CN107389638A - A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device - Google Patents
A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device Download PDFInfo
- Publication number
- CN107389638A CN107389638A CN201710613328.4A CN201710613328A CN107389638A CN 107389638 A CN107389638 A CN 107389638A CN 201710613328 A CN201710613328 A CN 201710613328A CN 107389638 A CN107389638 A CN 107389638A
- Authority
- CN
- China
- Prior art keywords
- image
- imaging
- microscopic
- light
- spectral imaging
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N2021/6417—Spectrofluorimetric devices
Abstract
The invention discloses a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method, the invention also discloses a kind of microscopic fluorescent spectral imaging marine phytoplankton original position Classification and Identification device, it is intended to fluorescence microscopy and spectral imaging technology being combined, development can apply the quick micro-fluorescence spectrum Detection Techniques of algae under marine environment, obtain the pattern matrix of close algae under different wave length, by the D coordinates value of embodiment of the spectrum in colorimetry under different excitation wavelengths, additional information as algae image, the different images of same species to being obtained under different wave length carry out feature extraction, train grader, the Classification and Identification and statistics of similar algae species in the form of realizing to inside same monoid.Imaging system is divided into lab scenario and underwater scheme, and two systems consider the actual requirement of the checking that can complement each other under different situations and different accuracy.
Description
Technical field
The present invention relates to a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device.
Background technology
The monitoring of phytoplankton species and quantity is a routine basic work in scientific research of seas.Quickly, it is accurate
Really, phytoplankton automatic identification in situ and the exploration of analysis method and its foundation of system, have important theoretical and actual
Meaning；Phytoplankton sort research can be not only enriched, to realize to halomereid with environmental change, generation, development, extinction
The real-time detection of process, and can be widely applied to the neck such as marine ecological investigation, environmental monitoring, aquaculture, red tide, geology
Domain.This is the target pursued jointly of going together both at home and abroad.
At present the various phytoplanktons developed automatically analyze with recognition methods：Absorption spectrometry is convenient, and cost is relatively low,
It can automatically analyze, but be influenceed greatly by environmental disturbances and algae kind physiological status, sensitivity is low, and resolution ratio is low, it is impossible to analyzes kind；Liquid
Phase chromatography is widely accepted, and can be automatically analyzed, but complex steps, and pre-treatment step is complicated, can only classify to main kind of,
Kind can not be analyzed；Flow cytometric methods are quick, high-volume, can be simultaneously qualitative, quantitative, main suitable for miniature phytoplankton
Analysis on Groups to be done, but sample must be kept fresh, needs professional to operate, determination data repeatability is low, and resolution ratio is not high, expensive,
Instrument is heavy；The species that molecular probe method can be difficult to differentiate to conventional meanses is identified, but process is cumbersome, only stops at present
In laboratory stage.
Based on what traditional microscopy grew up planktonic organism is carried out using image procossing and mode identification technology
Automatic classification with count be current study hotspot.With the quick hair of the method for various image characteristics extractions and grader
Exhibition, for the image information acquired in microscope, realizes the method and algorithm to the automatic Classification and Identification of algae of different shape
Research be current common Research Thinking, but it is this excited based on single light source under image-recognizing method for same monoid
The similar species identification of internal form still has larger difficulty, and automated graphics Classification and Identification is even more to be difficult to.
Spectral imaging technology just refers to that corresponding image sequence will be gathered at different wavelengths, a width at a particular wavelength
The different light values of each point are recorded on image, and the image sequence formed at different wavelengths then constitutes analyzed sample
The overall picture of optics behavior under different wave length.The technology can not only provide tradition using the image of upper sample and qualitative, quantitative point
Information is analysed, and composite distribution figure can be provided, so as to obtain positioning analysis information, fully meets the multidimensional that high and new technology proposes
Comprehensive analysis requirement.
Microspectrum imaging technique just refers to that corresponding pattern matrix will be gathered at different wavelengths under microscopy environment,
The different light values and chromatic value of each point are recorded on piece image at a particular wavelength, are formed at different wavelengths
Pattern matrix then constitute the overall picture of optics behavior under analyzed sample different wave length.What it was provided is not only measured object form
Upper information, while abundant spectral information can also be provided.
The present invention fluorescence microscopy and spectral imaging technology are combined, development can apply marine environment under algae it is fast
Fast micro-fluorescence spectrum Detection Techniques, the pattern matrix of close algae under different wave length is obtained, by spectrum under different excitation wavelengths
The D coordinates value of embodiment in colorimetry, as the additional information of algae image, to the same thing obtained under different wave length
The different images of kind carry out feature extraction, train grader, the similar algae species in the form of realizing to inside same monoid
Classification and Identification and statistics.
The content of the invention
The technical problem to be solved in the present invention is the deficiency for more than, there is provided a kind of microscopic fluorescent spectral imaging Marine Planktonic
Plant original position classifying identification method and device, fluorescence microscopy and spectral imaging technology can be combined, realize for
Swim under water quick, accurate, real-time automatic identification and quantitative analysis, solve the problem of the Classification and Identification of marine algae.
