CN1148574C - High resolution Adama conversion micro-image analyzer - Google Patents
High resolution Adama conversion micro-image analyzer Download PDFInfo
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- CN1148574C CN1148574C CNB011065362A CN01106536A CN1148574C CN 1148574 C CN1148574 C CN 1148574C CN B011065362 A CNB011065362 A CN B011065362A CN 01106536 A CN01106536 A CN 01106536A CN 1148574 C CN1148574 C CN 1148574C
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Abstract
The present invention discloses a novel high resolution and high sensitivity Adama conversion micro-image analyzer of which each image is composed of 26 ten thousand pixels. The present invention is provided with a 511 unit Adama template and a linear array CCD detector with high sensitivity and combines Adama conversion space multichannel imaging technique and microtechnique. An image pattern and an image quantitative analysis result of a tiny object can be supplied to analyze a micro fluorescence spectrum image and a micro transmitted light spectrum image, and the present invention establishes new, rapid, sensitive, accurate and applicable micro spectrum image quantitative analysis technology for the fields of cell molecular biology and other subjects. The present invention has the advantages that the measuring precision is 16 times higher than that of a convention image analyzer, the sensibility is higher than that of a charge coupled device (CCD) camera of 0.02 Lux per M <2> (Lux), and the present invention is particularly suitable for detecting a weak optical signal. The detection of the single cell pattern, cell fluorescence intensity, and the reproducibility of analysis results are good, the relative standard deviation of single measurement result, i.e. a variation coefficient (RSD), is smaller than 10%.
Description
Technical field
The present invention relates to a kind of high resolving power Aadamard's transformation micrograph analytical instrument, it is that 511 coding Hadamard transform hyperchannel aerial image technology are combined with microtechnic, with line array CCD as detector, under the measuring microscope, the spectrum picture new instrument of the microscopic fluorescence of small items and micro-transmitted light, be a kind of novel high sensitivity, high s/n ratio, be specially adapted to the surveying instrument that faint microspectrum image is analyzed.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.
Background technology
Hadamard transform (Hadamard Transform) spectrum is the nearly 30 years a kind of light modulation techniques that are similar to Fourier transform (Fourier Transform) spectrum that grow up.When measuring feeble signal, has the advantage [M.O.Harwit, N.J.A.Sloane, " Hadamard Transform Optics ", 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 Hadamard transform micro-Raman imaging [P.J.Treado that encodes 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, be that the instrument of one is only at the inventor patent of invention [patent No.: the ZL 94 1 07751.9 in early stage about Hadamard transform micro-fluorescence spectrum image and the technological incorporation of micro-transmitted light spectrum picture; International monopoly Main classification number: G01N21/31 authorizes 1998.7] report arranged.ZL 94 1 07751.9 patents, a kind of multi-functional Adama conversion micro-image instrument is disclosed, by light source, fluorescent microscope, compression of images imaging, Hadamard transform modulation template, monochromator, photomultiplier detector and computing machine are formed, this patent adopts 15 * 17 two dimension pattern plates, photomultiplier single-point to survey, and resolution is low, and every width of cloth image is made up of 255 pixels.
Summary of the invention
The objective of the invention is to utilize high resolving power Hadamard transform light modulation techniques, provide a kind of high resolving power Aadamard's transformation micrograph analytical instrument device that is used for the graphical analysis of faint microscopic field of view characteristic spectrum, for cellular elements biology and other ambits are set up a kind of quick, sensitive, accurate, suitable microspectrum image quantitative test new technology.
For realizing that the technical scheme that above-mentioned order invention is taked is: a kind of high resolving power Aadamard's transformation micrograph analytical instrument, the optical system components, photoelectric commutator and the computing machine that comprise light source, fluorescent microscope, have the compression of images imaging, Hadamard transform modulation and collimation, color separation are integrated, Hadamard transform modulation system adopts Hadamard transform 511 one-dimensional coding templates; Template includes sampling/hold amplifier, photoelectric commutator is to include the conversion of 16 figure place moulds at interior line array CCD.
Also configurable colour plane array charge-coupled device CCD, Flame Image Process interface board, i.e. frame memory on the above-mentioned fluorescent microscope.
