Chlorophyll fluorescence, as a probe for photosynthesis research, has been widely studied and applied. Chlorophyll fluorescence can not only reflect the initial reaction process of photosynthesis such as light energy absorption, excitation energy transfer and photochemical reaction, but also related to electron transfer, proton gradient establishment, ATP synthesis and CO2 fixation. Almost all changes in photosynthesis can be reflected by chlorophyll fluorescence, while fluorescence measurement technology does not need to break cells and harm organisms. Therefore, indirect study of photosynthesis changes by studying chlorophyll fluorescence is a simple, fast and reliable method. method. At present, chlorophyll fluorescence has been widely used in photosynthesis, plant stress physiology, aquatic biology, oceanography and remote sensing. Development History The automatic biochemical analyzer is an instrument that measures a specific chemical composition in body fluids according to the principle of photoelectric colorimetry. Due to its fast measurement speed, high accuracy and small consumption of reagents, it has been widely used in hospitals, epidemic prevention stations and family planning service stations at all levels. The combined use can greatly improve the efficiency and benefits of routine biochemical testing. Bio Chemistry Analyzer, Clinical Chemistry Analyzer, Blood Chemistry Analyzer,Urine Chemistry Analyzer Jilin Sinoscience Technology Co. LTD , https://www.contoryinstruments.com
principle
The automatic analyzer is to automatically run all or part of the steps of sampling, mixing, warm bath (37°C) detection, result calculation, judgment, display and printing results and cleaning in the original manual operation process. Today, biochemical tests are basically automated analysis, and there are fully automatic biochemical analysis systems designed for large or very large clinical laboratories and commercial laboratories, which can be arbitrarily configured according to the laboratory's testing volume.
Whether it is the fastest-running (9600Test/h) modular fully automatic biochemical analyzer today, or the original manual-operated photoelectric colorimeter for colorimetry, the principle is the use of absorption spectroscopy in spectroscopic technology. It is the most basic core of the biochemical instrument.
Optical system: is a key part of ACA. Older ACA systems used halogen tungsten lamps, lenses, color filters, and photocell assemblies. The optical part of the new ACA system has been greatly improved. ACA's beam splitting system can be divided into front splitting and rear splitting due to different light positions. The advanced optical components use a set of lenses between the light source and the cuvette to convert the original light source. The light projected by the lamp passes through the cuvette to bring the beam to the speed of light (unlike traditional wedge beams), so that the spot beam can pass through even the smallest cuvette. Compared with traditional methods, it can save reagent consumption by 40-60%. After the spot beam passes through the cuvette, the spot beam is restored to the original beam through this group of restoration lenses (wide difference correction system), and is divided into several fixed wavelengths (about 10 or more wavelengths) by the grating. The optical/digital signal direct conversion technology is used to directly convert the optical signal in the optical path into a digital signal. It completely eliminates the interference of electromagnetic waves to the signal and the attenuation in the process of signal transmission. At the same time, the optical fiber is used in the signal transmission process, so that the signal can achieve no attenuation, and the test accuracy is improved by nearly 100 times. The closed combination of the optical path system makes the optical path without any maintenance, and the light splitting is accurate and the service life is long.
Constant temperature system: Since the temperature of the biochemical reaction has a great influence on the reaction results, the sensitivity and accuracy of the constant temperature system directly affect the measurement results. The early biochemical instruments used the method of air bath, and later developed into a dry bath with constant temperature liquid circulation which combines the advantages of dry air bath and water bath. The principle is to design a constant temperature tank around the cuvette, and add a stable constant temperature liquid that is odorless, non-polluting, non-evaporating and non-deteriorating in the tank. The constant temperature liquid has a large capacity, good thermal stability and uniformity. The cuvette does not directly contact the constant temperature liquid, which overcomes the characteristics of the water bath type constant temperature being susceptible to pollution and the uneven and unstable air bath.
Sample reaction stirring technology and probe technology: The traditional reaction stirring technology adopts magnetic bead type and vortex stirring type. The current popular stirring technology is a stirring unit composed of multiple groups of stirring rods that imitate the manual cleaning process. When the first group of stirring rods is stirring the sample/reagent or mixed solution, the second group of stirring rods performs high-speed and high-efficiency cleaning at the same time. The set of stirring bars also undergoes a warm water washing and air drying process at the same time. In the design of a single stirring rod, a new type of spiral high-speed rotating stirring is adopted, and the rotation direction is opposite to the spiral direction, thereby increasing the stirring force, the stirred liquid does not foam, and reducing the scattering of light by microbubbles. Reagent and sample probes are based on the principle of early capacitive sensing, but slightly improved to increase the alarm of blood clots and protein clots, and re-test results according to the alarm level, reducing sample aspiration errors and improving the reliability of test results. . Large-scale biochemical instruments can detect more than 1,000 tests per hour, so automatic retesting is very important. Subjective evaluation of test results and manual retesting can no longer meet clinical needs.
