CN106248643A - A kind of system of the scanning laser optics chromatography imaging method of high spectral resolution - Google Patents
A kind of system of the scanning laser optics chromatography imaging method of high spectral resolution Download PDFInfo
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- CN106248643A CN106248643A CN201610561213.0A CN201610561213A CN106248643A CN 106248643 A CN106248643 A CN 106248643A CN 201610561213 A CN201610561213 A CN 201610561213A CN 106248643 A CN106248643 A CN 106248643A
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- 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
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
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- 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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N2021/1738—Optionally different kinds of measurements; Method being valid for different kinds of measurement
- G01N2021/174—Optionally different kinds of measurements; Method being valid for different kinds of measurement either absorption-reflection or emission-fluorescence
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- 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
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Abstract
The present invention provides the system of the scanning laser optics chromatography imaging method of a kind of high spectral resolution, including laser instrument, reflecting mirror M1, reflecting mirror M2, reflecting mirror M3, scanning galvanometer, turntable, lens L1, lens L2, lens L3, lens L4, fluorescence filter plate, dispersion original paper, detector 1, detector 2, detector 3, computer, the present invention organically combines high spectral resolution imaging and scanning laser optics tomography, photon utilization ratio is high, the advantage with the rapidly and efficiently imaging that sample is carried out various dimensions fluorescence imaging, the imaging precision isotropism of 3 D stereo, imaging system is compact, the advantage such as easy to use.
Description
Technical field
The present invention relates to photonic propulsion and biomedical skewing mechanism design field, particularly relate to a kind of photonic propulsion and biological doctor
Learn the field of the scanning laser optics chromatography imaging method intersected.
Background technology
Life sciences and medicinal pharmacology research are one of maximum focuses that development in science and technology of world and the mankind pay close attention to now.Biological
The new branch of subject that medical photonics intersects with biomedicine as photonic propulsion, relates to the energy that biosystem discharges with form of photons
Amount, from the photon detection process of biosystem, and the structure of the relevant biosystem entrained by photon and function information etc.
Various aspects.Owing to this technology has many prominent advantages, as good, highly sensitive in specificity, resolution is high, therefore become
The important research instrument that modern life science and biomedical research are increasingly relied on for counsel..
Biomedical research, in order to be better understood from vital movement, the disease the most pharmaceutically-active mechanism of development, increases day by day
It is added on complete biosystem the demand of the biological function information of high accuracy research.And traditional nuclear magnetic resonance, NMR (MRI), X-ray
Computed tomography (X-ray CT) and Positron emission computed tomography (PET) although etc. technology can obtain three
Dimension imaging, but imaging object primary limitation is at the biomedical sample of macroscopic view.They not optical image technologies simultaneously, it is impossible to profit
By technology such as dyeing/fluorescent labelinies extremely conventional on biologic medical, label distribution is carried out imaging.At biomedical light
Learning imaging field, traditional copolymerization Jiao/multi-photon optical image technology is limited by light field and sweep speed limits, and is typically only capable to three
Dimension scanning imagery tens is to the sample [1,2] of hundreds of μm, it is difficult to meet biomedical research in order to be better understood from vital movement,
Disease develops the most pharmaceutically-active mechanism, day by day increases the biological function information of high accuracy research on complete biosystem
Demand.Therefore, imaging depth in mm-cm scope and has novel Jie of high spatial resolution (μm magnitude) and sees three-dimensional light and study
As studying once proposition of technology, just by the extensive concern of world's biologic medical optical imagery researcher, advanced by leaps and bounds
Development, be one of the forward position focus of current biologic medical optical imagery research field.
Currently, it is achieved the approach of the sight three-dimensional optical imaging that is situated between mainly includes optical projection tomoscan (Optical
Projection Tomography, OPT) [3], scan laser optics tomography (Scanning Laser Optical
Tomography, SLOT) [4], mating plate frequency microscope (Light Sheet Microscopy) [5,6].SLOT technology is optics meter
A kind of form under the scope of calculation machine tomoscan (Optical Computed Tomography), uses single-point excite and detect,
Utilize laser scanning to form projection imaging, obtain the intensity projection images that a series of angle is relevant, be then based on computer three
Dimension restructuring receives the 3 D stereo strength information of sample, can the absorbing/fluorescent of collecting sample.But it is currently limited to intensity imaging,
And the distributed densities of ionization meter easy stimulated luminescence intensity, sample quencher and fluorescent dye or protein etc. are permitted multifactorial shadow
Ring, be generally only used as observation measurements, be difficult to sample is carried out quantitative measurement effectively, more cannot be carried out functional imaging.
