CN1039453C - Multifunction Aadamard's transformation micrograph analytical instrument - Google Patents
Multifunction Aadamard's transformation micrograph analytical instrument Download PDFInfo
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- CN1039453C CN1039453C CN94107751A CN94107751A CN1039453C CN 1039453 C CN1039453 C CN 1039453C CN 94107751 A CN94107751 A CN 94107751A CN 94107751 A CN94107751 A CN 94107751A CN 1039453 C CN1039453 C CN 1039453C
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Abstract
The present invention discloses a new method for providing an image and a quantitative analysis result of a tiny object by a detector (a photomultiplier) and by combining Aadamard's transformation space multichannel imaging technique and microtechnique. Three dimensional Aadamard's transformation microscopic fluorescence image analysis, microscopic Raman image analysis, microscopic luminosity image analysis and spectrum analysis can be carried out according to a developed multifunction Aadamard's transformation micrograph analytical instrument, and normal image analysis, micrograph analysis and microscopic luminosity analysis can be carried out. The present invention has the advantages that the measuring precision of the present invention is 16 times higher than that of a convention micrograph analytical instrument, the measured signal-noise ratio is enhanced by 8 to 20 times, the sensibility is higher than that of a 0.02 Lux CCD video camera, and the present invention is particularly suitable for detecting a weak light signal. The reproducibility of an analysis result of the fluorescence intensity of single cell and the image of the cell is good, and relative standard deviation (RSD) proportion is below 10%.
Description
The present invention relates to a kind of Multifunction Aadamard's transformation micrograph analytical instrument, it is that Hadamard transform hyperchannel aerial image technology is combined with microtechnic, new instrument with the three-dimensional microscopic fluorescence of a photomultiplier measurement microscopically small items, micro-Raman and micro-luminosity image, be a kind of novel high sensitivity, high s/n ratio, be specially adapted to the microspectrum image analytical instrument that faint light is measured.It belongs to medicine detector device technical field, belongs to the analysis and testing technology field, also belongs to technological field of biochemistry, is the product of multiple technologies combinations such as light modulation techniques, instrument manufacturing, computing machine and modern instrumental analysis.
Hadamard transform (Hadamard Transform) spectrum is a kind of light modulation techniques that is similar to Fourier transform (Fourier Transform) spectrum that grew up in twenties years in the past.When measuring feeble signal, has the advantage [M.O.Harwit, N.J.A.Sloane, " Hadamard TransformOptics ", Academic:New York, 1980] of high s/n ratio, multi channel imaging and energy distribution.The Hadamard transform imaging technique is one of forward position research topic in the world.
Microtechnic is a kind of most important, the most basic technology in cell biology and clinical medicine domain.At present, be based upon on the microtechnic basis, be used for cell or other small items are qualitative or the instrument of quantitative test has microspectrophotometer, microfluorophotometer and various micro-image analyzer.Preceding two kinds of instruments often are used to pair cell or other small items carry out quantitative test, and then a quasi-instrument then usually is used for the image morphology analysis of cell or small items.But these instruments are insensitive to faint optical signal, and all exist weak point at aspects such as quantitative test speed, analysis precisions.In addition, instrument such as microspectrophotometer, flow cytometer also is difficult to provide simultaneously the micro-image and the quantitative analysis results of analytical sample.
Once studied the micro-Raman imaging of one-dimensional coding Hadamard transform [P.J.Treado abroad, A.Govil, M.D.Morris, K.D.Sternitxke and R.L.Mccreery, Appl.Spectrosc., 44:1270,1990], but owing to problems such as having poor reproducibility, device bulky complex has stopped further research work.Up to now, about Hadamard transform microscopic fluorescence image and micro-luminosity image, and the instrument that these three kinds of image techniques combine together do not seen that as yet report is arranged.Obtain distortionless faint spectrum picture, its faint light intensity must keep relative stability within a certain period of time, this relatively stablely be content with very little for Raman spectrum, absorption spectrum.But,, increased the technical difficulty of gathering owing to be subjected to the influence of fluorescence lifetime, fluorescence decay for gathering fluoroscopic image.