Other aspects: barcode recognition of reagents and samples and computer login. Due to the lack of barcode recognition function of early biochemical instruments, there are more opportunities for errors. In recent years, both imported and domestic chemical instruments have adopted barcode detection. The use of this technology in biochemical instruments has provided technical support for the development of high-speed ACA, and also made the instrument quite supportive. The software development is simple and easy, therefore, barcode detection is the basis for the intelligence of the instrument. Open reagents, as an important factor for hospitals to choose models, whether the instrument supports open reagents is very important. After the reagents are opened, hospitals and scientific research units can choose their own reagent suppliers, and have a greater degree of freedom in measuring the price, the reliability of the test results, and the validity period of the reagents. Ion Selective Electrode Analysis Accessory (ISE), human serum and urine electrolyte indicators are very important, and hospitals can save money by adding ISE to the ACA system.
Development history and measurement principle of chlorophyll fluorescence technology
The chlorophyll fluorescence phenomenon was first discovered by the missionary Brewster. In 1834, Brewster discovered that when a strong sunlight passed through the ethanol extract of the bay leaf, the color of the solution turned into a green complementary color, red, and the color varied with the thickness of the solution, which is historically chlorophyll. The first record of fluorescence and its reabsorption. Later, Stokes (1852) realized that this was a phenomenon of light emission and used the term "fluorescence". In 1874, Müller found that after dilution of the chlorophyll solution, the fluorescence intensity was much stronger than that of the living leaves. Although Müller suggested that there may be an inverse relationship between chlorophyll fluorescence and photosynthesis, because his experiments were not controlled and the experimental conditions were not strictly controlled, the discovery of chlorophyll fluorescence induction (transient) phenomenon was not attributed to Müller.
Kautsky is recognized as a discoverer of chlorophyll fluorescence induction. In 1931, Kautsky and Hirsch visually observed and recorded chlorophyll fluorescence induction (Lichtenthaler, 1992; Govindjee, 1995). After illuminating the dark-adapted leaves, they found that the chlorophyll fluorescence intensity changed with time and was related to the fixation of CO2 (Fig. 3.1). The main conclusions they obtained were as follows: 1) Chlorophyll fluorescence rapidly rose to the highest point, then decreased, eventually reaching a steady state, and the whole process was completed in a few minutes. 2) The rise of the curve reflects the original photochemical reaction of photosynthesis and is not affected by temperature (0 ° C and 30 ° C) and HCN treatment. If the light is turned off at the highest point, the fluorescence drops rapidly. 3) The change in fluorescence intensity is inversely related to the fixation of CO2. If the fluorescence intensity decreases, the CO2 fixation increases. This means that when the fluorescence intensity is reduced, more light energy is used to convert into chemical energy. 4) The strange thing is that (after illuminating) the fixation of CO2 has a lag period, which seems to indicate that the "light dependent" process is necessary for the CO2 fixation process. Another unexplained phenomenon is that recovery of fluorescence levels takes a long time if light is turned off after the end of fluorescence induction. After the discovery of Kautsky, extensive and in-depth research on chlorophyll fluorescence induction has been carried out, and the theory of photosynthesis fluorescence induction has been gradually formed, which is widely used in photosynthesis research. Due to Kautsky's outstanding contribution, the chlorophyll fluorescence induction phenomenon is also known as the Kautsky Effect.
Quantum yield
The intracellular chlorophyll molecule transitions from the ground state (low energy state) to the excited state (high energy state) by directly absorbing light quantum or indirectly through the light-harvesting pigment to absorb light quantum energy. The shorter the wavelength, the higher the energy. Therefore, after the chlorophyll molecule absorbs red light, the electron transitions to the lowest excited state; after absorbing blue light, the electron transitions to a higher energy level (higher excited state) than the absorbed red light. The chlorophyll molecules in the higher excited state are very unstable. Within a few hundred femtoseconds (fs, 1 fs = 10-15 s), the heat is radiated to the surrounding environment by vibrational relaxation, returning to the lowest excited state (Fig. 3.2). The lowest excited state of the chlorophyll molecule can be stable for a few nanoseconds (ns, 1 ns = 10-9 s).
Chlorophyll molecules in a lower excited state can release energy back to a stable ground state in several ways. The ways in which energy is released are as follows (Fig. 3.3) (Campbell et al., 1998; RoháÄek & Barták, 1999; Malkin & Niyogi, 2000): 1) Re-release of a photon, returning to the ground state, ie, producing fluorescence. Since part of the excitation energy is dissipated as heat before the fluorescent photons are emitted, the wavelength of the fluorescence is longer than the wavelength of the absorbed light, and the chlorophyll fluorescence is generally located in the red region. 2) Do not emit photons and dissipate them directly in the form of heat (non-radiative energy dissipation). 3) Transfer energy from one chlorophyll molecule to another adjacent chlorophyll molecule. Energy is transferred between a series of chlorophyll molecules and finally reaches the reaction center. The chlorophyll molecule in the reaction center transfers energy to the electron acceptor through charge separation, thereby performing photochemistry. reaction. The above three processes are mutually competitive, and often the process with the highest rate is dominant. For many pigment molecules, fluorescence occurs in the nanosecond range, while photochemistry occurs at the ps level. Therefore, when photosynthetic organisms are in a normal physiological state, most of the light energy absorbed by the antenna pigment is used for photochemical reactions, and fluorescence is only used. a small part.
