CN106226276B - A kind of step angle laser aid - Google Patents
A kind of step angle laser aid Download PDFInfo
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- CN106226276B CN106226276B CN201610561212.6A CN201610561212A CN106226276B CN 106226276 B CN106226276 B CN 106226276B CN 201610561212 A CN201610561212 A CN 201610561212A CN 106226276 B CN106226276 B CN 106226276B
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
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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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
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Abstract
The present invention provides a kind of step angle laser aid, including laser, the first reflecting mirror (M1), the second reflecting mirror (M2), third reflecting mirror (M3), scanning galvanometer, turntable, the first lens (L1), the second lens (L2), the third lens (L3), the 4th lens (L4), fluorescence filter plate, dispersion element, adaptation, polling set-up, the first detector (1), the second detector (2), third detector (3), computer.The present invention organically combines high spectral resolution imaging and scanning laser optical tomography, photon utilization efficiency is high, has the advantages that carry out sample the rapidly and efficiently imaging of various dimensions fluorescence imaging, the imaging precision isotropism of 3 D stereo, the advantages that imaging system is compact, easy to use.
Description
Technical field
The present invention relates to photonics and biomedical skewing mechanism design fields more particularly to a kind of photonics and biology to cure
Learn the field for the scanning laser optical tomography method intersected.
Background technique
Life science and medicinal pharmacology research are one of the maximum hot spots of development in science and technology of world and mankind's concern now.Biology
The new branch of subject that medical photonics intersect as photonics with biomedicine, is related to the energy that biosystem is discharged with form of photons
It measures, the structure of related biosystem entrained by photon detection process and photon from biosystem and functional information etc.
Various aspects.Since the technology has the advantages that many protrusions, such as specific good, high sensitivity, resolution ratio height, therefore become
The important research tool that modern life science and biomedical research are increasingly relied on for counsel.
Biomedical research increasingly increases to better understand vital movement, disease development even pharmaceutically-active mechanism
It is added in the demand for studying biological functional information in complete biosystem in high precision.And traditional nuclear magnetic resonance (MRI), X-ray
Computed tomography (X-ray CT) and Positron emission computed tomography (PET) although etc. technologies can obtain three
Dimension imaging, but imaging object primary limitation is in the biomedical sample of macroscopic view.They are not optical image technology, Wu Fali simultaneously
Marker distribution is imaged with technologies such as dyeing/fluorescent markers extremely common on biologic medical.In biomedical light
Imaging field is learned, traditional copolymerization coke/multi-photon optical image technology is limited by light field and sweep speed limits, and is typically only capable to three
Tie up the sample of scanning imagery tens to several hundred microns, it is difficult to meet biomedical research to better understand vital movement, disease
Disease development even pharmaceutically-active mechanism increasingly increases the need for studying biological functional information in high precision in complete biosystem
It asks.Therefore, imaging depth in millimeter to cm range and there is novel Jie of high spatial resolution (micron dimension) to see three-dimensional
Optical image technology is studied once proposition, and just the extensive concern by world biologic medical optical imagery researcher, is dashed forward
Fly the development pushed ahead vigorously, is one of the forward position focus of current biologic medical optical imagery research field.
Currently, the approach for realizing the sight three-dimensional optical imaging that is situated between mainly includes optical projection tomoscan (Optical
Projection Tomography, OPT), scanning laser optical tomography (Scanning Laser Optical
Tomography, SLOT), mating plate frequency microscope (Light Sheet Microscopy).SLOT technology is optical computer tomography
A kind of form under the scope of (Optical Computed Tomography) is scanned, is excited and is detected using single-point, utilize laser
Scanning obtains a series of relevant intensity projection images of angles to form projection imaging, is then based on Computerized three-dimensional recombination and obtains
To the 3 D stereo strength information of sample, can collecting sample absorbing/fluorescent.But it is currently limited to intensity imaging, and intensity is surveyed
The influence of many factors such as the distributed density for being easy stimulated luminescence intensity, sample quenching and fluorescent dye or protein is measured, usually
It is only used as observational measurement, is difficult to carry out effectively quantitative measurment to sample, it is even more impossible to carry out functional imaging.