To solve above technical problem, the present invention uses following technical scheme：Float a kind of microscopic fluorescent spectral imaging ocean
Plant original position classifying identification method is swum, includes feature extraction and classifying identification step, the image of fluorescent microscopic imaging step, image
Colourity is composed and fluorescence spectrum correlation analysis step, the in site measurement parameter step that to determine sample collection relevant.
A kind of prioritization scheme, fluorescence microscopy and spectral imaging technology are combined, and development can be applied under marine environment
The quick micro-fluorescence spectrum Detection Techniques of algae, the pattern matrix of close algae under different wave length is obtained, by different excitation waves
The D coordinates value of long lower embodiment of the spectrum in colorimetry, as the additional information of algae image, to being obtained under different wave length
Same species different images carry out feature extraction, train grader, by realize to inside same monoid in the form of it is similar
The Classification and Identification and statistics of algae species.Imaging system is divided into lab scenario and underwater scheme, and two systems consider difference
The actual requirement of the checking that can complement each other in situation and different accuracy.
Further, fluorescent microscopic imaging step includes laboratory microscopic fluorescent spectral imaging step, and the laboratory shows
Micro- fluorescence spectrum image-forming step be tunable wave length light-pulse generator excite under fluoroscopic image system build and acquisition technique, pin
Enter the repacking of line light source, including the design of tunable excitation source to inverted fluorescence microscope, conduct, pass through via optical fiber beam
Irradiated by the light beam regulation of lens group, light beam；Fluorescent microscopic imaging and image collection system are arranged using matching somebody with somebody for fluorescence microscope
It is standby.Utilized in the laboratory microscopic fluorescent spectral imaging step and adjust Q Nd:YAG laser, wavelength is entered through nonlinear crystal
Row tuning or xenon lamp are as activating system, imaging and image capturing system using Leica DMILM fluorescence inverted microscopes.
Further, fluorescent microscopic imaging step includes underwater microscopic fluorescent spectral imaging step, described underwater micro- glimmering
Light light spectrum image-forming step, it is contemplated that stability, anti-interference, pressure-resistant corrosion-resistant, the compactedness of light path design of the light path of instrument
Etc. factor, while Flow Sampling system, including peristaltic pump, sample feeding pipe, flowing sample room and waste liquid displacer are realized in design；It sets
Meter is to realize needs that underwater in-situ detects in real time, need to consider sample cell structure, the speed of sample introduction, peristaltic pump when
Sequence designs, the influence of the factor such as transmitted light intensity.
Further, the feature extraction and classifying identification step of image, image chroma spectrum and fluorescence spectrum correlation are established
To realize the method for Spectra feature extraction；
Image chroma compose with fluorescence spectrum correlation analysis step, realize the similar species identification of form inside same monoid
And quantitative statisticses.
Further, exciting light uses face mode of excitation in the fluorescent microscopic imaging step, while utilizes Wavelength tunable
Humorous light source can obtain corresponding image sequence under different wavelength, reflect the colorimetry information of spatial points.
Further, the signal of transmission laser background and fluorescence passes through high-pass filter in the fluorescent microscopic imaging step
Afterwards, laser background removal can be carried out data acquisition using EMCCD sequential control systems are imaged, realize that dark background is imaged, its is glimmering
Optical information is reflected that form texture emerges from the form of images with RGB color and gray value.
Further, this method by establishing the colorimetry information of certain area on different excitation wavelength hypographs step by step
Intensity+ RGB value ~ λ and fluorescence spectrum correlation, determine the corresponding pass between colorimetry Information Spectrum and spectrum
System；
The certain area includes whole image, topography, single pixel point.
The present invention also provides a kind of microscopic fluorescent spectral imaging marine phytoplankton original position Classification and Identification device, including excites
Light-source system, flowing sample cell, CCD imaging systems；
The excitation light source system includes light supply apparatus and optical system；
The optical system includes grating, beam expanding lens, lens, micro imaging system and optical filter, and wherein excitation wavelength is in 220-
550nm is adjustable or optional, and can carry out face to sample in the range of 1mm × 1mm after beam expanding lens expands excites, through micro-imaging
It can be imaged after system in μm precision.
A kind of prioritization scheme, the light supply apparatus include excitation source, imaging and image capturing system；
The imaging and image capturing system use microscopic fluorescent spectral imaging system in situ, and use can send feux rouges, green glow
Pigtail-LEDs with blue light is as excitation source, using the digital camera of 1392 × 1040 pixels as detector；
The light that 2 red-light LEDs, 3 green light LEDs and 2 blue-ray LEDs are sent is converged into special optical material using micro optical fiber
On, then it is connected to excite the phytoplankton fluorescence in Flow Sampling sample cell with micro imaging system.
After the present invention uses above technical scheme, compared with prior art, there is advantages below：
1st, fluorescence microscopy and spectral imaging technology are combined by the present invention, are realized quick, accurate, real for swimming under water
When automatic identification and quantitative analysis, solve the problem of the Classification and Identification of marine algae.
2nd, the present invention is influenceed small, high sensitivity by environmental disturbances and algae kind physiological status, can analyze kind；
3rd, Flow Sampling system design is applied in micro imaging system, while the applicable Yu Haiyang condition of work of Aided design
Under light-pulse generator and EMCCD sequential control system.