Fluorescence self has many reference amounts characteristic in fact, and in addition to intensity described above, fluorescence spectrum, life-span and polarization etc. are joined
Number all contains abundant biological function information.Owing to fluorescence emission wavelengths is relevant to the level structure of fluorogen, therefore fluorescence
Spectral measurement combines from optical imagery can distinguish different fluorogens, and the biochemical function information imaging to spatial discrimination.
(such as confocal microscopic image, multi-photon shows current fluorescence spectrum imaging technique many combinations microscopic three-dimensional imaging technique both domestic and external
Micro-imaging even super-resolution fluorescence micro-imaging), and be mainly used in microcosmic sample (such as cell, unimolecule etc.), utilization be
Laterally (x, y) and axially (z) is scanned imaging, and imaging region is less than even much smaller than hundreds of μm, it is impossible to it is right to be applicable to
Biologic medical is situated between and sees the research of (mm-cm) sample (integrated biological systems: organ, tissue, embryo and model animal etc.).It addition,
In conjunction with these three-dimensional optical imaging techniques all by the anisotropic restriction of spatial accuracy, be easily caused laterally and axially resolution
The puzzlement that the inconsistent information retrieval caused is fuzzy.These various dimensions Imaging-PAMs are difficult to simply be transplanted to biologic medical and are situated between
Seeing three-dimensional optical imaging, a most serious technical bottleneck is exactly that after two kinds of technology combine the effective rate of utilization of excitation photon is not
High.
According to problem above, the present invention is compared to conventional traditional approach, and imaging of the present invention is efficiently quick, system compact, behaviour
Facilitate, imaging precision isotropism, sample can be carried out the spectrally resolved of 3 D stereo information, it is adaptable to sample is tied
Structure and functional analysis.Intactly biological tissue's systematic sample especially can provide the spectral information of 3 D stereo, and photon utilizes
Rate is high, and therefore, the present invention has more advantage in terms of spectral matching factor.
Summary of the invention
The technical problem to be solved in the present invention is: how to choose fluorescence spectrum resolution techniques at a high speed and combines scanning laser light
Learn tomography to form highly original know-why and scheme, it is achieved the most quickly pin of spatial accuracy isotropic
The 3 D stereo information of sample is carried out high spectral resolution, and realizes this fluorescence various dimensions quantitative imaging technique and system.This
Technology and system and device can be widely applied to fundamental biological knowledge, immunobiology, anatomical structure biology, brain science, medical science are ground
Study carefully and the field such as drug development, be a kind of novel various dimensions measurement technology for complete bio medical system sample, can send out
Exhibition is modern life science and the important research instrument of medicinal pharmacology research.
For solving the problems referred to above, the present invention provides techniques below scheme: the scanning laser optics layer of a kind of high spectral resolution
The system of analysis formation method, including laser instrument, reflecting mirror M1, reflecting mirror M2, reflecting mirror M3, scanning galvanometer, turntable, lens
L1, lens L2, lens L3, lens L4, fluorescence filter plate, dispersion original paper, detector 1, detector 2, detector 3, computer;
Detector 1, detector 2, detector 3 are connected with computer by electric circuit;
Computer is connected with scanning galvanometer and turntable;
Lens L3, lens L4, fluorescence filter plate one imaging system that can choose a specific wavelength of composition, fluorescence filters
Sheet is used for choosing specific wavelength;
Laser instrument sends laser, and by reflecting mirror M1, laser is squeezed into scanning galvanometer by reflecting mirror M2, by scanning galvanometer
Reflection is focused on the sample being positioned under turntable by lens L1 again;Laser passes through to visit by remaining laser after sample absorbance
Surveying device 3, computer is acquired according to instruction;Laser can pass through semi-transparent semi-reflecting lens BS by the fluorescence produced after sample, and half
Semi-reflective mirror BS is by the half of fluorescence through lens L3, fluorescence filter plate thoroughly, and after lens L4 squeezes into detector 2, computer is according to finger
Order is acquired;Second half of fluorescence focuses on dispersion original paper through lens L2 and carries out the resolution of spectral signal, further according to calculating
The instruction of machine utilizes detector 1 to carry out the collection of spectral signal;
Computer sends instruction triggers scanning galvanometer and does fast two-dimensional transversal scanning, thus enters sample under each angle
Row scanning, and be absorbed light or the intensity projection images of fluorescence and spectrally resolved information projection picture;
Computer sends instruction and controls to trigger turntable step angle, and repeat the above steps finally gives that angle is relevant
The intensity projection images of range of absorbent light fluorescence and spectrally resolved information projection picture;
After computer carries out data destructing to the information gathered, the projection signal of recycling multi-angle is by linear projection's layer
Analysis reestablishment imaging, finally can get the 3 D stereo strength information of sample and the EO-1 hyperion 3 D stereo information of sample;
Wherein, computer sends instruction, it is achieved the control of scan control, turntable, detector triggering, spectrum assignment, signal
Gather and storage function;Computer is connected with detector 1, detector 2, detector 3, and by trigger circuit and scanning galvanometer and
Turntable connects.
