CN104089937B - Marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection method in situ - Google Patents
Marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection method in situ Download PDFInfo
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Abstract
A kind of have wide range of applications, analyze fast and convenient and beneficially environmental protection marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection method in situ.Technical scheme is: it is characterized in that comprising the following steps: that (1) is by transport pump detected water sample solution；(2) water sample solution flows through Filter column；(3) flow through the water sample after Filter column and enter fluorescence pond；(4) the pulse excitation light source deep ultraviolet laser diode pumping solid laser utilizing high frequency produces the light source of continuous wave band, irradiates fluorescence pond, utilizes the spectrum device grating in detection side, fluorescence pond to carry out space light splitting, forms λ after dispersion1‑λ2Band；(5) it is positioned at the CCD photoelectric detector at detection window and gathers λ simultaneously1‑λ2Spectroscopic data, obtain wavelength light intensity two-dimension spectrum by inner transformation and time series integration；(6) data handling system analyzes marine bacteria abundance and multiformity through marine bacteria fluorescence identifying mode computation.
Description
Technical field
The invention belongs to marine bacteria in situ abundance and multiformity detection method field, a kind of have wide range of applications, analyze fast and convenient and beneficially environmental protection marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection method in situ.The fluorescence spectrum that the optical characteristics-the most various marine bacterias of " characteristic spectrum " that the fluorescence spectrum that this method utilizes laser-induced fluorescence (LIF) process to produce itself has with various marine bacterias produce during laser-induced fluorescence (LIF) is compared on different-waveband, in conjunction with marine bacteria fluorescence identifying pattern, analyze marine bacteria abundance and multiformity.
Background technology
Marine bacteria be marine ecosystems important component part.One of field of current Front Fields of Life Sciences is not only in the research of antibacterial in marine environment, is also the important component part to marine ecosystems structural research.Marine bacteria participates in degrade various marine pollutants and the process of poisonous substance, helps to maintain the balance of marine ecosystem and promotes ocean self-purification capacity；Marine bacteria is to produce new antibiotic, aminoacid, vitamin and the important Producer of other biological active substanceies；Antibacterial participates in the diagenesis of ocean, as participated in sulfur ore deposit and the formation etc. of deepwater station；During ocean becomes oil, becomes gas, antibacterial plays an important role；Sea water has bactericidal effect, is the antagonism due to marine bacteria and bacteriolysis, causes Lu Yuan pathogenic bacterium the most dead；Marine bacteria can be directly as the bait of marine economic animal；Antibacterial participates in burn into degeneration, filth and the destructive process to various oceans material；Some marine bacteria is human body or halobiontic pathogenic bacterium；Under given conditions, the accumulation of marine bacteria metabolite can poison breeding environment, and the accumulation such as ammonia and hydrogen sulfide endangers biological cultivation；The metabolic activity that can also utilize antibacterial improves the breeding environment poisoned, oxidation etc. such as ammonia, therefore to marine bacteria abundance and multiformity detection method research, for further appreciating that the ecologic structure of marine bacteria and the effect in marine carbon circulates have considerable meaning.
On research method, currently mainly using ocean scene sampling, the multiformity of marine bacteria is observed and studied to laboratory separation and Culture, the method for gene sequencing and fluorescence microscopy.Detection technique of fluorescence is constantly in considerable position owing to it has higher sensitivity in bacterial studies.The application in antibacterial of the most conventional detection technique of fluorescence mainly has fluorescence microscopy, fluidic cell fluorescence counting method, ATP fluorescence fast detection method and fluorescent protein labeling etc..Needing to be acquired sample and film-making owing to these technology exist, sample introduction requires strict, needs to make the defects such as fluorescent marker, is difficult to apply and detects antibacterial at ocean scene.Fluorescence microscopy and plating method research simultaneously proves, in oceanic environment, the most a small amount of marine bacteria can form bacterium colony by traditional fixed flat planar technology, some antibacterials are in viable but non-culturable state, the isolated culture method of application routine cannot reflect marine bacteria resource status and ecological functions comprehensively, is difficult to fully understand the abundance of antibacterial and multiformity and ecological significance thereof and the effect in marine carbon circulates.