4th, the feature extraction and classifying recognition methods that algae identification is carried out using fluoroscopic image array is studied, and establishes pattern colour
Degree spectrum to realize the method for Spectra feature extraction, realizes the similar thing of form inside same monoid with fluorescence spectrum correlation
Kind identification and quantitative statisticses.
The present invention is described in detail with reference to the accompanying drawings and examples.
Brief description of the drawings
The step of accompanying drawing 1 is recognition methods of the present invention is schemed；
Accompanying drawing 2 is another block diagram of recognition methods of the present invention；
The step of accompanying drawing 3 is 1 recognition methods of the embodiment of the present invention is schemed；
The step of accompanying drawing 4 is 2 recognition methods of the embodiment of the present invention is schemed；
Accompanying drawing 5 is the structural representation of identification device of the present invention；
In figure,
1- excitation light source systems, 11- light supply apparatuses, 12- optical systems, 2- flowing sample cells, 3-CCD imaging systems, 31- filtering
Piece, 32- object lens, 33- eyepieces, 34- detectors, 35- data processing equipments.
Embodiment
In order to which technical characteristic, purpose and the effect of the present invention is more clearly understood, now control illustrates this hair
Bright embodiment.
A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method of embodiment 1
As Figure 1-3, the present invention provides a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method, bag
Include following steps：
Step 1, fluorescent microscopic imaging step；
Step 2, the feature extraction and classifying identification step of algae image；
Step 3, image chroma spectrum and fluorescence spectrum correlation analysis step；
Step 4, in site measurement determines the relevant parameter step of sample collection.
Wherein,
Step 1, fluorescent microscopic imaging step：Including laboratory microscopic fluorescent spectral imaging step,
Including laboratory microscopic fluorescent spectral imaging step：By the use of xenon lamp as excitation source, fallen using Leica DMILM fluorescence
Put microscopical imaging and image capturing system.Exciting light uses face mode of excitation.Face mode of excitation excites whole sight simultaneously
Region is examined, while detects the fluorescence signal of whole region, image taking speed is fast, is adapted to the change procedure of reflection algae in real time.Together
Shi Liyong Wavelength tunable light sources can obtain corresponding image sequence under different wavelength, reflect the colorimetry letter of spatial points
Breath.
Optical system includes grating, beam expanding lens, lens, micro imaging system and optical filter etc. in addition to light source.Excitation wavelength
It is adjustable or optional in 220-550nm, sample progress face can be excited in the range of 1mm × 1mm after beam expanding lens expands, through aobvious
It can be imaged after micro- imaging system in μm precision, optical filter can filter to veiling glare.
Adjust Q Nd:YAG laser, is tuned through nonlinear crystal to wavelength or xenon lamp is as activating system, uses
IXon Imaging EMCCDs are as detecting system.Exciting light uses face mode of excitation.Face mode of excitation excites whole simultaneously
Viewing area, while the fluorescence signal of whole region is detected, image taking speed is fast, is adapted to the change procedure of reflection algae in real time.Together
Shi Liyong tunable wave lengths LASER Light Source can obtain corresponding image sequence under different wavelength, reflect the spectrum of spatial points
Information.This scheme can obtain high resolution, the image and spectral information of abundant, the quick algae of projection in real time of spectral information, obtain
To algae form, texture, spectral information, it is to realize to carry out frustule shell surface to be advantageous to by image segmentation and contours extract
Automatic identification, the content distribution of different algae kind two-dimensional spaces in observation area is obtained, while pass through its spectrum peak and intensity
Carry out the quantitative analysis of the suspension three dimensions content of algae.The method is due to laser and the volume and price and right of imaging
The factors such as environmental requirement, laboratory comparative study scheme and computer classes expert database can be used as.
Step 2, the feature extraction and classifying identification step of algae image：
To the light spectrum image-forming data of acquisition, what laboratory micro imaging system obtained is the sharp of different wave length in certain spectral region
The fluorescence distribution image for the series that lights, determine the point position-finding light quantity of point at different wavelengths for a certain on image respectively
Value, so that it may to obtain the curve of spectrum corresponding to the point that top is illustrated (abscissa is that wavelength, ordinate are light values).
According to the curve of spectrum of every bit position, it can not only determine that this on sample (thus can make to the light value of some determination wavelength
The quantitatively or semi-quantitatively analysis of sample this algae component), and can be sentenced according to the peak valley wavelength location of its curve of spectrum
The extreme value (thus qualitative analysis can be made) that this of reading sample is to different wavelengths of light scholarship and moral conduct.
Step 3, image chroma spectrum and fluorescence spectrum correlation analysis step
The present invention by establishing certain area on different excitation wavelength hypographs step by step（Whole image → topography → single picture
Vegetarian refreshments）Colorimetry information（Intensity+ RGB value ~ λ）With the correlation of fluorescence spectrum, colorimetry information is determined
Corresponding relation between spectrum and spectrum.
Step 4, in site measurement determines the relevant parameter step of sample collection, including：The determination of number of arrays and implementation,
The determination of data compression ratio and implementation.
