristics as the sample is adapted to the dark, a change in the sample chlorophyll fluorescence parameter appears in the background of working light having the same value as daylight (or in the background of the selected irradiation value) at the sample dividing position.

The generation of measurement areas of light that reproduces the intensity of daylight at the same splitting location allows to determine different types of extinction, and thus to calculate the light curve of electron transfer and to determine the optimal photosynthetic area.

As the impulse time increases above 1000 mcsec, several photosynthetic reactions begin, making it difficult to interpret the data and measuring one exact value of the parameter without adapting the sample for 10 minutes in the dark. It becomes impossible. The longer the impulse is, the longer it will take to adapt later in the dark.

After adaptation in the dark, the photosynthetic electron transfer chain is completely released from the electrons and the mechanism-regulating primary photosynthetic reaction is stopped. When light is turned on, light and dark reactions begin to control the photosynthetic response. The shorter the light impulse, the shorter the time, so that additional impulses can be supplied without changing the photosynthesis device. Therefore, initially supply a short impulse to measure F0 and then supply a longer impulse to measure another time-saving parameter for each measurement cycle. To ensure reliable statistical analysis of the data from the experiments, run several impulses (groups) at the same time.

The measurement data obtained by a single impulse from a single phytoplankton sample offers the possibility of creating a more accurate mathematical model of the photosynthetic process, thereby allowing the ratio of the constant rate of reaction to the photosynthetic apparatus of plankton algae not measured by direct methods. Calculate

The above method for measuring photosynthetic parameters of photoindependent nutritional organisms is shown in the specific example of studying phytoplankton cell chlorophyll fluorescence. This choice is based on the fact that nearly half of the photosynthetic organisms on Earth belong to phytoplankton.

Collection of water samples containing cells of phytoplankton is carried out by any method known based on the work and conditions of the basic study.

Divide the water sample into two samples, one of which is placed in a measuring chamber with light similar to the intensity of irradiation in the natural environment, the other study of individual cells in the medium and the contribution of individual algae species to the production characteristics of phytoplankton. Place it in a separate block to determine it.

We select the most beneficial fluorescence parameters for the proposed study and then develop a protocol for the realization and study of a device for the purpose of fluorescence excitation to specifically measure the fluorescence parameters of the PSA object functional state.

Chlorophyll fluorescence of all phytoplankton cells induced in response to light is measured by exposing the sample to an impulse of excitation light that varies with the selected algorithm time. The impulses have the same amplitude at all sample light exposure times.

Algorithms are measured simultaneously in a single sample to determine the fluorescence parameters in the above-described range when using three time impulses.

Create a constant light in the chamber with the same quantum number as the natural background irradiation at the sampling location, or operator according to the typical average value of the natural irradiation at the sampling location for the entire time of the first measurement step at the three indicated times. operator) to determine the fluorescence intensity (see FIG. 1).

1. Regime of chlorophyll fluorescence intensity, in which the impulse of the excitation light does not affect the state of the photosynthesis device.

Impulse time is 1-5 mcsec, interval between impulses is 50-100 ms, and average density of irradiation power is 0.4 Wtm ² or less. In this regime, the average value of chlorophyll fluorescence (Ft) is determined.

The number of measurements is selected depending on the need to reach the mistake of average set point automatically or by calculation of the operator.

If the average density of the irradiation capacity is 0 Jm ^-2 s ^-1 or less, an additional impulse may be provided at 10 to 100 mcsec since less than 1% of the RC is closed and the RC is returned to the open state for a closed time by a short impulse. have.

After regime 1 is completed, proceed to regime 2.

2. Growth crystal regime of chlorophyll fluorescence intensity during impulse action to measure the size of the light-collecting complex of pigments central to photosynthetic reaction.

To induce this measurement, an optical impulse with a time of 100 to 200 mcsec was supplied to the sample, and the chlorophyll fluorescence intensity was measured every 10 mcsec to determine the average growth of chlorophyll fluorescence intensity during the impulse as an intensity derivative over time. Calculate

The number of measurements is selected according to the need to achieve a set value of the average real number automatically or by operator calculation.

After regime 2 is completed, proceed to regime 3.