Above-mentioned Hadamard transform optical system components is by total reflection prism P, compression imaging lens group L1, M1, Hadamard transform template I and diaphragm, collimation lens set L2, color separation grating G, mirror M 2, cylindrical lens L3 constitute, optical pressure shortens into as lens combination L1, M1 sample is imaged on the template I, by the light signal collimation of L2 compound lens to template I modulation, grating G color separation, enter slit S through L3 cylindrical lens compression convergence, the Hadamard transform spectrum picture is finished collecting work by line array CCD.
The present invention adopts 511 one-dimensional coding templates, utilize 512 photosensitive units (line array CCD) to detect simultaneously, every width of cloth image is made up of 261632 pixels, the resolution height (improving one over thousands of times than 94 107751.9 patents) of image can carry out the quantitative test of microspectrum image quick, sensitive, exactly.
Compared with the prior art, the technique effect that reached of the present invention:
Gather curve of spectrum line array CCD commonly used, images acquired area array CCD commonly used both at home and abroad at present, inventor ZL 94 1 07751.9 patents combine images acquired with single detector (photomultiplier) with 15 * 17 two-dimensional encoded templates, obtain the low-resolution image that every width of cloth is made up of 255 pixels.If still combine images acquired information with 511 * 512 two-dimensional encoded templates, can obtain the full resolution pricture that every width of cloth is made up of a pixel surplus 260,000 with a photomultiplier.But in fact this coding templet is at aspects such as the modulation accuracy of XY direction of principal axis crisscross motion, measuring speeds, and prior art all is difficult to realize.
The present invention adopts the line array CCDs that can be used as the detection simultaneously of popping one's head in to combine with one dimension 511 coding templets more, only need be at a direction (X or Y-axis) rectilinear motion, utilize 512 photosensitive detectors (line array CCD) to carry out parallel detection, so just can solve problems such as mechanical modulation precision and quickening measuring speed.The image of gathering is made up of 261632 pixel/width of cloth, and its resolution improves one over thousands of times than ZL 94 1 07751.9 patented technologies.The design of miniaturization makes the present invention aspect machine volume and weight, reduce 20 surplus times and alleviate 20 surplus times.
The present invention adopts the high stability continuous light to make excitation source, and fluorescence lifetime is to measuring not influence; Since the operational speed of a computer surpass already 300,000,000 times/second (＞300M), adopt template rectilinear motion quick sampling, can avoid the slowly caused measuring error of decay of most of system fluorescence.Responsive especially as for those to light, 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 well those to the responsive especially fluorescence system of light 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 fluoroscopic image of high resolution Adama conversion and the transmitted light images 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, high 16 times (its calibration, image analyzer are 256 units to the strong measuring accuracy of photometry than image analyzer commonly used, 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, identical with existing micro image analysis system architecture, 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 colored charge coupled device ccd, Flame Image Process interface board (frame memory), computing machine and respective image process software.P shifts out light path with completely reflecting mirror, and colored charge-coupled image sensor (CCD) is in microscopical vertical optical path, can directly absorb the image of small items, to observe the colored pattern of sample.Store by Flame Image Process interface board (frame memory), and this image is 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 the image analyzer of one kind of multiple functions.From measuring sensitivity and accuracy of quantitative analysis aspect relatively, the colored CCD system is far away from the performance of the Aadamard's transformation micrograph analytical instrument of monochrome.
Description of drawings
Fig. 1 is a structural representation of the present invention.
Fig. 2 is that electricity connects block diagram between the parts of the present invention.
Embodiment
Among Fig. 1, microscope body comprises that halogen tungsten lamp, lens, condenser constitute microscopical transillumination system; Excitation source (high-pressure sodium lamp, xenon lamp), and the beam splitting dichroic mirror of microscope inside, completely reflecting mirror constitute microscope and fall to penetrating the formula lighting system.View field image enters the Hadamard transform optical system through microcobjective: P-beam steering prism, L1-convergent lens group, M1-turns to total reflective mirror, I-Hadamard transform template, L2-collimation lens set, G-grating, M2-turns to total reflective mirror, the L3-cylindrical lens, the S-slit is surveyed by linear charge-coupled array C1.L-is the imaging len that coloured image is analyzed the face battle array C2 in the optical system, C1-line array CCD, C2-area array CCD, W-object lens, Z-objective table, J-condenser, D1, D2-motor, H-mercury lamp, X-halogen tungsten lamp.