The objective of the invention is to utilize Hadamard transform two dimension light modulation techniques, develop a kind of new instrument that is used for the graphical analysis of faint microscopic field of view characteristic spectrum, for cellular elements biology and clinical medicine are set up a kind of quick, accurate, sensitive quantitative test new method.
Be the technical measures that realize that order of the present invention is taked:
Instrument of the present invention is formed by light source, fluorescent microscope, compression of images optical system, Hadamard transform two-dimensional modulation template and planar stepping motor drive system, grating monochromator, photomultiplier and by microcomputer-controlled sampling/hold amplifier, 12 (bit) digital-to-analog conversion plates and microcomputer, this is the main body of Aadamard's transformation micrograph analytical instrument, the configuration charge coupled device ccd, the Flame Image Process interface board, promptly frame memory constitutes micro image analysis, general image analyser.
Each parts reasonably combined, can carry out the three-dimensional microscopic fluorescence of Hadamard transform, micro-Raman and the analysis of micro-luminosity spectrum picture, can carry out normal image analysis, micro image analysis and microspectroscopy, make this instrument constitute a kind of multifunctional image analyser.
Technical characterictic of the present invention is with Hadamard transform spatial multichannel imaging technique and the coupling of microscopic fluorescence technology, to drawing the view field image of microscope light path, carry out the optical compression imaging, and with small-sized photoetching Hadamard transform two dimension pattern plate, adopt light modulation techniques that its image is carried out optical modulation, optical compression once more, the characteristic spectrum of monochromator sorting image, it is the characteristic spectrum of component to be measured, photomultiplier measurement amplifies the analog to digital conversion digitizing, demodulation is reduced into the microspectrum image of component to be measured, provides microscopically the observed qualitative and quantitative information relevant with small items.
Constitute microscopical transillumination system by microscope components bromine tungsten filament lamp, lens L6, condenser, the object lens that play condenser constitute and fall to penetrating the formula illuminator by excitation source (high-pressure sodium lamp, xenon lamp or laser), lens L5, dichromatic beam spectroscope M, simultaneously.Cell on the microscope stage or other small items under the irradiation of continuous light (exciting light or transmitted light) produce light signal (fluorescence signal, Raman signal or absorption signal), and the optical signals microcobjective that is produced is collected.
The interface of a band completely reflecting mirror P is installed on fluorescent microscope top, by the position of completely reflecting mirror " " or " not existing " microscope central light beam, select to determine horizontal optical path system or vertical optical path system.Completely reflecting mirror is inserted light path, and light is promptly rolled over to turn 90 degrees and is entered the horizontal optical path system, and completely reflecting mirror is shifted out light path, and light enters charge-coupled device (CCD) vertically upward, and images in the charge-coupled device (CCD) focal plane.
Optical pressure shortens picture lens combination L1, L2 into, and Hadamard transform diaphragm and Hadamard transform modulation template, optical pressure shorten picture lens combination L3, L4 into and form the horizontal optical path system.Wherein the lens combination of being made up of lens L1, L2 images on the template sample, by the lens combination that lens L3, L4 form the light signal via the template modulation is compressed, signal after the compression enters the entrance slit of grating monochromator, with the optical parametric coupling of grating monochromator, bear Hadamard transform optical modulation work.
The two dimension Hadamard transform template and diaphragm adopting quartz glass substrate metal-coated membrane, scribe small two-dimension coding templet and diaphragm with mask etching method photoetching making by the Hadamard transform coded sequence, wherein diaphragm maintains static, template can move on two-dimensional directional freely, so that light signal is carried out spatial modulation.
Two dimension Hadamard transform template is fixed on the two-dimentional machinery gearing, and this device is to adopt stepper motor to drive milscale to rotate, by milscale the rotation of motor is become linear translation, reaches to promote template displacement design.Mechanical driving device is by computer interface, software and the control of stepper motor power amplification plate.This machine driven system can be controlled three stepper motors simultaneously and turn round freely, and wherein two motors are used for the accurately displacement of control template two dimension, and one is used to control the grating monochromator spectral scan.