3. A regime that saturates the photosynthetic apparatus of phytoplankton cells with light to determine the kinetics and rectification levels of chlorophyll fluorescence intensity. To achieve this, an optical impulse with a time of 200-100 ms and an average density of irradiation capacity of 3000 Jm ^-2 sec ^-1 is produced. Chlorophyll fluorescence intensity is measured every 10 mcsec from the start of the impulse. The growth rate of chlorophyll fluorescence intensity is inversely proportional to the size of the quinone pool.

In all regimes of chlorophyll fluorescence intensity, the impulse of the excitation light is the same in amplitude. The average density of different values of the light irradiation ability to induce chlorophyll fluorescence is achieved through different impulse times.

4. Next, turn off the continuous background light similar to the light intensity of the natural environment. After adapting the same water sample in the dark, chlorophyll fluorescence is measured in regimes 1, 2 and 3, without constant background light.

Based on the measurement results with continuous light and the measurement results without continuous light, the fluorescence parameter is calculated.

Fo—Chlorophyll fluorescence intensity against an impulse of excitation light with a time of 1-5 mcsec after adapting the sample to constant background light and in the dark.

Fm-maximum value of chlorophyll fluorescence intensity for the impulse of excitation light with a time of 200-1000 mcsec, in the absence of constant background light, and after adapting the object to darkness.

Ft-value of chlorophyll fluorescence intensity for the impulse of excitation light with a time of 1-5 mcsec in constant background light.

F'm-The maximum value of the chlorophyll fluorescence intensity for an impulse of excitation light with a time of 200-1000 mcsec in constant background light.

From these additional parameters by equations are provided.

1. Primary quantum yield of charge separation in PS2 as ratio Fv / Fm = (Fm-Fo) / Fm.

This parameter is proportional to the quotient of the active RC in the dark.

2. Photochemical disappearance upon background irradiation-qP = (F'm-Ft) / (F'm-Fo ').

3. Non-photochemical disappearance at background investigation-NPQ = (Fm / F'm) -1.

4. The quantum yield of photochemical conversion of absorbed light energy at PS2 upon background irradiation as ratio Y = (Fm'-Ft) / Fm ', reflecting the acyclic movement of electrons during photosynthesis.

5. The absolute value of Fo as an index of aquatic phytoplankton abundance.

6. The relative size of the quinone pool, which can be calculated by the rate of electron transfer from Qa to Qb, the relative size of the light collection antenna complex, and the kinetics of chlorophyll fluorescence intensity growth from Fo to Fm.

The data obtained in the measurement period is introduced into the mathematical model of the photosynthesis process and other parameters not obtained by the direct method are calculated.

To define the contribution of individual species of algae in the production characteristics of phytoplankton, the second sample is concentrated by water filtration through a nuclear filter, and the self-formed concentrate is dispersed in one cell layer in a Najotte chamber with a volume of 70 mcl. Visually determine the cell species of phytoplankton organisms. Each cell of a specific type of plankton in the chamber is a measure of the fluorescence parameters by the method applied to measure the fluorescence of the first sample, which is then determined by the efficiency of the photosynthetic process and the relative content of the pigment in the cell (by value F0). Determine the distribution of bird species. In this way, the species of the cell to be examined first is determined by the microscope's field of view, and then its fluorescence characteristics are measured. These measurements are performed on dozens of individual cells of each phytoplankton mass species, and the measurement results determine the composition of this species and the number of cells in each phytoplankton algae population, the relative content of photosynthetic pigments and the efficiency of the primary photosynthetic process. Also determines the cell distribution of each type of algae.

For the measurement, a microfluorometric detached device consisting of a luminescence microscope with a fluorometer attachment and an LED light source connected to a supply system of fluorescence excitation light impulses is used.

Measurement of individual cells allows one to determine the heterogeneity of a population of individual species of algae.

The complex of chlorophyll fluorescence intensity values (Fo, Fm, Ft, F'm) and the kinetics of phytoplankton samples and Fo-Fm of mixed samples of individual cells are based on known dependencies and mathematical models, In one sample, the state of the photosynthetic device of the phytoplankton combination can be determined as a whole and the individual algal species constituting the composition can be determined.