To the pattern of lab analysis after additionally method uses field investigation and sampling, i.e. can not realize scene, the mode measured in real time, sample transportation and processing procedure are easily introduced other interfering materials, the accuracy of impact analysis.Marine bacteria abundance and multiformity detection relate to the process of certain environmental condition and complexity, therefore this process is not it cannot guarantee that have the probability that secondary is got dirty, the most important thing is due to the difficult cultivation property of marine bacteria and the inadaptability to oxygen environment, bigger plus difference between laboratory environment and marine environment, causing there is the biggest difference between measurement result and practical situation, accuracy and the reliability of its result are under suspicion.
Said method also exists following defect in various degree: 1, must complete in the lab, and application can not be on-the-spot real-time, and scope is restricted.2, analyze the persistent period long, at least need several days time.3, analyzing process numerous and diverse, condition is harsh, energy consumption is big, and the technical merit of experimenter is required height.
Due to particularity and the not Culturability of marine bacteria of marine environment, take Site Detection can reflect the situation of marine bacteria the most truly.And fluorescence measurement has measurement quick, convenient, continuous, need not the advantages such as cultivation, preferably antibacterial can be carried out in-site measurement.
nullIn recent years，Laser-induced fluorescence (LIF) (laser induced fluorescence，LIF) detection method is as a kind of novel high-sensitivity detection mode，It is developed rapidly in recent years and is widely applied，It it is the highest optical detecting method of sensitivity up to now，Remolding sensitivity 1-3 order of magnitude of common fluorescent height of Laser-Induced Fluorescence Detection Technology，The detection of fluorescent material is limited by it can reach a year nmol order of magnitude，The most even can realize Single Molecule Detection，Owing to it has highly sensitive、Quickly、Convenient、Measure continuously、Need not the advantages such as cultivation，Therefore，Local laser induced fluorescence technology is used preferably abundance and multiformity to marine bacteria to detect，Can be as marine bacteria abundance and a kind of important method of multiformity in situ detection.
Summary of the invention
The invention provides and a kind of have wide range of applications, analyze fast and convenient and beneficially environmental protection marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection method in situ.Laser Induced Fluorescence Technology is organically combined by the method with the variety classes of fluorescent material, content and ratio in bacterial body, set up marine bacteria abundance based on laser-induced fluorescence (LIF) and multiformity detection pattern, provide means for on-the-spot, fast monitored marine bacteria abundance and multiformity.
In order to reach to solve the purpose of above-mentioned technical problem, the present invention adopts the following technical scheme that
A kind of marine bacteria based on laser-induced fluorescence (LIF) abundance and multiformity detection method in situ, it is characterised in that the method step of the present invention is as follows:
(1) by transport pump detected water sample solution；
(2) water sample solution is under the effect of pump, flowing through Filter column, Filter column is internal fills MgO-Supported ferro-cobalt metal magnetic nano material, and has temperature regulating device, owing to water body containing the bigger phytoplankton of particle diameter, therefore can filter out, by Filter column, the phytoplankton that particle diameter is big.
(3) water sample after Filter column is flowed through, enter fluorescence pond, the window material in fluorescence pond, employing Nano diamond material near exciting light source, main elimination water body Raman scattering, the silica glass of graphene film material, the main fluorescent scattering eliminating water body are posted in employing near detection window side.