The beam splitting system of this instrument adopts commercial grating monochromator, detecting device adopts photomultiplier, its electric signal is amplified by two-stage integrated operational amplifier amplifying circuit, enlargement factor is 150-700 times, modulus/the digital-to-analog conversion plate is with analog signal digital to adopt 12, and overall process is finished by microcomputer.
Measurement for the characteristic spectrum curve (characteristic spectrum of component to be measured in the sample) of microscopic field of view image, by computer controlled automatic length scanning controller, the grating of rotation monochromator carries out spectral scan, gather spectral wavelength and intensity relevant information in real time, determine the characteristic spectrum of component to be measured in the image (sample) that desire selects, the i.e. characteristic spectrum of Adama conversion micro-image by image (sample) curve of spectrum that obtains.
Microscope is got vertical optical path, by charge-coupled device (CCD), Flame Image Process interface board (hardwood storer), microcomputer and respective image process software, bears the work of micro-image analyzer.
Adopt charge-coupled device (CCD) video camera, Flame Image Process interface board (hardwood storer), microcomputer and respective image process software, finish the function of normal image analyser.
Horizontal optical path system and be the core of Hadamard transform light modulation techniques with microcomputer-controlled two-dimensional modulation template mechanical driving device.
Above various parts can be formed Multifunction Aadamard's transformation micrograph analytical instrument by after the accompanying drawing assembling.On this instrument, can carry out Hadamard transform microscopic fluorescence image and micro-luminosity graphical analysis.Carry out the micro-Raman image of Hadamard transform when analyzing, change spherical shape ultra-high pressure mercury lamp excitation source into the argon laser light source and get final product.
Compared with the prior art, the technique effect that reached of the present invention:
The present invention has adopted two-dimentional Hadamard transform template that picture signal is carried out spatial modulation on horizontal optical path, adopt compression of images, monochromator optics branch is got a certain characteristic spectrum of image, adopts photomultiplier to make electrooptical device, makes this instrument can detect faint spectrum picture information.The present invention adopts continuous light to make excitation source, and fluorescence lifetime is to measuring not influence; Since machine calculate the machine arithmetic speed surpass already hundred million times/second (＞100M), adopt quick sampling, can avoid the slowly caused measuring error of decay of most of system fluorescence.As for those to photaesthesia, easily cause the system of fluorescence decay, as some Biosample, adopt and measure the fluorescence decay curve that fluorescence intensity weakens with linear relationship in time, utilize computing machine to carry out synchronous correction, can solve those photosensitive fluorescence systems well because of the caused measuring error of fluorescence decay, further improve the signal to noise ratio (S/N ratio) and the accuracy of quantitative analysis of instrument, thereby realized gathering the faint three-dimensional fluorescence image of Hadamard transform and the luminosity image of tiny sample in the world first.Because this multicenter detecting method, can detect the intensity of spectrum picture signal and background signal simultaneously, thereby can be from the spectrum picture signal background correction signal synchronously, make this method aspect analysis speed and accuracy of analysis, obviously be better than conventional micro-photometric analysis and microscopic fluorescence analytical approach, measure the high 8-20 of signal to noise ratio (S/N ratio) doubly, high 16 times of (its calibration of luminous intensity measurement ratio of precision image analyzer, image analyzer is 256 units, and this instrument is 4096 units), remolding sensitivity 0.02 lumen/rice
2(Lux) charge-coupled device (CCD) video camera height is particularly suitable for Feebleness Light Signal Examining.To the good reproducibility of analysis results of the fluorescence intensity image of individual cells, single measurement result's relative standard deviation, promptly coefficient of alteration RSD is lower than 10%.
In addition, same as the prior art, on the vertical direction of fluorescent microscope, the position of socket camera is equipped with camera system, promptly forms the micro image analysis system by charge coupled device ccd, Flame Image Process interface board (frame memory), computing machine and respective image process software.Completely reflecting mirror is shifted out light path, and charge-coupled device (CCD) is in microscopical vertical optical path, can directly absorb the image of small items, to observe the pattern of sample.Be stored in computing machine by Flame Image Process interface board (frame memory), and this image handled, extract sample intuitive image information, play a part micro-image analyzer with corresponding software.Also can directly absorb X-ray sheet, photo or other objects by the charge-coupled device (CCD) video camera, thereby make this instrument also possess the function of normal image analyser, further widen the function and the usable range of instrument, constituted a kind of multifunctional image analyser.