Accompanying drawing explanation
Fig. 1 is the system assumption diagram of the present invention;
Fig. 2 is the workflow diagram of the present invention;
Fig. 3 is the system assumption diagram of another embodiment of the present invention;
Detailed description of the invention
In order to make the technical problem to be solved, technical scheme and beneficial effect clearer, below tie
Closing drawings and Examples, the present invention will be described in detail.It should be noted that, specific embodiment described herein is only used
To explain the present invention, being not intended to limit the present invention, the product that can realize said function belongs to equivalent and improvement, all comprises
Within protection scope of the present invention.Concrete grammar is as follows:
Specific embodiment 1:
The invention discloses the system of the scanning laser optics chromatography imaging method of a kind of high spectral resolution.Utilize spot scan
Laser excitation sample obtain fluorescence, carry out high light spectrum image-forming by the fluorescence that single-point is sent, carry out spectrally resolved;Utilization is swept
System of retouching carries out the transversal scanning of two dimension to sample, rotates sample in conjunction with turntable, obtains Gao Guang under all angles
The two-dimensional projection image that spectrum is differentiated, by carrying out computing of recombinating, it is achieved to sample three to the projection picture under these all angles
The high spectral resolution of dimension steric information.
The present invention relates to a kind of scanning laser optics chromatography imaging method that sample is carried out high spectral resolution and system dress
Put, including laser instrument, reflecting mirror M1, reflecting mirror M2, reflecting mirror M3, scanning galvanometer, turntable, lens L1, lens L2, lens
L3, lens L4, fluorescence filter plate, dispersion original paper, detector 1, detector 2, detector 3, computer;Detector 1, detector 2,
Detector 3 is connected with computer by electric circuit;Computer is connected with scanning galvanometer and turntable;Lens L3, lens L4, glimmering
Light filter plate one imaging system that can choose a specific wavelength of composition, fluorescence filter plate is used for choosing specific wavelength;
Formation method includes: on the laser light incident of (1) spot scan to sample, it is thus achieved that fluorescence signal;(2) by dispersion original paper
With the spectral information that detector accepts single-point fluorescence;(3) manipulation laser carries out the transversal scanning of two dimension;(4) manipulation turntable enters
Row angle rotates;(5), after computer carries out data destructing to spectral information, the projection signal of recycling multi-angle is thrown by straight line
Shadow tomography;Described device by laser instrument, dispersion original paper, detector, turntable, laser scanning system, external trigger signal source,
Data collecting card, computer form;Detector, data collecting card, computer are electrically connected successively;External trigger signal source and detection
Device, data collecting card connect, and are connected with laser scanning system and turntable by triggering circuit;
Laser instrument sends laser, and by reflecting mirror M1, laser is squeezed into scanning galvanometer by reflecting mirror M2, by scanning galvanometer
Reflection is focused on the sample being positioned under turntable by lens L1 again;Laser passes through to visit by remaining laser after sample absorbance
Surveying device 3, computer is acquired according to instruction;Laser can pass through semi-transparent semi-reflecting lens by the absorption fluorescence produced after sample
The half of absorption fluorescence through lens L3, fluorescence filter plate, after lens L4 squeezes into detector 2, is calculated by BS, semi-transparent semi-reflecting lens BS
Machine is acquired according to instruction;Second half absorption fluorescence focuses on dispersion original paper through lens L2 and carries out dividing of spectral signal
Distinguishing, recycling detector 1 carries out the collection of spectral signal according to the instruction of computer;
Computer sends instruction triggers scanning galvanometer and does fast two-dimensional transversal scanning, thus enters sample under each angle
Row scanning, and the intensity projection images of the fluorescence that is absorbed and spectrally resolved information projection picture;
Computer sends instruction and controls to trigger turntable step angle, and repeat the above steps finally gives that angle is relevant
The intensity projection images of range of absorbent fluorescence and spectrally resolved information projection picture;
After computer carries out data destructing to the information gathered, the projection signal of recycling multi-angle is by linear projection's layer
Analysis reestablishment imaging, finally can get the 3 D stereo strength information of sample and the 3 D stereo information of EO-1 hyperion;
Wherein, computer sends instruction, it is achieved the control of scan control, turntable, detector triggering, spectrum assignment, signal
Gather and storage function;Computer is connected with detector 1, detector 2, detector 3, and by trigger circuit and scanning galvanometer and
Turntable connects.