Fig. 2 comprises following functional circuit: i.e. instruction by computing machine 1 moves linearly Hadamard transform template I by controllor for step-by-step motor 2 control template displacement motors 3; Computing machine 1 rotates grating G by controllor for step-by-step motor 4 control grating rotary electric machines 5.Sample by line array CCD controller 6 control linear array C1 by computing machine 1 instruction; Sample by area array CCD frame memory 8 chain of command battle array C2 by computing machine 1.
Referring to Fig. 1, Fig. 2, the present invention is by light source, fluorescent microscope, include the special-purpose optical system that compression of images imaging, Hadamard transform modulation and collimation, color separation are integrated, include sampling/hold amplifier, 16 (bit) digital-to-analog conversions in interior line array CCD detection system, and the computing machine composition, this is the main body of Aadamard's transformation micrograph analytical instrument.Other is equipped with colour plane battle array ccd detector and is used to gather micro-coloured image.
Different parts reasonably combined can be carried out Hadamard transform microscopic fluorescence and analysis of micro-transmitted light spectrum picture and microspectroscopy, can carry out conventional coloured image analysis, makes this instrument constitute a kind of image analyzer that contains multiple function.
Technical characterictic of the present invention is with Hadamard transform spatial multichannel imaging technique and the coupling of microscopic fluorescence technology, adopt 511 Hadamard transform sequences, with the small-sized template of mask etching method, photoetching making as optical modulation component, by computer controlled automatic, microscopic field of view image (light signal) is carried out Hadamard transform (modulation).Image (light signal) through ovennodulation is chosen its characteristic spectrum through collimation, color separation, i.e. the characteristic spectrum of sample to be tested component.Survey detection with line array CCD, the one group of Hadamard transform electric signal that is obtained promptly is reduced into microscopic field of view characteristic spectrum image (component image to be measured in the sample) through digitizing, demodulation, accurately provide microscopically the observed qualitative and quantitative information relevant, also can obtain the more information of observed object by computing machine to Flame Image Process and statistics with small items.
Constitute microscopical transillumination system by microscope components halogen tungsten lamp and condenser etc., by microscope components excitation source (high-pressure sodium lamp, xenon lamp), beam splitting dichroic mirror M, play that the formations such as object lens of condenser are microscopical to fall to penetrating the formula illuminator 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 or optical signal transmissive), and the optical signals microcobjective that is produced is collected.
The Hadamard transform optical system components is compressed imaging lens group L1, M1, L2 by total reflection prism P, Hadamard transform template I and diaphragm, and collimation lens set L2, color separation grating G, mirror M 2, cylindrical lens L3 constitutes.Optical pressure shortens into as lens combination L1, M1 sample is imaged on the template I, by the light signal collimation of L2 compound lens to template I modulation, grating G color separation enters slit S through L3 cylindrical lens compression convergence, by line array CCD the Hadamard transform spectrum picture is finished collecting work.Below the total reflection prism P of optical system components on the casing, use be camera lens with C-Mount standard plug receptacle, can pass through the standard plug receptacle with microscopical the connection, can arbitrarily discharge as the replacing camera lens and patch.
In fluorescent microscope upper arm standard interface place's socket Hadamard transform optical system components, by the position of total reflection prism P " " or " not existing " microscope central light beam, select to determine horizontal optical path system or vertical optical path system.The total reflection prism is inserted light path, and light is promptly rolled over the horizontal optical path system that enters that turn 90 degrees; Completely reflecting mirror is shifted out light path, and light utilizes imaging len L that view field image is focused on colored charge-coupled image sensor (CCD) focal plane vertically upward, and coloured image is finished collecting work.
Adopt photoetching technique to make optical element Hadamard transform template I and diaphragm, be the substrate metal-coated membrane promptly with quartz or optical glass, press the s-matrix of M sequence structure, become 511 seam unit Hadamard transform circulation templates with multilayer mask etching method lithography process, form the bar code of a series of light and shades, every unit seam is wide to be 12.5 μ m.Wherein diaphragm maintains static, and template moves according to computer software instructions, and light signal is carried out spatial modulation.