The apparatus for realizing the present invention has a configuration as shown in FIG.

The measurement chamber 1, which is optically connected to the light source 2 and the fluorescence detector 3, and whose output part is connected to the control block 5 via the signal processing block 4, includes a data registration and processing block 6, For example, it is connected to a computer, in particular a personal computer. The input of the light source 2 is connected to the output of the current stabilizer 7 of the light source which is connected to the output of the control block 5, which can be represented by a microprocessor. The sensor 8 of working light is connected to the input of the control block 5 and can be designed as a photodiode with a correction light filter system.

The signal processing block 4 may comprise at least one intensifier 9 which is connected to the analog to digital converter 11 via a synchronous detector 10.

A preferred variant is that the signal processing block 4 comprises an operational intensifier 9 connected in series, with each output between them via an synchronous detector 10 and an analog to digital converter 11. Is connected to the input of the control block 5 and to the output of the control block 5 and to the personal computer 6.

The current stabilizer 7 for the light source 2 is for conversion of the control voltage supplied from the control block 5 to a current which ensures light source irradiation of the required intensity in accordance with the setting parameter. The current stabilizer 7 is designed as several independent channels of the number corresponding to the number of light sources 2, each of which outputs is connected to the electrical input of the corresponding light source 2.

Each channel of the ballast 7 can be made, for example, as a dead resistance connected to the field transistors connected in series and the control output of the control block 5.

The fluorescent detector 3 can be designed, for example, as an optical amplifier connected to the independent high voltage power supply 12 (manufactured by TRACO) of the modules MHV 12 to 1.5. As the optical amplifier 3, for example, an optoelectronic amplifier φεy-79 having a limited optical filter KC 18 that allows registration of irradiation having a wavelength of 680 nm or more can be used.

The computer and control block may be equipped with independent DC power supplies or supplied from a multichannel voltage converter (not shown).

The light source 2 and the optical amplifier 3 comprise a corresponding optical system.

LEDs, each of which has the function of measuring and / or saturation and / or constant light, for example the powerful LED L400CWO12K, T4 round (Ledtronics) with an irradiation wavelength of up to 612 nm can be used as the light source.

An even number of light sources 2 are evenly located around the axis of one plane of the window of the measuring chamber 1. The light sources 2 are provided in pairs on opposite sides of each other, and each of them is designed with the possibility of absorbing light from the light source located on the opposite side thereof.

The skeletal structure 13 of the measurement chamber 1 (see FIG. 3) includes a window 14 for a light source, which is provided with an optical system of a light source. Each optical system comprises a spherical lens 15, an optical filter 16 and a focus lens 17, the light source 2 being a heatsink connected to the framework 13 in the area of the window 14. on the sink 18. In the window 19 for the optical amplifier is situated the optical system of the optical amplifier 3. The window 14 for the light source 2 is provided around the axis of the framework 13 with the possibility of arranging the light sources 2 in pairs on a plane perpendicular to the axis of the framework 13. The focusing of the lens 17 is performed such that the light patch diameter of the opposite illuminator does not exceed the diameter of this illuminator. The window 19 for the optical amplifier is located on the same axis as the optical system of the optical amplifier 3, with each axis coinciding with the axis of the framework 13.

The block of the device for measuring the fluorescence parameter of the first sample is located in a rigid sealing frame with electronic and optical measurement systems, which together with the power block provide a built-in fluorometer or a submersible sensing device fluorometer.

The device is connected to a sampling pump connected to a collector (not shown in the figure), which supplies two samples, one to the measurement chamber 1 and the other to a sample concentration system. Disperse

The measurement block of chlorophyll fluorescence parameters of phytoplankton individual cells is concentrated in a Najotte chamber mounted on a люMaM-и3 type luminescent microscope table with a fluorometer attachment of type φMθл1-A modified by an LED light source, current stabilizer, control Block and computer. For registration and measurement of cell fluorescence, the fluorescence registration module described above is used.

The device works in the following manner.