(4) the pulse excitation light source-deep ultraviolet laser diode pumping solid laser utilizing high frequency produces continuous wave band, irradiates fluorescence pond, utilizes the spectrum device-grating in detection side, fluorescence pond to carry out space light splitting, forms λ after dispersion1-λ2Band；
(5) it is positioned at the CCD photoelectric detector at detection window and gathers λ simultaneously1-λ2Spectroscopic data, obtain wavelength-light intensity two-dimension spectrum by inner transformation and time series integration；
(6) first, the detection error may brought for picphytoplankton in water body, utilize the pulse excitation light source of high frequency, excite employing 400nm, launch and use 685nm, CCD photoelectric detector gathers spectroscopic data, as algae and water factor of influence；
(7) secondly, for the impact of CDOM in water body, the characteristic fluorescence (exciting 340/ transmitting 430) of oneself is had according to CDOM, also with the pulse excitation light source utilizing high frequency, excite employing 340nm, launching and use 430nm, CCD photoelectric detector gathers spectroscopic data, as water body CDOM factor of influence；
(8) last, pulse excitation light source-deep ultraviolet laser the diode pumping solid laser utilizing high frequency produces the light source 275-450nm of continuous wave band, irradiate fluorescence pond, spectrum device-the grating in detection side, fluorescence pond is utilized to carry out space light splitting, the band of 280-535nm is collected after dispersion, it is positioned at the CCD photoelectric detector at detection window and gathers the spectroscopic data of 280-535nm simultaneously, obtain wavelength-light intensity two-dimension spectrum by inner transformation and time series integration；
(9) data handling system uses the marine bacterial diversity fluorescence identifying pattern of foundation, analyzes marine bacteria abundance and multiformity through marine bacteria fluorescence identifying mode computation.
In the present invention, also there is techniques below feature: water sample flow is 5.0-10.0ml/min.
In the present invention, also there is techniques below feature: Filter column is internal fills MgO-Supported ferro-cobalt metal magnetic nano material, temperature range 10-15 DEG C of temperature regulating device.
In the present invention, also there is techniques below feature: utilize the pulse excitation light source-deep ultraviolet laser diode pumping solid laser of high frequency, there is volume little, without water-cooled, feature, the high-frequency range 100-150KHz such as fluctuation noise is little, power supply is simple.Additionally use continuous agitation formula pulse excitation light source, improve certainty of measurement, typical fluorescent material in employing deep ultraviolet excites and can excite bacterial body simultaneously, it is not necessary to carry out the steps such as dyeing to a certain extent.
In the present invention, also there is techniques below feature: the marine bacterial diversity fluorescence identifying pattern of described foundation is to be contained within some compositions that can excite generation fluorescence based on bacterial body, use in the middle of the process of via Laser-induced Fluorescence Method detection marine bacteria, " characteristic spectrum " will be sent, these compositions different classes of, content or the difference of ratio, cause fluorescence peak that bigger difference occurs, by to the collection of fluorescence signal and analysis, Phylogenetic diversity of bacteria is differentiated by the method using main constituent fluorescent matrix, simultaneously take account of environmental factors such as algae, CDOM, and inorganic particle likely produces impact to fluorescence, use main constituent fluorescence spectrum analysis method, set up marine bacterial diversity fluorescence identifying pattern.
In the present invention, also having techniques below feature: pump is peristaltic pump, pipeline uses polytetrafluoroethylmaterial material to make.
In the present invention, also there is techniques below feature: CCD detection element uses U.S.'s marine optics back-illuminated type two-dimensional array CCD spectrogrph.
In the present invention, also there is techniques below feature: microcomputer Data Analysis Services system uses marine bacterial diversity fluorescence identifying pattern, this model has good correcting action for the factor of influence such as algae and CDOM, can eliminate these factors of influence to computational analysis marine bacteria abundance and multifarious impact.
The effect of the present invention is:
The present invention uses via Laser-induced Fluorescence Method detection marine bacteria abundance and multiformity, it it is the important component part in current marine eco-environment monitoring system, method utilizes fluorescent material in marine bacteria body all can send the phenomenon of " characteristic spectrum ", by laser Induced Fluorescence Technology and the variety classes of fluorescent material in bacterial body, content and ratio organically combine, the plane dispersion map with wavelength as abscissa with spectral sequence as vertical coordinate is obtained by beam splitting system, import CCD detection element, optical signal processes through photodetection and is converted to signal of telecommunication output, the output signal of telecommunication is changed through weak signal amplification circuit, being amplified to certain voltage amplitude send the A/D ALT-CH alternate channel of data processing section to quantify, entirely composed after time series Integral Processing.
By the full spectrum obtained after time series Integral Processing, according to " characteristic spectrum " of fluorescent material in various bacterial bodies, through being analyzed obtaining marine bacteria abundance and multiformity to full spectrum through marine bacterial diversity fluorescence identifying mode software.