Fluorescence itself has many reference amounts characteristic in fact, other than intensity described above, the ginseng such as fluorescence spectrum, service life and polarization
Number all contains biological function information abundant.Since fluorescence emission wavelengths are related to the level structure of fluorogen, fluorescence
Spectral measurement can distinguish different fluorogens in conjunction with optical imagery, and to the biochemical function information imaging of spatial discrimination.
Current fluorescence spectrum imaging technique both domestic and external combines microscopic three-dimensional imaging technique more, and (such as confocal microscopic image, multi-photon are aobvious
Micro- imaging even super-resolution fluorescence micro-imaging), and it is mainly used in microcosmic sample (such as cell, unimolecule), what is utilized is
It is scanned imaging in transverse direction (x, y) and axial (z), imaging region, which is less than, is even much smaller than several hundred a microns, can not be suitable for
It is situated between to biologic medical and sees the research of (millimeter-centimetre) sample (integrated biological systems: organ, tissue, embryo and model animal etc.).
In addition, these three-dimensional optical imaging techniques combined are easily led to laterally and axially all by the anisotropic limitation of spatial accuracy
The fuzzy puzzlement of information extraction caused by resolution ratio is inconsistent.These various dimensions Imaging-PAMs are not easy simply to be transplanted to biology
Medical treatment, which is situated between, sees three-dimensional optical imaging, wherein a serious technical bottleneck is exactly effective benefit that two kinds of technologies combine rear excitation photon
It is not high with rate.
According to problem above, for the present invention compared to previous traditional approach, efficiently quick, system compact, behaviour is imaged in the present invention
Facilitate, imaging precision isotropism, the spectrally resolved of 3 D stereo information can be carried out to sample, suitable for tying to sample
Structure and functional analysis.The spectral information of 3 D stereo can be especially provided completely biological tissue's systematic sample, and photon utilizes
Rate is high, and therefore, the present invention has the advantages that more in terms of spectral matching factor.
Summary of the invention
The technical problem to be solved by the present invention is how to choose the fluorescence spectrum resolution techniques combination scanning laser light of high speed
Tomography is learned to form height original technical principle and scheme, realizes efficiently the quickly needle of spatial accuracy isotropic
High spectral resolution is carried out to the 3 D stereo information of sample, and realizes this fluorescence various dimensions quantitative imaging technique and system.This
Technology and systems device can be widely applied to fundamental biological knowledge, immuno-biology, anatomical structure biology, brain science, medicine and grind
Study carefully and the fields such as drug development, is a kind of novel various dimensions measuring technique for complete bio medical system sample, can send out
Exhibition is the important research tool that modern life science and medicinal pharmacology are studied.
To solve the above problems, the present invention is the following technical schemes are provided: a kind of step angle laser aid, including laser
It is device, reflecting mirror M1, reflecting mirror M2, reflecting mirror M3, scanning galvanometer, turntable, lens L1, lens L2, lens L3, lens L4, glimmering
Light filter plate, dispersion element, detector 1, detector 2, detector 3, computer；
Detector 1, detector 2, detector 3 are connect by electric circuit with computer；
Computer is connect with scanning galvanometer and turntable；
Adaptation is connected with computer and detector 1；
Polling set-up is connected with computer and detector 3；
Lens L3, lens L4, fluorescence filter plate form the imaging system that can choose a specific wavelength, fluorescence filtering
Piece is for choosing specific wavelength；
Adaptation is matched according to the instruction of computer according to the type for the sample being previously entered, matched
Rule is:
Sample is numbered, and the information of the number of sample is stored with more helical structures；Wherein, more spiral knots
Structure includes: the laser spectrum region formed with sample, and the region in each region in the laser spectrum region that sample is formed is marked
Know and form single linked list for column, and each area identification is associated as well to a circular linked list, circular linked list includes to belong to laser spectrum
One or more neighbours' laser spectrum area identification of area identification corresponding region；Polling set-up carries out inquiring in computer simultaneously anti-
It feeds detector 3, detector 3 generates control information according to the instruction of polling set-up, believes some unwanted angles of sample
Breath is removed, to complete the image formed according to specific user instruction and dimensional information, forms personalized difference；
Laser issues laser, and by reflecting mirror M1, laser is squeezed into scanning galvanometer by reflecting mirror M2, passes through scanning galvanometer
Reflection is focused on the sample under turntable by lens L1 again；Laser passes through spy by laser remaining after sample absorbance
Device 3 is surveyed, computer is acquired according to instruction；Laser passes through the fluorescence generated after sample can be by semi-transparent semi-reflecting lens BS, partly
The half of fluorescence is passed through lens L3 by saturating semi-reflective mirror BS, and fluorescence filter plate, after lens L4 squeezes into detector 2, computer is according to finger
Order is acquired；The other half of fluorescence focuses on the resolution that dispersion element carries out spectral signal by lens L2, further according to calculating
The instruction of machine carries out the acquisition of spectral signal using detector 1；
Computer issue instruction triggers scanning galvanometer do fast two-dimensional transversal scanning, thus to sample under each angle into
Row scanning, and intensity projection images and the spectrally resolved information projected image of be absorbed light or fluorescence；
Computer issues instruction control triggering turntable step angle, repeats the above steps and finally obtains angle relevant one
The intensity projection images of range of absorbent light fluorescence and spectrally resolved information projected image；
After computer carries out data destructing to the information of acquisition, the projection signal of multi-angle is recycled to pass through linear projection's layer
Reestablishment imaging is analysed, the 3 D stereo strength information of sample and the EO-1 hyperion 3 D stereo information of sample finally can be obtained；
Wherein, computer issues instruction, realizes scan control, turntable control, detector triggering, spectrum assignment, signal
Acquisition and storage function；Computer is connect with detector 1, detector 2, detector 3, and by trigger circuit and scanning galvanometer and
Turntable connection.
Detailed description of the invention
Fig. 1 is system assumption diagram of the invention；
Fig. 2 is work flow diagram of the invention；
Specific embodiment
In order to which technical problems, technical solutions and advantages to be solved are more clearly understood, tie below
Accompanying drawings and embodiments are closed, the present invention will be described in detail.It should be noted that specific embodiment described herein is only used
It to explain the present invention, is not intended to limit the present invention, the product for being able to achieve said function belongs to equivalent replacement and improvement, includes
Within protection scope of the present invention.The specific method is as follows:
Specific embodiment 1:
The invention discloses a kind of step angle laser aids.Fluorescence is obtained using the laser excitation sample of spot scan, is led to
It crosses and high light spectrum image-forming is carried out to the fluorescence that single-point issues, carry out spectrally resolved；Two-dimensional cross is carried out to sample using scanning system
To scanning, sample is rotated in conjunction with turntable, the two-dimensional projection image of the high spectral resolution under all angles is obtained, passes through
Recombination operation is carried out to the projected image under these all angles, realizes the high spectral resolution to sample 3 D stereo information.