The medium sample under analysis taken by the pump goes to the collector, where the water sample containing phytoplankton is divided into two samples, one of which goes to the working volume of the measuring chamber (1) and the other is phytoplankton Enter the concentration system of the measuring block of chlorophyll fluorescence of the individual cells, after which the second sample enters the Najotte chamber.

The sensor 8 measures daylight irradiation at the position of the water sample (natural underwater irradiation) and supplies it to the control block 5. The measurement of natural irradiation provides a signal corresponding to the intensity of the acting light, from the control block 5 to the light source 2 through the current stabilizer 7, thereby corresponding to the natural light emission in the measurement chamber 1. Light emission is formed.

The intensity of natural light emission is measured by the number of light protons per unit area in the range of 400 to 700 nm corresponding to the range of photosynthetic emission. The emission of algal fluorescence occurs in the wavelength range of 680 to 740 nm. In this way, there is a region of natural light that is characteristic of chlorophyll fluorescence, where low fluorescence emission (about 2%) is directly under the influence of natural light to measure the fluorescence intensity for low (characteristic of the environment) algae. Do not allow it. Therefore, in the above-described apparatus, the quantum number is the same as natural irradiation, but background illumination similar to natural irradiation occurs in the range of 400 to 600 nm.

The control block 5 is located in the measurement chamber 1 (under the possibility of change of the fluorescence signal), and the irradiation intensity is equal to the condition of natural irradiation of the number of photons at the sampling position.

Samples of phytoplankton located in the measurement chamber 1 are exposed to light impulse irradiation according to the selected time diagram (irradiation in the measurement chamber is the same as the natural environment of the sampling location) (FIG. 1).

Because of the even number of light sources and the arrangement of the pairs facing each other, the optical system of each light source is a "trap" to the light of the opposite light source. Excluding the possibility, this reduces the parasite exposure of the fluorescence detector (optical amplifier) by excitation light.

The light from the light source 2 by the spherical lens 15 collects in a parallel bunch, passes through the optical filter 16 and has the potential to fall on the sample with scattered light in the opposing optical system, with the focus lens. The focus is on the weakly converging beam by (17) (FIG. 4).

An optical signal of sample fluorescence through an optical system including an optical filter for selecting the spectral range of chlorophyll fluorescence is registered by the optical amplifier 3. The output signal of the optical amplifier 3, which contains information about the chlorophyll fluorescence value through the signal processing block 4, is supplied to the re-registration and data processing block 6, in this case a personal computer.

In natural environments, the concentration of phytoplankton varies in a large range from 0.01 mcg / l to 100 mcg / l. In this way, its concentration and the value of the electrical signal received at the output of the optical amplifier 3 are unknown. When the concentration of phytoplankton is low, the electrical signal is removed from the amplifier 9 and the synchronous detector 10 having a low and maximum amplification coefficient. As the concentration increases, and then the electrical signal becomes large, the signal on the amplifier 9 becomes larger than the maximum value of this amplifier and cannot be measured. For this reason, it is preferable to make the block of an input signal into the chain 9-9 ³ of an amplifier which has a known amplification coefficient. The amplification factor of the amplifier 9 is chosen to be 4 because Fm is four times larger than Fo, which is able to measure the fluorescence signal in one impulse without adjusting the intensifying tract. Thus, when the concentration of phytoplankton is low, the electrical signal is low and is removed from the amplifier 9 ³ and the synchronous detector 10 ³ having the maximum amplification coefficient. As the concentration increases, and then the electrical signal becomes large, the signal on the amplifier 9 ³ becomes larger than the maximum value of this amplifier and cannot be measured. In a cascade design, the signal is removed from the one amplifier ^(92), when the fluorescent signal is further large, the amplifier ⁽⁹¹⁾ and the synchronous detector including a synchronous detector (10 ¹⁾ includes a synchronous detector (10 ²⁾ ( The signal is removed from the amplifier 9 with 10). The output signal of the synchronous detector 10 by the analog to digital converter 11 is converted into a digital form. Control block 5 (microprocessor) selects the unsaturated channel closest to the final step of the amplifier to measure the fluorescence signal value.