The efflux of chlorophyll b was not detected in the living cells due to the transfer of excitation energy from chlorophyll b to chlorophyll a almost 100%. At room temperature, the vast majority (about 90%) of the chlorophyll fluorescence of living organisms comes from the antenna pigment system of PSII, and the energy absorbed by photosynthetic organs is only about 3% to 5% for fluorescence (Lin Shiqing, 1996; Krause & Weis, 1991) ).
How does the saturation pulse technology work?
The so-called saturation pulse technique is to turn on a strong light with a short duration (generally less than 1 s) to turn off all electronic gates (photosynthesis is temporarily suppressed), so that the chlorophyll fluorescence is maximized. Saturation Pulse (SP) can be seen as a special case of actinic light. The stronger the actinic light, the more electrons are released by the PS II, and the more electrons accumulate at the PQ, that is, the more electronic gates in the closed state, the higher the F. When the actinic light reaches the intensity that all electronic gates are closed (no photosynthesis is possible), it is called a saturation pulse.
When the saturation pulse is turned on, the electronic gate that is originally in an open state converts the energy for photosynthesis into chlorophyll fluorescence and heat, and F reaches a maximum value.
After full dark adaptation, all electronic gates are in an open state, turn on the measurement light to get Fo, and then give a saturation pulse. All the electronic gates convert the energy used for photosynthesis into fluorescence and heat. The chlorophyll fluorescence obtained was Fm. According to Fm and Fo, the maximum quantum yield of PS II Fv/Fm=(Fm-Fo)/Fm can be calculated, which reflects the potential maximum photosynthetic capacity of plants.
When photosynthesis is carried out under illumination, only some of the electronic gates are in an open state. If a saturation pulse is given, the electron gate, which is originally in an open state, converts the energy for photosynthesis into chlorophyll fluorescence and heat, and the chlorophyll fluorescence obtained at this time is Fm'. According to Fm' and F, the actual primary light energy capture efficiency under the condition that the PSII reaction center is partially closed under illumination conditions = ΦPSII=ΔF/Fm'=(Fm'-F)/Fm', which reflects the current plant Actual photosynthetic efficiency.
When photosynthesis is carried out under illumination, only some of the electronic gates are in the off state, and the real-time fluorescence F is lower than Fm, that is, fluorescence quenching occurs. The light energy absorbed by plants has only three pathways: photosynthesis, chlorophyll fluorescence and heat. According to energy conservation: 1 = photosynthesis + chlorophyll fluorescence + heat. It can be concluded that chlorophyll fluorescence = 1 - photosynthesis - heat. That is to say, the decrease (quenching) of chlorophyll fluorescence production may be caused by an increase in photosynthesis or an increase in heat dissipation. Fluorescence quenching caused by photosynthesis is called photochemical quenching (qP); fluorescence quenching caused by heat dissipation is called non-photochemical quenching (qN or NPQ). Photochemical quenching reflects the photosynthetic activity of plants; non-photochemical quenching reflects the ability of plants to dissipate excess light energy into heat, that is, photoprotective capacity.
When the saturation pulse is turned on under illumination, the electronic gate is completely closed, and photosynthesis is temporarily suppressed, that is, photochemical quenching is completely suppressed, but the fluorescence value is still lower than Fm, that is, there is fluorescence quenching. The remaining fluorescence quenching is non-photochemical quenching. The calculation formula of the quenching coefficient is: qP=(Fm'-Fs)/Fv'=1-(Fs-Fo')/(Fm'-Fo'); qN=(Fv-Fv')/Fv=1- (Fm'-Fo')/(Fm-Fo); NPQ=(Fm-Fm')/Fm'=Fm/Fm'-1.
When F reaches steady state, the actinic light is turned off, and Far-red Light (FL) is turned on (about 3-5 s) to promote PS I to quickly absorb the electrons accumulated at the electronic gate, so that the electronic gate is Returning to the open state in a short time, F returns to the vicinity of the minimum fluorescence Fo, and the fluorescence obtained at this time is Fo'. Since it is inconvenient to measure Fo' in the field, most of the field-modulated fluorometers (except PAM-2100 and WATER-PAM) are not equipped with far-red light. At this time, it is possible to directly calculate the qP and qN by using Fo instead of Fo'. Although the obtained parameter values ​​are slightly different, the trends of qP and qN are consistent with those calculated by Fo'. Since the calculation of NPQ does not require Fo', it has been more and more widely used in the past 10 years.