Description of drawings:
Accompanying drawing is the structured flowchart (schematic diagram) of Multifunction Aadamard's transformation micrograph analytical instrument.
In the accompanying drawing, 1 is bromine tungsten filament lamp; Lens L1 ... L6; 2 is condenser, and by 1 bromine tungsten filament lamp, lens L6,2 condensers constitute microscopical transillumination system; 3 is microscope stage; 4 is microcobjective; 5 is excitation source (high-pressure sodium lamp, xenon lamp or laser), dichromatic beam spectroscope M, completely reflecting mirror P, charge coupled device ccd; 6 are the Hadamard transform diaphragm; 7 are the Hadamard transform template; 8 is grating monochromator; 9 is photomultiplier; 10 are sampling/hold amplifier; 11 are number conversion (AD/DA) plate; 12 is microcomputer; 13 are two-dimensional modulation template mechanical driving device and computer interface; 14 is Flame Image Process interface board (frame memory), and 15 is grating monochromator length scanning self-actuated controller.
Below in conjunction with embodiment the present invention is described in further detail.
Embodiment 1, Aadamard's transformation micrograph analytical instrument:
Analytical sample places on the microscope stage, completely reflecting mirror P on the fluorescent microscope is pushed into light path, the picture signal of analytical sample (transmitted light, fluorescence or Raman image) is collected by microcobjective, view field image is promptly rolled over and is turn 90 degrees, enter by lens L1, lens L2, Hadamard transform template and diaphragm, lens L3, lens L4, i.e. horizontal optical path system as shown in the figure.Wherein the lens combination of lens L1, L2 formation is compressed the microscopic field of view picture, blur-free imaging is on the Hadamard transform stencil plane, fixed Hadamard transform diaphragm is used for limited images spatial modulation scope, and two dimension (X, Y-axis) mobile Hadamard transform template is carried out spatial modulation to picture signal.The picture signal of modulation, warp and the lens combination that lens L3, L4 that the grating monochromator optical parametric adapts constitute are compressed into picture once more, enter the grating monochromator entrance slit.For the selection of microscopic field of view characteristics of image spectrum, available computers is controlled grating monochromator automatically and is carried out spectral scan and obtain the curve of spectrum, determines the characteristic spectrum of microscopic field of view image by the curve of spectrum.Through the grating monochromator sorting, convert electric signal to by photomultiplier, amplify by amplifying circuit.The electric signal digitizing of modulation is carried out analog to digital conversion by 12 moduluses/digital-to-analog conversion plate, and is stored in the computing machine, finishes a complete sampling process.Digitized modulation signal is through the fast handmard transform demodulation, be reduced into the three-dimensional microspectrum image (being the component image to be measured in the sample) of Hadamard transform of image, this characteristic spectrum image comprises spectral intensity dimension of two space peacekeepings, with self-programmed software this image is handled to obtain required qualitative, quantitative information.
Carry out micro-Raman image when analyzing, change spherical shape ultra-high pressure mercury lamp excitation source into the argon laser light source, can on this instrument, carry out the micro-Raman image analysis of Hadamard transform.
Embodiment 2, human breast cancer cell fluoroscopic image and nucleus pattern and dna content quantitative test:
Tumor locus cell quantitative with this instrumental analysis 100 routine HBT patients, 20 cells of the minimum analysis of each patient, each cell is surveyed two quantitative datas: nucleus DNA content (is unit with arbitrary unit) and pattern (survey area is a unit with pixel).The measurement data explanation, the Hadamard transform spectrum picture can correctly reflect the basic fluorescence distribution of cell, obtains quantitative fluorescence intensity and distributed intelligence.Compare with standard cell lines, can obviously find the difference between nucleus DNA ploidy and area and the normal cell, can accurately identify normal cell, sick cell and cancerous tumor cell.The histogram of measurement data provides the character of tumour, the concluding observation that aspects such as the grade malignancy of canceration are extremely useful intuitively for clinical diagnose.