In this way, in a single measurement cycle (one impulse), the “non off-scale” amplifier is selected and the concentration is taken into account in consideration of its amplification factor. This information in signal processing design extends the dynamic measurement range and allows measurements to be made at the early stages of luminescence in the early stages of the induction process, giving the possibility of working in the full range of phytoplankton concentrations found in the natural environment. .

Management of the measurement cycles is carried out by the microprocessor control block 5, from which the signal containing information on the chlorophyll fluorescence intensity enters the personal computer 6. By fluorescence parameter measurement results introduced into a mathematical model of a photosynthetic device with a special program, a constant value of the movement velocity of electrons not measured in a direct experiment is calculated.

According to the selected measurement program, the personal computer 6 transmits to the control block 5 the instructions of the measurement section, in particular the algorithm of the current stabilizer 7 and the light source 2, and also the sensor 8 of the acting light (sample sampling). Probe measurement in position).

To determine the contribution of individual algal species in the production characteristics of phytoplankton, a second sample of the medium under investigation concentrated by water filtration through the nuclear filter 20 is used. Concentrated algae are placed in a Najotte chamber of 70 mcl in volume and dispersed in one layer of cells.

With low luminescence to ensure visual control of the sample in the Najotte chamber, each cell of the field of view is located by substage in a photometric zone of 37.5 mcm in diameter. At the operator's command, a cycle is initiated from the computer that consists of fluorescence measurements in response to a series of optical impulses of the LEDs. Based on the measurement results, the average value of Fo and Fm is calculated, which indicates the relative content of pigment cells and the efficiency of the photosynthetic process, thereby obtaining information on the distribution of different algal species cells.

As an example, a study of fluorescence characteristics on mass algal species on the Black Sea coast is provided.

Based on the data of the initial study, mass species of phytoplankton were selected, and cell distribution was analyzed by photosynthetic efficiency. During the investigation, representative binary Th. nizsehioides and Rh. fragilissima was in a restrained condition. About half of these cells had low levels of photosynthetic efficiency. Growth of the algal cell number was made based on algal culture and data obtained from natural contamination with mean value (Fm-Fo) / Fm level of less than 0.3. For this reason, a decrease in the number of these species is predicted by the relative variable fluorescence. The majority of cells, Nitzschia sp. And G. wulffii, a representative dinoflagellate, have high photosynthetic activity levels that demonstrate favorable conditions for them and allow them to predict the growth of the number of these species. Two weeks after initiation, Nitzschia sp. Cells develop less and Th. nitzsehioides and Rh. fragilissima cells began to appear temporarily.

Table 1 shows the frequency of appearance of the microalgal species, the average value of fluorescence intensity (Fo) corresponding to the photosynthetic pigment content of individual cells, and the average relative variable (Fm-Fo) of algae fluorescence indicating the efficiency of the primary process of bio photosynthesis. Provide / Fm. Water samples were taken in the September 2002 wastage (44 ⁰ 32.8 na 37 ⁰ 57.7 el) and the content of the pigment delivered in Xл was calculated by the fluorescence intensity value (Fo).

Application of the microfluorescence method in this way allows us to evaluate the functional state of the photosynthetic device of microalgae cells and obtain data for short-term prediction of the kinetics of individual phytoplankton species in this phytocenosis. The high sensitivity and performance of these methods allow accurate measurements of plants in their natural environment, especially in the study of natural phytoplankton in the least productive marine regions.

The present invention is not limited to applications that measure fluorescence using photosynthetic organisms, and can be used in any case where detailed investigation of the process can be performed as a chemical or biological assay based on fluorescence change by a series of flashes. .

Species of algae, contribution of algal biomass to the total biomass of phytoplankton, content of photosynthetic pigments and variable fluorescence of single cells {(Fm-Fo) / Fm}

Table 1

1 is a time chart for fluorescence measurement.

2 is a block diagram of an apparatus for measuring phytoplankton fluorescence parameters.

3 is a layout view of the measurement chamber.