Imaging of the present invention is efficiently quick, and system compact is easy to operate, imaging precision isotropism, sample can be carried out three
Tie up the spectrally resolved of steric information, it is adaptable to sample is carried out 26S Proteasome Structure and Function analysis.Especially can be to intactly biological tissue
Systematic sample provides the spectral information of 3 D stereo, and photon utilization rate is high, almost without phototoxicity and photobleaching, and simultaneously can be to structure
Information and function information detect, can based on biology medicinal pharmacology research important research instrument, such as study form
Grow and embry ogenesis, allelotaxis, anatomical structure tissue morphology, plant and halobiontic architectural characteristic and functional characteristic
Deng.
Specific embodiment 2:
Such as workflow and the structural representation of Fig. 3 of Fig. 2, wherein detector 2 is fluorescence intensity detector, detector 3
For absorption intensity detector, this two-way optics can add according to demand or cancel.
This device combines SLOT projection imaging system and spectrally resolved system, by system hardware report control platform, it is achieved inhale
Receive (3)/fluorescence (2)/fluorescence spectrum (1) relevant data acquisition.As above, shown in figure experimental provision, wherein 2 roads and 3 tunnels can be according to need
Ask increase and decrease.
(1) laser instrument (semiconductor laser of e.g.488nm, 200mw) is as the excitation source of imaging, in the optical path,
By on laser focusing to sample, and done quickly by scanning system (e.g. two-dimensional scanning mirrors 2-D Scanning Mirror)
Two-dimensional transversal scans.The fluorescence that sample is sent enters spectrally resolved system (e.g. dispersion original paper or spectrogrph), carries out light
Spectrum resolution is then fed into detector (e.g.CCD detector or detector array or sCMOS detector);Can also pass through
2 tunnel image opticss are collected, and send into detector 2 (e.g.Photo-multiplier Tube) and carry out the collection of fluorescence intensity;Also may be used
Absorption signal collection is carried out absorbing light to be sent in detector 3 (e.g Photon Detector) by No. 3 light paths.
(2) intensity/spectrum projection image in each angle is to be controlled to be scanned (x, y) shape by laser scanning system
Become.
(3) by turntable step angle, repeat the above steps finally gives a series of intensity projection figures that angle is relevant
As (x, z, θ)/spectral information projection picture (x, y, λ, θ);
Wherein, the hardware report control platform (hardware that e.g.Labview or Micromanager writes of experimental provision is utilized
Control program), it is achieved the merits such as the control of above-mentioned scan control, turntable, detector triggering, spectrum assignment, signals collecting and storage
Energy.External trigger signal source is connected with detector, data collecting card, and connects with laser scanning system and turntable by triggering circuit
Connect.
This device combines SLOT projection imaging system and spectrally resolved system, by computer to triggering circuit, scanning and shake
Mirror, turntable and each detector are controlled, thus realize relevant data acquisition.Described laser instrument produces swashing of spot scan
Light, by reflecting mirror M1, reflecting mirror M2, scanning galvanometer, the sample that incides on turntable, it is thus achieved that fluorescence signal;Fluorescence is believed
Number carrying out spectrally resolved by dispersion original paper, produce the signal with spectrum analysis, computer allows detector 1 gather by instruction
Accept fluorescence signal and produce the signal with spectrum analysis;
Computer operating scanning galvanometer carries out the transversal scanning of two dimension;
Computer operating turntable carries out angle rotation;
After computer carries out data destructing to spectral information, the projection signal of recycling multi-angle is chromatographed by linear projection
Imaging;
Wherein detector 2 is fluorescence intensity detector, and detector 3 is absorption intensity detector, and this two-way optics can basis
Demand adds or cancels.
This device combines SLOT projection imaging system and spectrally resolved system, by system hardware report control platform, it is achieved inhale
Receive (3)/fluorescence (2)/fluorescence spectrum (1) relevant data acquisition.As above, shown in figure experimental provision, wherein 2 roads and 3 tunnels (light path) can
Increase and decrease according to demand.