The present invention relates to the scanning laser optical tomography method and system dresses that a kind of pair of sample carries out high spectral resolution
It sets, including laser, reflecting mirror M1, reflecting mirror M2, reflecting mirror M3, scanning galvanometer, turntable, lens L1, lens L2, lens
L3, lens L4, fluorescence filter plate, dispersion element, detector 1, detector 2, detector 3, computer；Detector 1, detector 2,
Detector 3 is connect by electric circuit with computer；Computer is connect with scanning galvanometer and turntable；It is lens L3, lens L4, glimmering
Light filter plate forms the imaging system that can choose a specific wavelength, and fluorescence filter plate is for choosing specific wavelength；
Imaging method includes: to obtain fluorescence signal on the laser light incident to sample of (1) spot scan；(2) pass through dispersion element
Receive the spectral information of single-point fluorescence with detector；(3) manipulation laser carries out two-dimensional transversal scanning；(4) manipulation turntable into
The rotation of row angle；(5) after computer carries out data destructing to spectral information, the projection signal of multi-angle is recycled to throw by straight line
Shadow tomography；Described device by laser, dispersion element, detector, turntable, laser scanning system, external trigger signal source,
Data collecting card, computer composition；Detector, data collecting card, computer are successively electrically connected；External trigger signal source and detection
Device, data collecting card connection, and connect by trigger circuit with laser scanning system and turntable；
Laser issues laser, and by reflecting mirror M1, laser is squeezed into scanning galvanometer by reflecting mirror M2, passes through scanning galvanometer
Reflection is focused on the sample under turntable by lens L1 again；Laser passes through spy by laser remaining after sample absorbance
Device 3 is surveyed, computer is acquired according to instruction；Laser, which passes through the absorption fluorescence generated after sample, can pass through semi-transparent semi-reflecting lens
The half for absorbing fluorescence is passed through lens L3 by BS, semi-transparent semi-reflecting lens BS, and fluorescence filter plate after lens L4 squeezes into detector 2, calculates
Machine is acquired according to instruction；The other half absorption fluorescence focuses on point that dispersion element carries out spectral signal by lens L2
It distinguishes, detector 1 is recycled to carry out the acquisition of spectral signal according to the instruction of computer；Adaptation and 1 phase of computer and detector
Even；Polling set-up is connected with computer and detector 3；Lens L3, lens L4, fluorescence filter plate composition one can choose a spy
The long imaging system of standing wave, fluorescence filter plate is for choosing specific wavelength；Adaptation according to the instruction of computer, and according to
The type for the sample being previously entered is matched, and matched rule is: sample being numbered, and by the letter of the number of sample
Breath is stored with more helical structures；Helical structure includes the laser spectrum region formed with sample, and is swashed what sample was formed
The area identification in each region of light SPECTRAL REGION is that column form single linked list, and each area identification is associated as well to circular linked list,
Circular linked list includes one or more neighbours' laser spectrum area identification for belonging to laser spectrum area identification corresponding region；Poll dress
It sets and detector 3 is inquired and fed back in computer, detector 3 generates control information according to the instruction of polling set-up, right
The angle information of sample is screened, to complete the image formed according to specific user instruction and dimensional information, is formed a
Property difference；
Computer issue instruction triggers scanning galvanometer do fast two-dimensional transversal scanning, thus to sample under each angle into
Row scanning, and the intensity projection images for the fluorescence that is absorbed and spectrally resolved information projected image；
Computer issues instruction control triggering turntable step angle, repeats the above steps and finally obtains angle relevant one
The intensity projection images of range of absorbent fluorescence and spectrally resolved information projected image；
After computer carries out data destructing to the information of acquisition, the projection signal of multi-angle is recycled to pass through linear projection's layer
Reestablishment imaging is analysed, the 3 D stereo strength information of sample and the 3 D stereo information of EO-1 hyperion finally can be obtained；
Wherein, computer issues instruction, realizes scan control, turntable control, detector triggering, spectrum assignment, signal
Acquisition and storage function；Computer is connect with detector 1, detector 2, detector 3, and by trigger circuit and scanning galvanometer and
Turntable connection.
Efficiently quick, system compact is imaged in the present invention, and easy to operate, imaging precision isotropism can carry out three to sample
The spectrally resolved of steric information is tieed up, is suitable for carrying out structure and function analysis to sample.It especially can be to completely biological tissue
Systematic sample provides the spectral information of 3 D stereo, and photon utilization rate is high, almost without phototoxicity and photobleaching, while can be to structure
Information and functional information detect, and can be used as the important research tool of fundamental biological knowledge medicinal pharmacology research, such as research form
Development and embry ogenesis, allelotaxis, anatomical structure tissue morphology, the architectural characteristic and functional characteristic of plant and marine organisms
Deng.