Claims (23)

As a method of measuring fluorescence of photosynthetic parameters of photoautotrophic organisms, Exposing the sample of the analytical medium to light by excitation light having sufficient energy to excite the chlorophyll fluorescence of the sample, Further measuring the fluorescence intensity to determine the photosynthetic parameter of the object under study, The impulse of the excitation light has the same amplitude and variable time, and at the same time, the chlorophyll fluorescence characteristic is measured under constant background illumination, similar to the object irradiation intensity during irradiation and after adapting the sample to darkness, and the full range of fluorescence intensity values A method for measuring fluorescence of photosynthetic parameters of a photoindependent nutritional organism, characterized by defining the parameters of the photosynthetic device state. The method of claim 1, wherein the impulse time is selected from 1 to 5 mcsec, with an interval between pulses of 5 to 100 ms, in order to define a chlorophyll fluorescence intensity crystal in which the impulse of the excitation light does not affect the state of the photosynthesis device. A method of measuring fluorescence of photosynthesis parameters of photoindependent nutritional organisms. The chlorophyll fluorescence intensity according to claim 1, wherein the sample is supplied with an optical impulse of 100 to 200 mcsec at a time to measure the relative dimension of the light-harvesting complex of the pigment at the center of the photosynthetic reaction, and the chlorophyll fluorescence intensity during the working time of the impulse. A method for fluorescence measurement of photosynthetic parameters of a photoindependent nutritional organism, characterized in that further calculations of growth are performed to measure chlorophyll fluorescence intensities of less than 10 mcsec each from the start of the light impulse. 2. Three consecutive groups according to claim 1, wherein the amplitudes are the same and in each group the time is 1-5 mcsec, 100-200 ms and 200-1000 mcsec, respectively, with an average density of at least 3000 Jm ² s ^−1. In order to evaluate the regeneration rate of the constituents in the receptor portion of PS2 in the optical impulse of the object, the change in fluorescence intensity is individually measured under the influence of each of these impulses, and responded at an impulse time of 1 to 5 mcsec. The fluorescence intensity corresponds to the chlorophyll fluorescence intensity in which the impulse of the excitation light does not affect the state of the photosynthesis device, and the inclination angle of the main portion of the induction curve of the fluorescence intensity change in response to the impulse of the excitation light having a time of 100 to 200 mcsec, Determine the relative dimensions of the light harvesting complex and determine the relative values of the quinone pool dimensions by the inclination angle of the induction curve of the fluorescence intensity change in response to the impulse of the excitation light having a time of 200 to 1000 ms. A method for measuring fluorescence of photosynthetic parameters of photoindependent nutritional organisms, characterized by limiting the number. The method of claim 1, wherein the maximum level of chlorophyll fluorescence is determined by supplying light scintillation to the sample at a time of 200 to 500 ms and an irradiation power density of 3000 wt / m ² . How to measure fluorescence. The method of claim 1, wherein the action of exposing the probe sample by light scintillation at a time of 300 to 1000 v ms or its action of less than 1 ms to determine a constant value of the rate of electron transfer in the chain of photosynthetic electron transfer. During the measurement of chlorophyll fluorescence intensity, and obtaining the reaction kinetics (induction curve) of the fluorescence change by the measurement result. The method according to claim 1, wherein a fluorescence kinetics measurement result is used to create a mathematical model of the photosynthetic device, and through this mathematical model, to determine constant values and quality parameters of electron transfer reactions not defined in the experiment. Characterized in that the photosynthesis parameters of the photoindependent nutritional organisms fluorescence measurement. The method of claim 1, wherein the measurement of the fluorescence parameter is performed on the same probe sample of the assay medium through the continuous conversion of the excitation impulse regime after the measurement of each parameter, wherein the irradiation time at each successive regime is greater than before. A method for measuring fluorescence of photosynthetic parameters of a photoindependent nutritional organism, characterized in that it is fixed at a value. 9. The method of claim 1, which is used to determine photosynthetic characteristics of phytoplankton. 10. The photoindependent organism of claim 1, wherein the selected medium is used simultaneously to define the contribution of each type of individual algae to the production characteristics of phytoplankton and also to the heterogeneity of individual cell populations. Method for fluorescence measurement of photosynthesis parameters. 