The present invention utilizes via Laser-induced Fluorescence Method detection marine bacteria abundance and multiformity, and method can accurately, continuously, quickly be analyzed and test marine bacteria abundance and multiformity, can in rugged environment long-term reliably working.
The present invention is described further with embodiment below in conjunction with the accompanying drawings.
Accompanying drawing explanation
Fig. 1 is the inventive method operation principle flow chart；
Fig. 2 is the structure of the detecting device schematic diagram that the inventive method is used；
Fig. 3 is the laser-induced fluorescence (LIF) recognition mode figure of marine bacterial diversity.
Label declaration in figure: 1. water sample；2. water sample pump；3. pulse excitation light source；4. Nano diamond；5. fluorescence pond；6. post the silica glass of Graphene；7.CCD photodetector；8. control part；9. data processing section；10. waste collection；11. Filter columns；12. spectrum light-splitting devices.
Detailed description of the invention
See Fig. 1, Fig. 2,
The method step of the present invention is as follows:
(1) detected water sample solution is carried by pump 2；
(2) water sample solution is under the effect of pump 2, flowing through Filter column 11, Filter column 11 is internal fills MgO-Supported ferro-cobalt metal magnetic nano material, and has temperature regulating device, owing to water body containing the bigger phytoplankton of particle diameter, therefore can filter out, by Filter column, the phytoplankton that particle diameter is big.
(3) water sample after Filter column is flowed through, enter fluorescence pond 5, the window material in fluorescence pond, employing Nano diamond 4 material near exciting light source, main elimination water body Raman scattering, the silica glass 6 of graphene film material, the main fluorescent scattering eliminating water body are posted in employing near detection window side.
(4) the pulse excitation light source 3-deep ultraviolet laser diode pumping solid laser utilizing 100KHz produces continuous wave band, irradiate fluorescence pond 5, utilize the spectrum device-grating in detection side, fluorescence pond to carry out space light splitting, after dispersion, form the band of λ 1-λ 2；
(5) it is positioned at the CCD photoelectric detector 7 at detection window and gathers the spectroscopic data of λ 1-λ 2 simultaneously, obtain wavelength-light intensity two-dimension spectrum by inner transformation and time series integration；
(6) first, the detection error may brought for picphytoplankton in water body, utilize the pulse excitation light source 3 of 100KHz, excite employing 400nm, launch and use 685nm, CCD photoelectric detector 7 gathers spectroscopic data, as algae and water factor of influence；
(7) secondly, for the impact of CDOM in water body, the characteristic fluorescence (exciting 340/ transmitting 430) of oneself is had according to CDOM, also with the pulse excitation light source 3 utilizing 100KHz, excite employing 340nm, launching and use 430nm, CCD photoelectric detector 7 gathers spectroscopic data, as water body CDOM factor of influence；
(8) last, the pulse excitation light source 3-deep ultraviolet laser diode pumping solid laser utilizing 100KHz produces the light source 275-450nm of continuous wave band, irradiate fluorescence pond, the spectrum device 12-grating detecting side in fluorescence pond 5 is utilized to carry out space light splitting, the band of 280-535nm is collected after dispersion, it is positioned at the CCD photoelectric detector 7 at detection window and gathers the spectroscopic data of 280-535nm simultaneously, obtain wavelength-light intensity two-dimension spectrum by inner transformation and time series integration；
(9) data handling system uses the marine bacterial diversity fluorescence identifying pattern of foundation, analyzes marine bacteria abundance and multiformity through marine bacteria fluorescence identifying mode computation.
Described method water sample flow is 5.0-10.0ml/min.
Described method Filter column is internal fills MgO-Supported ferro-cobalt metal magnetic nano material, and has temperature regulating device, temperature range 10-15 DEG C.
Described method utilizes the pulse excitation light source-deep ultraviolet laser diode pumping solid laser of 100KHz, there is volume little, without water-cooled, the feature such as fluctuation noise is little, power supply is simple, additionally use continuous agitation formula 100KHz pulse excitation light source, improve certainty of measurement to a certain extent, typical fluorescent material in employing deep ultraviolet excites and can excite bacterial body simultaneously, it is not necessary to carry out the steps such as dyeing.