Claims (3)
1, a kind of Multifunction Aadamard's transformation micrograph analytical instrument, it comprises Hadamard transform hyperchannel aerial image technology is combined with microscopy, it is characterized in that: the main body of Aadamard's transformation micrograph analytical instrument is by light source, fluorescent microscope, form horizontal optical path system and planar stepping motor drive system by compression of images optical system, Hadamard transform two-dimensional modulation template, grating monochromator, photomultiplier, sampling/hold amplifier, 12 figure place mould change-over panels and microcomputer are formed; To drawing the view field image of microscope light path, carry out the optical compression imaging, and, adopt light modulation techniques that its image is carried out optical modulation, optical compression once more with small-sized photoetching Hadamard transform two dimension pattern plate, the characteristic spectrum of monochromator sorting image, be the characteristic spectrum of component to be measured, photomultiplier measurement amplifies, analog to digital conversion digitizing, demodulation are reduced into the microspectrum image of component to be measured; The configuration charged coupled device CCD camera, the Flame Image Process interface board, promptly frame memory constitutes micro image analysis, general image analyser.
2. by the described Multifunction Aadamard's transformation micrograph analytical instrument of claim 1, it is characterized in that: the horizontal optical path system is the light signal of completely reflecting mirror P reflection, through lens combination compression and image on the template, by template picture signal is carried out spatial modulation, signal after the modulation through compressing with the lens combination of monochromator optical match, enters the monochromator entrance slit again.
3. by the described Multifunction Aadamard's transformation micrograph analytical instrument of claim 1, it is characterized in that: mechanical driving device and computer control interface and driving circuit that the template two dimension of hyperchannel aerial image technology moves, by designed interface and driving circuit, general-purpose computers are controlled three stepper motor runnings freely, wherein two are used for two dimension pattern plate and move, and another is used for spectral scan.
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| CN94107751A CN1039453C (en) | 1994-06-23 | 1994-06-23 | Multifunction Aadamard's transformation micrograph analytical instrument |
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| CN94107751A CN1039453C (en) | 1994-06-23 | 1994-06-23 | Multifunction Aadamard's transformation micrograph analytical instrument |
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| CN1039453C true CN1039453C (en) | 1998-08-05 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2009109078A1 (en) * | 2008-03-06 | 2009-09-11 | 中国科学院西安光学精密机械研究所 | A hadamard transform interference spectrum imaging method and device |
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| CN101706433B (en) * | 2009-07-27 | 2011-06-08 | 中国环境科学研究院 | Method for calibrating three-dimensional fluorescence |
| CN101718585B (en) * | 2009-11-13 | 2011-11-02 | 武汉大学 | Hadamard transform modulation method for enhancing detection sensitivity of array photoelectric detector |
| CN104297155B (en) * | 2014-09-28 | 2016-11-23 | 中国科学院长春光学精密机械与物理研究所 | A kind of multi-channel parallel spectrum investigating system |
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| CN107655570B (en) * | 2017-09-12 | 2020-01-10 | 安徽理工大学 | Synchronous mobile mechanical template type Hadamard spectrometer |
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| WO1989001622A1 (en) * | 1987-08-14 | 1989-02-23 | D.O.M. Associates, Inc. | Raman spectrometer having hadamard electrooptical mask and diode detector |
| US5050989A (en) * | 1989-09-21 | 1991-09-24 | The United States Of America As Represented By The Secretary Of The Air Force | Single Hadamard mask spectrograph system |
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Patent Citations (5)
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|---|---|---|---|---|
| US4007989A (en) * | 1975-09-29 | 1977-02-15 | International Business Machines Corporation | Hadamard filter design |
| US4553165A (en) * | 1983-08-11 | 1985-11-12 | Eastman Kodak Company | Transform processing method for reducing noise in an image |
| JPS63212862A (en) * | 1987-02-27 | 1988-09-05 | Sumitomo Electric Ind Ltd | Method and device for monitoring cell proliferation status |
| WO1989001622A1 (en) * | 1987-08-14 | 1989-02-23 | D.O.M. Associates, Inc. | Raman spectrometer having hadamard electrooptical mask and diode detector |
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| Publication number | Priority date | Publication date | Assignee | Title |
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