The determination of number of arrays and implementation：The training required for effective recognition result can be reached by experimental study acquisition
The minimum samples and pattern matrix number of sample（Number of wavelengths）, the wave-length coverage of tunable optical source is determined, wavelength interval, is realized
Enough spectral informations are obtained with minimum number of wavelengths.
The determination of data compression ratio and implementation：The data compression ratio of multidimensional image array is determined, to meet data
The data compression ratio of minimum load in transmission engineering.
Embodiment 2：A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method
As shown in figure 4, the present invention provides a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method, including
Following steps：
Step 1, fluorescent microscopic imaging step；
Step 2, the feature extraction and classifying identification step of algae image；
Step 3, image chroma spectrum and fluorescence spectrum correlation analysis step；
Step 4, in site measurement determines the relevant parameter step of sample collection.
Wherein：
Step 1, fluorescent microscopic imaging step：Including underwater fluorescent microscopic imaging step；
Underwater fluorescent microscopic imaging step uses microscopic fluorescent spectral imaging system in situ, use can send feux rouges, green glow and
The pigtail-LEDs of blue light is as excitation source, using the digital camera of 1392 × 1040 pixels as detector.Using micro-
Optical fiber converges to the light that 2 red-light LEDs, 3 green light LEDs and 2 blue-ray LEDs are sent on special optical material, Ran Houyu
Micro imaging system is connected to excite the phytoplankton fluorescence in Flow Sampling sample cell.So, can both send respectively it is red,
Green or blue light, mixed white light can be obtained again, be the core component of microscopic fluorescent spectral imaging.This scheme is expected to be carried in
On AUV or ROV, ocean real-time in-situ detection is realized.Four color excitation sources of red, green, blue and white simultaneously, can fetching portion spectrum letter
Breath, then add partial spectrum information on the basis of fluorescent microscopic imaging, and this information and tunable laser source are obtained
Full spectral information is compareed, and is extracted three spectral informations and is carried out quantifying for algae content.It as shown is the light of LEDS light sources
Lu Tu.
Optical system includes beam expanding lens, lens, micro imaging system and optical filter etc. in addition to light source.Optical maser wavelength is in 350-
550nm is adjustable or optional, and can carry out face to flowing sample room in the range of 1mm × 1mm after beam expanding lens expands excites, through aobvious
It can be imaged after micro- imaging system in μm precision, optical filter can filter to veiling glare.
After the signal of transmission laser background and fluorescence is by high-pass filter, laser background removal can be utilized imaging
EMCCD carries out data acquisition.Most of ambient light can so be restrained, can be achieved dark background imaging, its fluorescence information with
（RGB）Color and gray value are reflected that form texture emerges from the form of images.Control and data handling system use
Two kinds of softwares of MetaMorph and MetaFluor.With very strong dynamic image pro cess function, suitable for captured in real-time.
Step 2, the feature extraction and classifying identification step of algae image；
To the light spectrum image-forming data of acquisition, what microscopic fluorescence system in situ obtained is the fluorescence distribution figure of limited individual wavelength exciting light
Picture.For specific algae component interested, the image for choosing certain wave strong point makees the qualitative, quantitatively of institute a little position on image
(sxemiquantitative) is analyzed, so that it may to obtain the distributed image of the analyzed sample specific component content (concentration) everywhere.
Simple to be difficult to carry out Classification and Identification work to close algae using image or merely using spectrum, utilization is glimmering
Light image array, the information of the wavelength resolution of fluorescence spectrum is added on the basis of image spatial resolution, information content
On the one hand increase increases the precision of Classification and Identification, but also necessarily cause the difficulty of data characteristics extraction and Classification and Identification, intends pin
To information such as the form, texture and the colors that reflect in algae image, obtained by the methods of Gabor, Wavelet, statistical analysis
Two dimensional image characteristic quantity under a certain wavelength, then the change of its chrominance information is turned to outer disturb to multiple classification under different wavelength
Device（Artificial neural network and SVMs etc.）Experiment is trained, to obtain the optimal classification that can effectively identify all kinds of algae
Scheme.
Step 3, image chroma spectrum and fluorescence spectrum correlation analysis step,
The present invention by establishing certain area on different excitation wavelength hypographs step by step（Whole image → topography → single picture
Vegetarian refreshments）Colorimetry information（Intensity+ RGB value ~ λ）With the correlation of fluorescence spectrum, it may be determined that colorimetry
Corresponding relation between Information Spectrum and spectrum.
Step 4, in site measurement determines the relevant parameter step of sample collection, including：The determination of number of arrays and implementation,
The determination of data compression ratio and implementation.
The determination of number of arrays and implementation：The training required for effective recognition result can be reached by experimental study acquisition
The minimum samples and pattern matrix number of sample（Number of wavelengths）, the wave-length coverage of tunable optical source is determined, wavelength interval, is realized
Enough spectral informations are obtained with minimum number of wavelengths.
The determination of data compression ratio and implementation：The data compression ratio of multidimensional image array is determined, to meet data
The data compression ratio of minimum load in transmission engineering.