The window material in described method fluorescence pond, near the employing Nano diamond material of exciting light source, main elimination water body Raman scattering, the silica glass of thin-film material-Graphene, the main fluorescent scattering eliminating water body are posted in the employing near detection window side.
The marine bacterial diversity fluorescence identifying pattern of described foundation is to be contained within some compositions that can excite generation fluorescence based on bacterial body, use in the middle of the process of via Laser-induced Fluorescence Method detection marine bacteria, " characteristic spectrum " will be sent, these compositions different classes of, content or the difference of ratio, cause fluorescence peak that bigger difference occurs, by to the collection of fluorescence signal and analysis, Phylogenetic diversity of bacteria is differentiated by the method using main constituent fluorescent matrix, simultaneously take account of environmental factors such as algae, CDOM, and inorganic particle likely produces impact to fluorescence, use main constituent fluorescence spectrum analysis method, set up marine bacterial diversity fluorescence identifying pattern.
Described method pump is peristaltic pump, and pipeline uses polytetrafluoroethylmaterial material to make.
Described method CCD detection element uses U.S.'s marine optics back-illuminated type two-dimensional array CCD spectrogrph.
Described method microcomputer Data Analysis Services system uses marine bacterial diversity fluorescence identifying pattern, this model has good correcting action for the factor of influence such as algae and CDOM, can eliminate these factors of influence to computational analysis marine bacteria abundance and multifarious impact.
The operation principle of the present invention is:
Use via Laser-induced Fluorescence Method that antibacterial classification is differentiated, it is primarily due to bacterial body and is contained within some compositions that can excite generation fluorescence, such as tryptophan, tyrosine, phenylalanine, DNA, RNA, NADH, NADPH, FAD and exoenzyme etc., different classes of, the content of these compositions or the difference of ratio, cause fluorescence peak bigger difference occur.The excitation wavelength range of these materials is from 250-450nm, and the transmitting wavelength peak-peak producing fluorescence occurs between 280-540nm.
The maximum excitation wavelength of marine bacteria inside and outside difference fluorescent target thing and transmitting wavelength, such as table 1:
Table 1: fluorescent target thing optical signature
By using Ultra-Violet Laser induced fluorescence technology for detection marine bacteria to excite the characteristic fluorescence signal of lower generation at different wave length, by the collection of fluorescence signal and analysis, finding out the fluorescent differences between different bacterium, it is achieved the multiformity of marine bacteria is detected.Detection needs to set up marine bacterial diversity fluorescence identifying pattern, and marine bacteria fluorescence identifying pattern mainly uses the method for main constituent fluorescent matrix to differentiate Phylogenetic diversity of bacteria.
The computational methods that main employing is following:
If=φ I0·(1-10- ε· l · c)
In formula, IfBeing fluorescence intensity, φ is quantum yield, I0Being incident intensity, ε is molar absorptivity, and l is the optical path of sample, and c is the molar concentration of fluorescent material.
Simultaneously, in view of environmental factors such as algae, CDOM and inorganic particle, fluorescence likely produced impact, use main constituent fluorescence spectrum analysis method, it is analyzed processing to the fluorescence signal gathered, find the best identified index of marine bacteria fluorescence classification, set up the laser-induced fluorescence (LIF) recognition mode (such as Fig. 3) of marine bacterial diversity.
According to above-mentioned reaction principle, use in the middle of the process of via Laser-induced Fluorescence Method detection marine bacteria, all can send " characteristic spectrum ".Laser Induced Fluorescence Technology organically combines with the variety classes of fluorescent material, content and ratio in bacterial body, the plane dispersion map with wavelength as abscissa with spectral sequence as vertical coordinate is obtained by beam splitting system, again by CCD detection element, the most entirely composed with spectrum direct access mode, be analyzed marine bacteria abundance and multiformity being detected to full spectrum through software.