The Hadamard transform template is fixed on the mechanical driving device, and this device adopts stepper motor, by turbine the rotation of motor is become linear translation, reaches the movable platen displacement.Two stepper motors, one is used for control template and accurately is shifted, and another is used to control grating spectrum scanning, finishes the sampling overall process according to computer software instructions.
Measurement for the characteristic spectrum curve (characteristic spectrum of component to be measured in the sample) of microscopic field of view image, by the computer software control stepper motor, rotating grating G 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 colored charge-coupled image sensor (CCD), Flame Image Process interface board (hardwood storer), computing machine and respective image process software, bears the work that photo chromic microimage is analyzed, and finishes the function of normal image analyser.
To press the accompanying drawing assembling with upper-part, can form Multifunction Aadamard's transformation micrograph analytical instrument, microscopic fluorescence and micro-transmitted light will be carried out image quantitative analysis.
Embodiment 1, Hadamard transform microspectrum image analyser.
See Fig. 1, Fig. 2.The research that analytical sample places the optical instrument factory, Chongqing to produce falls to penetrating on the formula fluorescent microscope objective table, the picture signal of analytical sample (transmitted light or fluorescence) is collected by microcobjective, Hadamard transform optical system components completely reflecting mirror P on the fluorescent microscope is pushed into light path, view field image is promptly rolled over and is turn 90 degrees, enter by convergent lens group L1, mirror M 1, Hadamard transform template I and diaphragm, collimation lens set L2, grating G, mirror M 1, cylindrical lens L3, pass through slit S, i.e. Hadamard transform microspectrum image analyser as shown in the figure.Wherein lens combination L1 compresses the microscopic field of view picture, through mirror M 1 blur-free imaging on Hadamard transform template I plane, fixed Hadamard transform diaphragm is used for limited images spatial modulation scope, and mobile Hadamard transform template I can carry out spatial modulation to picture signal.The picture signal of modulation is assembled through collimation, beam split, cylindrical lens, enters slit, is surveyed by line array CCDs such as the Japanese TCD1208UD of Toshiba type or Japanese shore pine S3904 types, finishes a complete sampling process.For the selection of microscopic field of view characteristics of image spectrum, available computers is controlled grating G 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.Digitized modulation signal is through the fast handmard transform demodulation, be reduced into Hadamard transform microspectrum image (being the component image to be measured in the sample), 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.
Fig. 3 is when using the 40x object lens, the Hadamard transform microspectrum image of two beautiful curtain pollen cells that obtained, the any fluorescence probe reagent dyeing of pollen because its cell membrane contains fluorescent material, therefore can send bright fluorescence (530nm) under strong exciting light (royal purple light) irradiation.Fig. 3 (a) is its autofluorescence image, the transmitted light images of Fig. 3 (b) for obtaining under identical wavelength.The all high-visible pollen cell wall of two accompanying drawings, the sharpness of its image (signal to noise ratio (S/N ratio)) is gratifying.The real space size (being spatial resolution) of each the pixel representative in the image depends on the enlargement ratio and the imaging effect of object lens, measures with result of calculation to show that when using the 40x object lens, spatial resolution can reach 0.6 μ m/pixel.
Embodiment 2: human breast cancer cell fluoroscopic image and nucleus pattern and dna content quantitative test.
Get tumor of breast puncture fluid direct smear, with the normal epithelium cell in normal person's peripheral vein blood lymphocyte or the tumor of breast sample is the standard diploid cell, with acridine orange (AO, 50 μ g/mL, contain 1%Triton X-100) be the cell DNA fluorescence probe, dna content (ploidy) with Hadamard transform microspectrum image analysis-e/or determining HBT cell, 20 tumor locus 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 identify normal cell, sick cell and cancerous tumor cell.The measurement data histogram provides the character of tumour, the aspect utmost point Useful Informations such as grade malignancy of canceration for clinical diagnose intuitively.
Embodiment 3: the cell cycle nonspecific drug carries out the cancer therapy drug preliminary screening.