Embodiment 3：A kind of microscopic fluorescent spectral imaging marine phytoplankton original position Classification and Identification device
As shown in figure 5, the present invention provides a kind of microscopic fluorescent spectral imaging marine phytoplankton original position Classification and Identification device, bag
Include：
Including excitation light source system 1, flowing sample cell 2, CCD imaging systems 3.
The excitation light source system 1 includes light supply apparatus 11 and optical system 12.
The light supply apparatus 11 includes excitation source, imaging and image capturing system.
The LASER Light Source is using xenon lamp as excitation source；Excitation source uses face mode of excitation.Face mode of excitation is
Whole viewing area is excited simultaneously, while detects the fluorescence signal of whole region, image taking speed is fast, is adapted to reflection algae in real time
Change procedure.Corresponding image sequence can be obtained under different wavelength using Wavelength tunable light source simultaneously, reflects space
The colorimetry information of each point.
The imaging and IMAQ system of the imaging and image capturing system using Leica DMILM fluorescence inverted microscopes
System；
Optical system 12 includes grating, beam expanding lens, lens, micro imaging system and optical filter etc..The wavelength of excitation source 1 exists
220-550nm is adjustable or optional, and can carry out face to sample in the range of 1mm × 1mm after beam expanding lens expands excites, through micro-
It can be imaged after imaging system in μm precision, optical filter can filter to veiling glare.
Sample cell 2 is flowed, for containing seawater sample to be detected.
CCD imaging systems 3 include filter plate 31, object lens 32, eyepiece 33, detector 34 and data processing equipment 35.
Excitation source footpath optical system 12 will excite light irradiation Flow sample product pond 2, flow seawater sample to be detected in sample cell 2
Product irradiate through excitation source, and irradiation light injects CCD imaging systems 3 through flowing sample cell 2, and CCD imaging systems 3 are to through Flow sample
The irradiation light that product pond 2 is injected is detected and detection data is analyzed.
Adjust Q Nd:YAG laser, is tuned through nonlinear crystal to wavelength or xenon lamp is as activating system, uses
IXon Imaging EMCCDs are as detecting system.Exciting light uses face mode of excitation.Face mode of excitation excites whole simultaneously
Viewing area, while the fluorescence signal of whole region is detected, image taking speed is fast, is adapted to the change procedure of reflection algae in real time.Together
Shi Liyong tunable wave lengths LASER Light Source can obtain corresponding image sequence under different wavelength, reflect the spectrum of spatial points
Information.This scheme can obtain high resolution, the image and spectral information of abundant, the quick algae of projection in real time of spectral information, obtain
To algae form, texture, spectral information, it is to realize to carry out frustule shell surface to be advantageous to by image segmentation and contours extract
Automatic identification, the content distribution of different algae kind two-dimensional spaces in observation area is obtained, while pass through its spectrum peak and intensity
Carry out the quantitative analysis of the suspension three dimensions content of algae.The method is due to laser and the volume and price and right of imaging
The factors such as environmental requirement, laboratory comparative study scheme and computer classes expert database can be used as.
The feature extraction and classifying identification of algae image, to the light spectrum image-forming data of acquisition, laboratory micro imaging system
What is obtained is the fluorescence distribution image of the exciting light series of different wave length in certain spectral region, respectively for a certain true on image
The fixed point position-finding light value of point at different wavelengths, so that it may bent to obtain the spectrum corresponding to the point illustrated top
Line (abscissa is that wavelength, ordinate are light values).According to the curve of spectrum of every bit position, can not only determine should on sample
Point is to the light value (thus can make the quantitatively or semi-quantitatively analysis of sample this algae component) of some determination wavelength, Er Qieke
Using according to the peak valley wavelength location interpretation of its curve of spectrum go out this of sample to different wavelengths of light scholarship and moral conduct as extreme value (thus can make
Qualitative analysis).
Image chroma is composed and fluorescence spectrum correlation research, and the present invention by establishing on different excitation wavelength hypographs step by step
Certain area（Whole image → topography → single pixel point）Colorimetry information（Intensity+ RGB value ~
λ）With the correlation of fluorescence spectrum, the corresponding relation between colorimetry Information Spectrum and spectrum is determined.
In site measurement determines the relevant parameter of sample collection, the determination of number of arrays and implementation：Obtained by experimental study
The minimum samples and pattern matrix number of the training sample required for effective recognition result can be reached by taking（Number of wavelengths）, it is determined that can
The wave-length coverage of tuning source, wavelength interval, realize and enough spectral informations are obtained with minimum number of wavelengths.
The determination of data compression ratio and implementation：The data compression ratio of multidimensional image array is determined, to meet data
The data compression ratio of minimum load in transmission engineering.
Citing described above for best mode for carrying out the invention, wherein the part do not addressed in detail is that this area is common
The common knowledge of technical staff.Protection scope of the present invention is defined by the content of claim, any technology based on the present invention
The equivalent transformation for enlightening and carrying out, also within protection scope of the present invention.
Claims (10)
- A kind of 1. microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method, it is characterised in that：Show including fluorescence Micro- image-forming step, the feature extraction and classifying identification step of image, image chroma spectrum and fluorescence spectrum correlation analysis step, original Position measurement determines the relevant parameter step of sample collection.