nullIn recent years，Laser-induced fluorescence (LIF) (laser induced fluorescence，LIF) detection method is as a kind of novel high-sensitivity detection mode，It is developed rapidly in recent years and is widely applied，It it is the highest optical detecting method of sensitivity up to now，Remolding sensitivity 1-3 order of magnitude of common fluorescent height of Laser-Induced Fluorescence Detection Technology，The detection of fluorescent material is limited by it can reach a year nmol order of magnitude，The most even can realize Single Molecule Detection，Owing to it has highly sensitive、Quickly、Convenient、Measure continuously、Need not the advantages such as cultivation，Therefore，Local laser induced fluorescence technology is used preferably abundance and multiformity to marine bacteria to detect，Can be as marine bacteria abundance and a kind of important method of multiformity in situ detection，It is a kind of ideal means，There is scene、Quickly，Easy，Sensitive feature.
CCD (Charge Coupled Devices) i.e. charge coupling device, it is that one represents light intensity magnitude with the quantity of electric charge, close mode with Rhizoma Nelumbinis and transmit the device of the quantity of electric charge, it has self-scanning, spectral region width, dynamic range is big, volume is little, low in energy consumption, life-span length, high reliability. and CCD two dimension linear array being placed on spectrum face, single exposure is achieved with whole spectrum. and it has the special feature that:
1. spectral region width, quantum efficiency is high (up to more than 90%), and dark current is little, noise is low, can realize multiple tracks and gather data simultaneously, its construction features, data can be gathered, it is thus achieved that Wavelength strength two dimension spectrogram as the full spectrum direct-reading detecting element in spectroanalysis instrument simultaneously.
2. can detect the faintest spectral signal by time series integration.Obtain time-resolved spectral signal, and then realize the quick analysis of spectrum.Spectral signal can be acquired by being combined CCD detection element with laser-induced fluorescence (LIF) light path system, after time series integration, the spectral intensity two-dimension spectrum figure with wavelength change can be obtained, use marine bacterial diversity fluorescence identifying pattern can analyze marine bacteria abundance and multiformity by processing software combination.
CCD detection element advantage is that all of pixel (N number of) exposes simultaneously, whole spectrum can obtain simultaneously, fast N times of the total time of same section of spectrum is detected than general single channel spectroscopic system, during absorbing whole spectrum, need not spectrogrph carry out mechanical scanning, there is not the unduplicated error of wavelength caused due to mechanical system；Decrease the relative line intensity error that intensity of light source instability causes；Can measure spectrum change dynamic process.
Whole system is light, mechanical, electrical, the Photodetection system of calculation integration.It is divided into six parts by operational module: Part I is laser-induced fluorescence (LIF), is primarily directed to bacterial body and is contained within some compositions that can excite generation fluorescence；Part II is optical spectroscopic components of system as directed, is mainly spatially separated from out by fluorescence bands by light-splitting device, it then follows optical dispersion principle；Part III is opto-electronic conversion and amplifier section, based on photoelectric effect principle, the most separate optical signal is transformed into the signal of telecommunication, and we mainly use CCD detection element；Part IV is data acquisition, record part, and this is partially completed the collection of the signal of telecommunication, A/D conversion, transmits and store: Part V is control part, the sequencing contro during main responsible photoelectric signal collection；Part VI is software processing part, is analyzed mainly in combination with marine bacterial diversity fluorescence identifying pattern and calculates.
The composition that can excite generation fluorescence carries out real-time qualitative, the method for quantitative analysis is current very effective marine bacteria abundance and multiformity in-situ detection method, is important developing direction by integrated laser induced fluorescence, light path system, CCD detecting element, bacterial body to be contained within some.
The above, be only presently preferred embodiments of the present invention, is not the restriction that the present invention makees other form, and any those skilled in the art are changed possibly also with the technology contents of the disclosure above or are modified as the Equivalent embodiments of equivalent variations.But every without departing from technical solution of the present invention content, any simple modification, equivalent variations and remodeling above example made according to the technical spirit of the present invention, still fall within the protection domain of technical solution of the present invention.