With human peripheric venous blood lymphocyte or mouse liver cell is material, with fluorescence probe AO (acridine orange) spike judge medicine can enter nucleus and with the binding ability of nuclear DNA, proposing a kind of is the new method that the cell cycle nonspecific agent (CCNSA) of target spot carries out the cancer therapy drug preliminary screening with the nucleus DNA, specific practice is with fluorescence probe AO labelling human peripheral vein blood lymphocyte or mouse liver cell, detects the fluorescence intensity of unicellular nuclear AO-DNA compound at emission peak 530nm place with Hadamard transform microscopic fluorescence image analyzer.With medicine pair cell DNA-AO compound fluorescence inhibiting rate ((F
0-F)/F
0* 100%) height, i.e. the interaction ability power of medicine and cell DNA, the foundation of judging as primary dcreening operation.If the fluorescence inhibiting rate reaches 50%, can judge that this medicine has certain active anticancer.Example is taken known CDDP, mitomycin C, HN
2Deng the cancer therapy drug test, its result conforms to the action principle of medicine.This method is to disturbing the synthetic cell cycle specific cancer therapy drug of biomacromolecule DNA insensitive.At some CCNS cancer therapy drugs of external non-activity, it is very low to show as the fluorescence inhibiting rate, is not the object of this method screening.
Claims (3)
1, a kind of high resolution Adama conversion micro-image analyzer, it comprises light source, fluorescent microscope, has the compression of images imaging, Hadamard transform modulation and collimation, the optical system components that color separation is integrated, opto-electronic conversion detecting device and computing machine is characterized in that Hadamard transform modulation employing 511 one-dimensional coding templates; The opto-electronic conversion detecting device is to include the conversion of sampling/hold amplifier and 16 figure place moulds at interior line array CCD.
2, by the described analyser of claim 1, it is characterized in that the colored charge coupled device ccd of configuration on fluorescent microscope, the Flame Image Process interface board, promptly frame memory constitutes general photo chromic microimage analyser.
3, by claim 1 or 2 described analysers, it is characterized in that the Hadamard transform optical system components is by total reflection prism P, compression imaging lens group L1, M1, Hadamard transform template I and diaphragm, collimation lens set L2, color separation grating G, mirror M 2, cylindrical lens L3 constitutes, and optical pressure shortens into as lens combination L1, M1 sample is imaged on the template I, by the light signal collimation of L2 compound lens to template I modulation, grating G color separation enters slit S through L3 cylindrical lens compression convergence, by line array CCD the Hadamard transform spectrum picture is finished collecting work.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101526400B (en) * | 2008-03-06 | 2010-11-03 | 中国科学院西安光学精密机械研究所 | Hadamard transform interferometric spectral imaging method and Hadamard transform interferometric spectral imaging equipment |
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| CN1296699C (en) * | 2003-12-19 | 2007-01-24 | 武汉大学 | Microscopic multispectral marrow and its peripheral blood cell auto-analyzing instrument and method |
| CN103712638B (en) * | 2013-02-27 | 2016-12-28 | 叶秋诗 | A kind of color absolute raster sensing device |
| 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 |
| CN104796609B (en) * | 2015-04-17 | 2018-01-05 | 南京理工大学 | Large visual field high resolution micro imaging method based on optimal Hadamard coding |
| CN104820994B (en) * | 2015-04-20 | 2017-11-28 | 陕西科技大学 | A kind of analysis method suitable for continuous high-resolution-ration transmission electric-lens image |
| CN105738365B (en) * | 2016-01-27 | 2018-11-06 | 马靖华 | Pathological diagnosis automatic staining analytical equipment |
| CN106204486B (en) * | 2016-07-08 | 2019-04-19 | 广东工业大学 | A kind of micro- cell imaging device and method of near-infrared fluorescent based on Hadamard transform |
| CN107941696A (en) * | 2017-11-28 | 2018-04-20 | 华中科技大学 | A kind of cyclic J-integral imaging system and its imaging method |
| CN112957011B (en) * | 2021-02-01 | 2022-06-24 | 西安电子科技大学 | High-sensitivity weak fluorescence signal detection system, method, storage medium and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101526400B (en) * | 2008-03-06 | 2010-11-03 | 中国科学院西安光学精密机械研究所 | Hadamard transform interferometric spectral imaging method and Hadamard transform interferometric spectral imaging equipment |
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