- 2. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：Fluorescence microscopy and spectral imaging technology are combined, development can apply marine environment under algae it is quick micro- Fluorescence spectrum Detection Techniques, the pattern matrix of close algae under different wave length is obtained, by spectrum under different excitation wavelengths in colourity The D coordinates value of embodiment in, as the additional information of algae image, to the same species that are obtained under different wave length not Feature extraction is carried out with image, trains grader, the classification of similar algae species in the form of realizing to inside same monoid Identification and statistics；Imaging system is divided into lab scenario and underwater scheme, two systems consider under different situations and different accuracy can be with Complement each other the actual requirement of checking.
- 3. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：Fluorescent microscopic imaging step includes laboratory microscopic fluorescent spectral imaging step, the laboratory micro-fluorescence spectrum Image-forming step be tunable wave length light-pulse generator excite lower fluoroscopic image system build and acquisition technique, for inversion fluorescence Microscope enters the repacking of line light source, including the design of tunable excitation source, is conducted via optical fiber beam, via lens group Light beam regulation, light beam irradiation；Fluorescent microscopic imaging and image collection system use the corollary equipment of fluorescence microscope；The experiment Utilized in the microscopic fluorescent spectral imaging step of room and adjust Q Nd:YAG laser, is tuned or xenon lamp through nonlinear crystal to wavelength As activating system, imaging and image capturing system using Leica DMILM fluorescence inverted microscopes.
- 4. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：Fluorescent microscopic imaging step includes underwater microscopic fluorescent spectral imaging step, the underwater microscopic fluorescent spectral imaging Step, it is contemplated that the stability of the light path of instrument, anti-interference, pressure-resistant corrosion-resistant, the factor such as compactedness of light path design, simultaneously Flow Sampling system, including peristaltic pump, sample feeding pipe, flowing sample room and waste liquid displacer are realized in design；Its design is for reality The needs that existing underwater in-situ detects in real time, need to consider sample cell structure, the speed of sample introduction, the timing Design of peristaltic pump, thoroughly Penetrate the influence of the factors such as light intensity.
- 5. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：The feature extraction and classifying identification step of image, image chroma spectrum is established with fluorescence spectrum correlation to realize spectrum The method of feature extraction；Image chroma compose with fluorescence spectrum correlation analysis step, realize the similar species identification of form inside same monoid And quantitative statisticses.
- 6. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：Exciting light uses face mode of excitation in the fluorescent microscopic imaging step, while can be using Wavelength tunable light source Corresponding image sequence is obtained under different wavelength, reflects the colorimetry information of spatial points.
- 7. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：After the signal of transmission laser background and fluorescence is by high-pass filter in the fluorescent microscopic imaging step, it will can swash Light background removal, using be imaged EMCCD sequential control systems carry out data acquisition, realize dark background be imaged, its fluorescence information with RGB color and gray value are reflected that form texture emerges from the form of images.
- 8. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method as claimed in claim 1, it is special Sign is：This method by establishing the colorimetry information Intensity+ of certain area on different excitation wavelength hypographs step by step RGB value ~ λ and fluorescence spectrum correlation, determine the corresponding relation between colorimetry Information Spectrum and spectrum；The certain area includes whole image, topography, single pixel point.
- A kind of 9. microscopic fluorescent spectral imaging marine phytoplankton original position Classification and Identification device, it is characterised in that：Including excitation light source system（1）, flowing sample cell（2）, CCD imaging systems（3）；The excitation light source system（1）Including light supply apparatus（11）And optical system（12）；The optical system（12）Including grating, beam expanding lens, lens, micro imaging system and optical filter, wherein excitation wavelength exists 220-550nm is adjustable or optional, and can carry out face to sample in the range of 1mm × 1mm after beam expanding lens expands excites, through micro- It can be imaged after imaging system in μm precision.