Claims (7)
1. a marine bacteria based on laser-induced fluorescence (LIF) abundance and multifarious detection side in situ
Method, it is characterised in that comprise the following steps:
(1) by transport pump detected water sample solution；
(2) water sample solution is under the effect of pump, flows through Filter column, and Filter column is internal fills oxygen
Change magnesium-supported ferro-cobalt metal magnetic nano material, and there is temperature regulating device, owing to water body containing
There is the bigger phytoplankton of particle diameter, therefore can filter out big the swimming of particle diameter by Filter column and plant
Thing；
(3) flow through the water sample after Filter column, enter fluorescence pond, the window material in fluorescence pond,
Near the employing Nano diamond material of exciting light source, main elimination water body Raman scattering, lean on
The silica glass of graphene film material is posted in the employing of nearly detection window side, mainly eliminates water body
Fluorescent scattering；
(4) the pulse excitation light source-deep ultraviolet laser diode-pumped solid laser of high frequency is utilized
Device produces continuous wave band, irradiates fluorescence pond, utilizes the spectrum light splitter in detection side, fluorescence pond
Part-grating carries out space light splitting, forms λ after dispersion1-λ2Band；
(5) it is positioned at the CCD photoelectric detector at detection window and gathers λ simultaneously1-λ2Spectrum
Data, obtain wavelength-light intensity two-dimension spectrum by inner transformation and time series integration；
(6) data handling system uses the marine bacterial diversity fluorescence identifying pattern of foundation,
Marine bacteria abundance and multiformity, described sea is analyzed through marine bacteria fluorescence identifying mode computation
Ocean Phylogenetic diversity of bacteria fluorescence identifying pattern allows for environmental factors and fluorescence likely produces shadow
Ring, use main constituent fluorescence spectrum analysis method, be analyzed processing to the fluorescence signal gathered,
Find the best identified index of marine bacteria fluorescence classification, set up the laser of marine bacterial diversity
Induced fluorescence recognition mode.
Marine bacteria based on laser-induced fluorescence (LIF) the most according to claim 1 is the richest
Degree and multifarious detection method, it is characterised in that the pulse excitation light source of described high frequency, excite
Using 400nm, launch and use 685nm, CCD photoelectric detector gathers spectroscopic data, as
Algae and water factor of influence；
The pulse excitation light source of described high frequency, excites employing 340nm, launches and use 430nm,
CCD photoelectric detector gathers spectroscopic data, as water body CDOM factor of influence；
The pulse excitation light source of described high frequency-deep ultraviolet laser diode pumping solid laser produces
Raw continuous wave band 275-450nm, irradiates fluorescence pond, utilizes the spectrum in detection side, fluorescence pond
Light-splitting device-grating carries out space light splitting, collects the band of 280-535nm, position after dispersion
CCD photoelectric detector at detection window gathers the spectroscopic data of 280-535nm simultaneously, logical
Cross inner transformation and time series integration obtains wavelength-light intensity two-dimension spectrum.
Marine bacteria based on laser-induced fluorescence (LIF) the most according to claim 1 is the richest
Degree and multifarious detection method, it is characterised in that described water sample flow is
5.0-10.0ml/min。
Marine bacteria based on laser-induced fluorescence (LIF) the most according to claim 1 is the richest
Degree and multifarious detection method, it is characterised in that described Filter column is internal fills MgO-Supported
Ferro-cobalt metal magnetic nano material, temperature range 10-15 DEG C of temperature regulating device.
Marine bacteria based on laser-induced fluorescence (LIF) the most according to claim 1 is the richest
Degree and multifarious detection method, the described pulse excitation light source-deep ultraviolet utilizing high frequency swashs
The high-frequency range 100-150KHz of optical diode light-pumped solid state laser.
Marine bacteria based on laser-induced fluorescence (LIF) the most according to claim 1 is the richest
Degree and multifarious detection method, it is characterised in that described pump is peristaltic pump, pipeline uses poly-
Tetrafluoroethene material is made.
Marine bacteria based on laser-induced fluorescence (LIF) the most according to claim 1 is the richest
Degree and multifarious detection method, it is technically characterized in that CCD detection element uses U.S. ocean
Optical back illuminated two-dimensional array CCD spectrogrph.
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