Specific embodiment 2:
Such as the workflow of Fig. 2, the present invention chooses the fluorescence spectrum resolution techniques combination scanning laser optical chromatography of high speed
To form height original technical principle and scheme, that realizes efficient quickly spatial accuracy isotropic is directed to sample for imaging
3 D stereo information carry out high spectral resolution, be a kind of fluorescence various dimensions quantitative imaging technique and system.This technology and systems
Device can be widely applied to fundamental biological knowledge, immuno-biology, anatomical structure biology, brain science, medical research and drug and open
The fields such as hair are a kind of novel various dimensions measuring techniques for complete bio medical system sample, can be developed as modern times life
The important research tool of life science and medicinal pharmacology research.This device combination SLOT projection imaging system and spectrally resolved system,
Trigger circuit, scanning galvanometer, turntable and each detector are controlled by computer, to realize that related data is adopted
Collection.The laser generates the laser of spot scan, by reflecting mirror M1, reflecting mirror M2, scanning galvanometer, is incident on turntable
On sample, fluorescence signal is obtained；Fluorescence signal carries out spectrally resolved, signal of the generation with spectrum analysis by dispersion element,
Computer allows detector 1 to acquire the signal for receiving fluorescence signal generation and having spectrum analysis by instruction；
Computer operating scanning galvanometer carries out two-dimensional transversal scanning；
Computer operating turntable carries out angle rotation；
After computer carries out data destructing to spectral information, the projection signal of multi-angle is recycled to chromatograph by linear projection
Imaging；
Wherein detector 2 is fluorescence intensity detector, and detector 3 is absorption intensity detector, this two-way optics can basis
Demand is added or cancels.
This device combination SLOT projection imaging system and spectrally resolved system are realized and are inhaled by system hardware report control platform
Receive (3)/fluorescence (2)/fluorescence spectrum (1) relevant data acquisition.Shown in figure experimental provision as above, wherein 2 roads and 3 tunnels (optical path) can
Increase and decrease according to demand.
Laser (488 nanometers, the semiconductor laser of 200 milliwatts) will swash in the optical path as the excitation light source of imaging
Light focuses on sample, and does fast two-dimensional transverse direction by scanning system (two-dimensional scanning mirrors 2-D Scanning Mirror)
Scanning.The fluorescence that sample is issued enters spectrally resolved system (dispersion element or spectrometer), carries out spectrally resolved and then send
Enter to detector (ccd detector or detector array or sCMOS detector)；It can also be collected by 2 tunnel image optics,
It is sent into the collection that detector 2 (Photo-multiplier Tube) carries out fluorescence intensity；It can also will be absorbed by No. 3 optical paths
Light, which is sent into detector 3 (Photon Detector), carries out absorption signal collection.
It is to be scanned (x, y) by laser scanning system control to be formed in intensity/spectrum projection image of each angle.
By turntable step angle, repeats the above steps and finally obtain a series of relevant intensity projection images of angle
(x, z, θ)/spectral information projected image (x, y, λ, θ)；
Wherein, the hardware report control platform (hardware controls that Labview or Micromanager write of experimental provision are utilized
Program), realize the functions such as above-mentioned scan control, turntable control, detector triggering, spectrum assignment, signal acquisition and storage.Outside
Trigger signal source is connect with detector, data collecting card, and is connect by trigger circuit with laser scanning system and turntable.
Design advantage mainly has: the present invention organically combines high spectral resolution imaging and scanning laser optical tomography, light
Sub- utilization efficiency is high, has the advantages that carry out sample the rapidly and efficiently imaging of various dimensions fluorescence imaging, the imaging of 3 D stereo
The advantages that precision isotropism, imaging system is compact, easy to use.