2. A nuclear filter according to claim 1, for defining the contribution of each type of individual algae to the production characteristics of phytoplankton and also to the heterogeneity of the individual cell populations of the primarily selected sample. Extract the secondary sample of the more concentrated analytical medium by filtration of water through, then place the concentrate in one cell layer, for example in a Najotte chamber, and then visually evaluate the cells of the phytoplankton organism. A method for fluorescence measurement of photosynthetic parameters of a photoindependent nutritional organism, characterized by defining a composition. The method according to claim 1, wherein the fluorescence parameters are measured according to the method according to any one of claims 1 to 8, at the same time as assessing the type of population, the efficiency of the photosynthesis process and the relative content of pigments in the cells. The distribution of cells in the population is determined, and after the sample is continuously adapted in the dark, the impulse of the excitation light in the absence of constant illumination is determined by the value of the chlorophyll fluorescence intensity which does not affect the state of the photosynthetic device. , Fluorescence measurement of photosynthetic parameters of photoindependent nutritional organisms. An apparatus for fluorescence determination of photosynthetic parameters of photoindependent nutritional organisms, Photo independent nutrient bio photosynthesis, consisting of a measurement chamber, a light source, a sample fluorescence measurement module, and a control block connected to a computer, which are optically connected to the measurement chamber and designed with the potential to induce sample fluorescence. The apparatus for fluorescence determination of parameters At least one light source, the output of which is connected to an electrical port of the light source, wherein the number of light sources optically connected to the measuring chamber is even, and the port controls a current stabilizer and a control block of the light source connected to the control block. An apparatus for fluorescence determination of photosynthetic parameters of a photoindependent nutritional organism comprising a sensor of natural irradiation coupled to a current stabilizer of a light source. 14. The apparatus of claim 13, wherein the light sources are identically manufactured and each light source is used as a measuring and / or saturating and / or working light source. The optical amplifier of claim 13, wherein the fluorescence measurement module comprises a fluorescence detector, eg, a signal processing block, connected to an independent high power source, and an optical amplifier connected through a control block with a registration device (e.g., a personal computer). A device for fluorescence determination of photosynthetic parameters of a photoindependent nutritional organism, which can be prepared with a photo-amplifier. The signal processing block of claim 13, wherein the signal processing block may include at least one intensifier connected to an analog and digital converter having an output connected to a control block through a synchronous detector. A device for fluorescence determination of photosynthetic parameters of a photoindependent nutritional organism. 14. The photoindependent nutritional method according to claim 13, wherein the signal processing block of four consecutively connected operational multipliers, each port of which is connected to an analog and digital converter, is connected to a control block. An apparatus for fluorescence determination of photosynthetic parameters of an organism. The device of claim 13, wherein in a preferred variant, the apparatus for fluorescence determination of photoindependent nutritional bio photosynthesis parameters, A pump, a collector having a first output connected to the measurement chamber and a second output connected to a concentration system of the secondary sample of the medium, and an additional measurement chamber for the determination of the fluorescence parameters of the individual cells (second measurement It is suitable to use a Najotte chamber as a chamber), a microfluorescence desorption apparatus consisting of a luminescent microscope with a fluorometric attachment, and an LED light source connected to the control block through a current stabilizer. An apparatus for fluorescence determination of photosynthetic parameters of a photoindependent nutritional organism, which may comprise. 19. The apparatus of claim 18, wherein the fluorometer attachment can be made with a fluorescence measurement module of the type described in claim 15. 19. The apparatus of claim 18, wherein the Najotte chamber is used as a further measurement chamber. As a measuring chamber, Includes an inlet and an outlet designed to have the possibility of feeding into and out of the chamber a light source, a fluorescence detector, and an irradiated sample, respectively, in a skeleton, a window of the framework; Wherein the measuring chamber comprises at least one additional light source opposite to the first light source with the possibility of absorbing light from the light source located opposite. 22. The system of claim 21, wherein the measurement chambers comprise two or more even light sources positioned in pairs opposite each other on a plane perpendicular to the axis of the framework, each light source absorbing light from opposite light sources. A measuring chamber, characterized in that it is designed with the potential. The measuring chamber of claim 21, wherein the fluorescence detector is an optical amplifier, and the axis of the optical system coincides with the skeletal axis.

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