- 10. a kind of microscopic fluorescent spectral imaging marine phytoplankton original position Classification and Identification device as claimed in claim 9, it is special Sign is：The light supply apparatus（11）Including excitation source, imaging and image capturing system；The imaging and image capturing system use microscopic fluorescent spectral imaging system in situ, and use can send feux rouges, green glow Pigtail-LEDs with blue light is as excitation source, using the digital camera of 1392 × 1040 pixels as detector；The light that 2 red-light LEDs, 3 green light LEDs and 2 blue-ray LEDs are sent is converged into special optical material using micro optical fiber On, then it is connected to excite the phytoplankton fluorescence in Flow Sampling sample cell with micro imaging system.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710613328.4A CN107389638A (en) | 2017-07-25 | 2017-07-25 | A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710613328.4A CN107389638A (en) | 2017-07-25 | 2017-07-25 | A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107389638A true CN107389638A (en) | 2017-11-24 |
Family
ID=60336789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710613328.4A Pending CN107389638A (en) | 2017-07-25 | 2017-07-25 | A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107389638A (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732142A (en) * | 2018-02-09 | 2018-11-02 | 秦皇岛红燕光电科技有限公司 | Phytoplankton & Suspension red tide based on three-dimensional fluorescence spectrum and detection method of toxicity |
| CN108765420A (en) * | 2018-07-04 | 2018-11-06 | 安图实验仪器(郑州)有限公司 | Fluorescence microscopy images result method of discrimination |
| CN110274899A (en) * | 2019-07-31 | 2019-09-24 | 中国科学院海洋研究所 | A kind of halomereid phosphorimager |
| CN111443028A (en) * | 2020-04-26 | 2020-07-24 | 长江流域水环境监测中心 | Automatic monitoring equipment and method for floating algae based on AI technology |
| CN113395461A (en) * | 2020-03-12 | 2021-09-14 | 平湖莱顿光学仪器制造有限公司 | Fluorescence-related intelligent two-dimensional video device and two-dimensional video playing method thereof |
| CN113391437A (en) * | 2020-03-12 | 2021-09-14 | 平湖莱顿光学仪器制造有限公司 | Fluorescence microscope imaging system and fluorescence imaging method thereof |
| CN113539368A (en) * | 2021-08-30 | 2021-10-22 | 长春理工大学 | Fluorescent image signal data storage and color classification method |
| CN114509866A (en) * | 2021-12-28 | 2022-05-17 | 广州博冠光电科技股份有限公司 | Underwater fluorescence microscope capable of automatically identifying and classifying |
| CN114646627A (en) * | 2022-05-23 | 2022-06-21 | 中国海洋大学 | Device and method for classifying and detecting seawater spilled oil by using spectral analysis technology |
| WO2023040310A1 (en) * | 2021-09-14 | 2023-03-23 | 河北地质大学 | Fluorescence sensing technology-based planktonic algae concentration monitor |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1645108A (en) * | 2004-12-27 | 2005-07-27 | 天津大学 | Red tide algae image analyzing system and method |
| CN1916604A (en) * | 2006-09-01 | 2007-02-21 | 中国科学院安徽光学精密机械研究所 | Method and device for classified detecting density of phytoplankton under water in site |
| CN101151623A (en) * | 2005-01-27 | 2008-03-26 | 剑桥研究和仪器设备股份有限公司 | Classifying image features |
| US20100323345A1 (en) * | 2008-02-13 | 2010-12-23 | Gary Borisy | Methods and compositions for identifying cells by combinatorial fluorescence imaging |
| CN103868901A (en) * | 2014-03-14 | 2014-06-18 | 中国科学院合肥物质科学研究院 | Discrete three-dimensional fluorescence spectrum-based phytoplankton identification and measurement method and discrete three-dimensional fluorescence spectrum-based phytoplankton identification and measurement device |
| CN104614353A (en) * | 2015-01-28 | 2015-05-13 | 中国科学院半导体研究所 | Two channel-based multi-spectrum fluorescent imaging microscopic system and method |
| CN106841072A (en) * | 2017-03-30 | 2017-06-13 | 莱森光学（深圳）有限公司 | A kind of method differentiated for algae with algae proliferation non-destructive monitoring situation |
-
2017
- 2017-07-25 CN CN201710613328.4A patent/CN107389638A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1645108A (en) * | 2004-12-27 | 2005-07-27 | 天津大学 | Red tide algae image analyzing system and method |
| CN101151623A (en) * | 2005-01-27 | 2008-03-26 | 剑桥研究和仪器设备股份有限公司 | Classifying image features |
| CN1916604A (en) * | 2006-09-01 | 2007-02-21 | 中国科学院安徽光学精密机械研究所 | Method and device for classified detecting density of phytoplankton under water in site |
| US20100323345A1 (en) * | 2008-02-13 | 2010-12-23 | Gary Borisy | Methods and compositions for identifying cells by combinatorial fluorescence imaging |
| CN103868901A (en) * | 2014-03-14 | 2014-06-18 | 中国科学院合肥物质科学研究院 | Discrete three-dimensional fluorescence spectrum-based phytoplankton identification and measurement method and discrete three-dimensional fluorescence spectrum-based phytoplankton identification and measurement device |
| CN104614353A (en) * | 2015-01-28 | 2015-05-13 | 中国科学院半导体研究所 | Two channel-based multi-spectrum fluorescent imaging microscopic system and method |
| CN106841072A (en) * | 2017-03-30 | 2017-06-13 | 莱森光学（深圳）有限公司 | A kind of method differentiated for algae with algae proliferation non-destructive monitoring situation |