Laser instrument (488 nanometers, the semiconductor laser of 200 milliwatts) is as the excitation source of imaging, in the optical path, will swash
Light focuses on sample, and it is horizontal to do fast two-dimensional by scanning system (two-dimensional scanning mirrors 2-D Scanning Mirror)
Scanning.Fluorescence that sample is sent enters spectrally resolved system (dispersion original paper or spectrogrph), carries out spectrally resolved then sending
Enter to detector (ccd detector or detector array or sCMOS detector);Can also be collected by 2 tunnel image opticss,
Send into detector 2 (Photo-multiplier Tube) and carry out the collection of fluorescence intensity;To can also be absorbed by No. 3 light paths
Light is sent in detector 3 (Photon Detector) and is carried out absorption signal collection.
Intensity/spectrum projection image in each angle is to be controlled to be scanned (x, y) formation by laser scanning system.
By turntable step angle, repeat the above steps finally gives a series of intensity projection images that angle is relevant
(x, z, θ)/spectral information projection picture (x, y, λ, θ);
Wherein, the hardware report control platform (hardware controls that Labview or Micromanager writes of experimental provision is utilized
Program), it is achieved the functions such as the control of above-mentioned scan control, turntable, detector triggering, spectrum assignment, signals collecting and storage.Outward
Trigger signal source is connected with detector, data collecting card, and is connected with laser scanning system and turntable by triggering circuit.
Design advantage mainly has: the present invention organically combines high spectral resolution imaging and scanning laser optics tomography, light
Sub-utilization ratio is high, the advantage with the rapidly and efficiently imaging that sample carries out various dimensions fluorescence imaging, the imaging of 3 D stereo
Precision isotropism, the advantages such as imaging system is compact, easy to use.
Claims (1)
1. the system of the scanning laser optics chromatography imaging method of a high spectral resolution, it is characterised in that: include laser instrument, anti-
Penetrate mirror M1, reflecting mirror M2, reflecting mirror M3, scanning galvanometer, turntable, lens L1, lens L2, lens L3, lens L4, fluorescence filtering
Sheet, dispersion original paper, detector 1, detector 2, detector 3, computer;
Described detector 1, described detector 2, described detector 3 are connected with described computer by electric circuit;
Described computer is connected with described scanning galvanometer and described turntable;
Described lens L3, described lens L4, described fluorescence filter plate one imaging system that can choose a specific wavelength of composition,
Described fluorescence filter plate is used for choosing specific wavelength;
Described laser instrument sends laser, by described reflecting mirror M1, described reflecting mirror M2, laser is squeezed into described scanning galvanometer, logical
The reflection crossing described scanning galvanometer is focused on the sample being positioned under described turntable by described lens L1 again;Described laser leads to
After crossing described sample absorbance, remaining laser passes through detector 3, and described computer is acquired according to instruction;Described laser passes through
The fluorescence produced after sample can pass through semi-transparent semi-reflecting lens BS, described semi-transparent semi-reflecting lens BS by the half of described fluorescence through described
Lens L3, described fluorescence filter plate, after described lens L4 squeezes into described detector 2, described computer is acquired according to instruction;
Second half of described fluorescence focuses on described dispersion original paper through described lens L2 and carries out the resolution of spectral signal, further according to described
The instruction of computer utilizes described detector 1 to carry out the collection of spectral signal;
Described computer sends scanning galvanometer described in instruction triggers and does fast two-dimensional transversal scanning, thus to described sample each
It is scanned under angle, and obtains described absorbing light or the intensity projection images of fluorescence and spectrally resolved information projection picture;
Described computer sends instruction and controls to trigger turntable step angle, and repeat the above steps finally gives that angle is relevant
The intensity projection images of the described absorbing light fluorescence of series and spectrally resolved information projection picture;
After described computer carries out data destructing to the information gathered, the projection signal of recycling multi-angle is by linear projection's layer
Analysis reestablishment imaging, finally can get the 3 D stereo strength information of sample and the EO-1 hyperion 3 D stereo information of sample;
Wherein, described computer sends instruction, it is achieved the control of scan control, turntable, detector triggering, spectrum assignment, signal
Gather and storage function;Described computer is connected with described detector 1, described detector 2, described detector 3, and by triggering
Circuit is connected with described scanning galvanometer and described turntable.
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CN108836275A (en) * | 2018-07-10 | 2018-11-20 | 天津工业大学 | A kind of fluorescent molecular tomography system under free space |
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Cited By (4)
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CN108836275A (en) * | 2018-07-10 | 2018-11-20 | 天津工业大学 | A kind of fluorescent molecular tomography system under free space |
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