Claims (1)
1. a kind of step angle laser aid, it is characterised in that: including laser, the first reflecting mirror (M1), the second reflecting mirror
(M2), third reflecting mirror (M3), scanning galvanometer, turntable, the first lens (L1), the second lens (L2), the third lens (L3),
Four lens (L4), fluorescence filter plate, dispersion element, adaptation, polling set-up, the first detector (1), the second detector (2),
Three detectors (3), computer；
First detector (1), second detector (2), the third detector (3) pass through electric circuit and the meter
The connection of calculation machine；
The computer is connect with the scanning galvanometer and the turntable；
The adaptation is connected with the computer and first detector (1)；
The polling set-up is connected with the computer and the third detector (3)；
The third lens (L3), the 4th lens (L4), fluorescence filter plate composition one can choose a certain wave
Long imaging system, the fluorescence filter plate is for choosing specific wavelength；
The adaptation is matched according to the instruction of computer according to the type for the sample being previously entered, wherein first
Detector (1) realizes the acquisition of the first fluorescence data；Second detector (2) is fluorescence intensity detector, realizes the second fluorescence
Spectrum data gathering；Third detector (3) is absorption intensity detector, realizes the acquisition to absorption spectra data；
Absorption, the first fluorescence are realized by system hardware report control platform in conjunction with SLOT projection imaging system and spectrally resolved system
With the second fluorescence spectrum relevant data acquisition；
The laser is 488 nanometers, and 200 milliwatt semiconductor lasers in the optical path, will swash as the excitation light source of imaging
Light focuses on sample, and does fast two-dimensional transversal scanning by scanning galvanometer；The fluorescence that sample is issued enters the dispersion
Element, progress is spectrally resolved to be then fed into first detector (1)；
The laser issues laser, and by first reflecting mirror (M1), laser is squeezed into institute by second reflecting mirror (M2)
Scanning galvanometer is stated, first lens (L1) are passed through by the reflection of the scanning galvanometer again and are focused under the turntable
Sample on；The laser by remaining laser after the sample absorbance by third detector (3), the computer according to
Instruction is acquired；The laser passes through the fluorescence generated after sample can be described semi-transparent semi-reflecting by semi-transparent semi-reflecting lens (BS)
Mirror (BS) beats the half of the fluorescence by the third lens (L3), the fluorescence filter plate, the 4th lens (L4)
After entering second detector (2), the computer is acquired according to instruction；The other half of the fluorescence passes through described second
Lens (L2) focus on the resolution that the dispersion element carries out spectral signal, and the instruction further according to the computer utilizes described the
The acquisition of one detector (1) progress spectral signal；
The computer issues scanning galvanometer described in instruction triggers and does fast two-dimensional transversal scanning, thus to the sample each
It is scanned under angle, and intensity projection images and the spectrally resolved information projected image of be absorbed light or fluorescence；
The computer issues instruction control triggering turntable step angle, finally obtains a series of relevant absorptions of angle
The intensity projection images and spectrally resolved information projected image of light or fluorescence；
After the computer carries out data destructing to the information of acquisition, the projection signal of multi-angle is recycled to pass through linear projection's layer
Reestablishment imaging is analysed, the 3 D stereo strength information of sample and the EO-1 hyperion 3 D stereo information of sample finally can be obtained；
Wherein, it using the hardware report control platform of experimental provision, realizes above-mentioned scan control, turntable control, detector triggering, expose
Photocontrol, signal acquisition and store function；External trigger signal source and first detector (1), the second detector (2), third
Detector (3), data collecting card connection, and connect by trigger circuit with the scanning galvanometer and the turntable.
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| CN107796333B (en) * | 2017-11-20 | 2020-04-24 | 北京航空航天大学 | Optical tomography system based on scanning galvanometer |
| CN108982448B (en) * | 2018-07-20 | 2021-11-02 | 郑州迈迪迅医疗科技有限公司 | Fluorescence immunoassay instrument and detection method thereof |
| CN111338096B (en) * | 2020-03-26 | 2021-06-08 | 吉林大学 | Exciting light three-dimensional focusing scanning system and image scanning method thereof |
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Effective date of registration: 20190829 Address after: 518000 Lantian Road 3002, Shijing Street, Pingshan District, Shenzhen City, Guangdong Province Patentee after: Shenzhen Technical University Address before: Room 611, Science and Technology Building, Shenzhen University, 3688 Nanhai Avenue, Nanshan District, Shenzhen City, Guangdong Province, 518000 Patentee before: Chen Lingling |