Non-Patent Citations (5)
| Title |
|---|
| 叶朝辉主编: "《武汉光电论坛系列文集 第3辑》", 31 October 2016, 华中科技大学出版社 * |
| 杨爱玲等: "单个油气包裹体的紫外-可见显微荧光光谱及色度研究", 《光学学报》 * |
| 范世福 等: "光谱成像技术及其在生物医学研究中的应用", 《现代科学仪器》 * |
| 范世福 等: "显微荧光光谱成像仪的研究与设计", 《仪器仪表学报》 * |
| 陈庆光等: "利用荧光光谱的牙菌斑成像评价", 《信号处理在生仪2014学术年会论文集》 * |
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108732142A (en) * | 2018-02-09 | 2018-11-02 | 秦皇岛红燕光电科技有限公司 | Phytoplankton & Suspension red tide based on three-dimensional fluorescence spectrum and detection method of toxicity |
| CN108732142B (en) * | 2018-02-09 | 2021-03-02 | 秦皇岛红燕光电科技有限公司 | Three-dimensional fluorescence spectrum-based seawater algae red tide and toxicity detection method |
| CN108765420A (en) * | 2018-07-04 | 2018-11-06 | 安图实验仪器(郑州)有限公司 | Fluorescence microscopy images result method of discrimination |
| CN108765420B (en) * | 2018-07-04 | 2021-06-08 | 安图实验仪器(郑州)有限公司 | Fluorescence microscope image result judging method |
| CN110274899A (en) * | 2019-07-31 | 2019-09-24 | 中国科学院海洋研究所 | A kind of halomereid phosphorimager |
| CN113391437A (en) * | 2020-03-12 | 2021-09-14 | 平湖莱顿光学仪器制造有限公司 | Fluorescence microscope imaging system and fluorescence imaging method thereof |
| CN113395461A (en) * | 2020-03-12 | 2021-09-14 | 平湖莱顿光学仪器制造有限公司 | Fluorescence-related intelligent two-dimensional video device and two-dimensional video playing method thereof |
| CN111443028A (en) * | 2020-04-26 | 2020-07-24 | 长江流域水环境监测中心 | Automatic monitoring equipment and method for floating algae based on AI technology |
| CN111443028B (en) * | 2020-04-26 | 2022-09-13 | 生态环境部长江流域生态环境监督管理局生态环境监测与科学研究中心 | Automatic monitoring equipment and method for floating algae based on AI technology |
| CN113539368A (en) * | 2021-08-30 | 2021-10-22 | 长春理工大学 | Fluorescent image signal data storage and color classification method |
| WO2023040310A1 (en) * | 2021-09-14 | 2023-03-23 | 河北地质大学 | Fluorescence sensing technology-based planktonic algae concentration monitor |
| CN114509866A (en) * | 2021-12-28 | 2022-05-17 | 广州博冠光电科技股份有限公司 | Underwater fluorescence microscope capable of automatically identifying and classifying |
| CN114646627A (en) * | 2022-05-23 | 2022-06-21 | 中国海洋大学 | Device and method for classifying and detecting seawater spilled oil by using spectral analysis technology |
| CN114646627B (en) * | 2022-05-23 | 2022-08-23 | 中国海洋大学 | Device for classifying and detecting sea water spilled oil by utilizing spectral analysis technology |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107389638A (en) | A kind of microscopic fluorescent spectral imaging marine phytoplankton original position classifying identification method and device | |
| US20210209337A1 (en) | Deep learning-enabled portable imaging flow cytometer for label-free analysis of water samples | |
| Gӧrӧcs et al. | A deep learning-enabled portable imaging flow cytometer for cost-effective, high-throughput, and label-free analysis of natural water samples | |
| AU2007352394B2 (en) | Quantitative, multispectral image analysis of tissue specimens stained with quantum dots | |
| Davis et al. | Real-time observation of taxa-specific plankton distributions: an optical sampling method | |
| US10180398B2 (en) | Trajectory-based triggering system for hyperspectral imaging flow cytometer | |
| CN106226247A (en) | A kind of cell detection method based on EO-1 hyperion micro-imaging technique | |
| JP2003529747A (en) | Method and apparatus for determining characteristics of a culture solution | |
| US9797836B1 (en) | Hyperspectral imaging flow cytometer | |
| CN115032196B (en) | Full-scribing high-flux color pathological imaging analysis instrument and method | |
| CN104897583A (en) | Trace bloodstain nondestructive testing device and method | |
| CN106841072A (en) | A kind of method differentiated for algae with algae proliferation non-destructive monitoring situation | |
| CN205844201U (en) | A kind of device of quick identification scene vestige | |
| CN110118758A (en) | A kind of scattering fluorescent dual module state flow-type imaging system | |
| Dondi et al. | An interactive tool for speed up the analysis of UV images of Stradivari violins | |
| US20220228968A1 (en) | Hyperspectral quantitative imaging cytometry system | |
| Liu et al. | Polarization staining and high-throughput detection of marine microalgae using single cell average Mueller matrices | |
| Zhou et al. | Profile probing of suspended particles in water by Stokes vector polarimetry | |
| CN217359583U (en) | Multispectral multi-dimensional dark field imaging system with high signal-to-noise ratio for aquatic organisms | |
| WO2023063877A2 (en) | Multi-color pulse-amplitude modulated handheld fluorescence imager | |
| Lin et al. | Portable in vivo measurement of apple sugar content based on mobile phone | |
| Akiba et al. | Design and testing of an underwater microscopy for the study of zooplankton distribution | |
| Lunven et al. | A new device for in situ video and fluorescence analysis of marine particles. Applications to phytoplankton studies. | |
| CN115308174A (en) | Plankton in-situ imaging system and method applied to open scene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171124 |
|
| RJ01 | Rejection of invention patent application after publication |