CN104677872A - Multi-photon excited multidirectional lighting microscopy imaging system - Google Patents
Multi-photon excited multidirectional lighting microscopy imaging system Download PDFInfo
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- 238000003384 imaging method Methods 0.000 title claims abstract description 67
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- 238000005286 illumination Methods 0.000 claims abstract description 53
- 230000005284 excitation Effects 0.000 claims description 50
- 230000013011 mating Effects 0.000 claims description 47
- 238000012545 processing Methods 0.000 claims description 17
- 238000001914 filtration Methods 0.000 claims description 3
- 230000004313 glare Effects 0.000 claims description 3
- 238000013519 translation Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 abstract description 11
- 238000011161 development Methods 0.000 abstract description 3
- 230000001537 neural effect Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 39
- 210000004027 cell Anatomy 0.000 description 7
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- 230000009286 beneficial effect Effects 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
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- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
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- 238000004621 scanning probe microscopy Methods 0.000 description 1
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Abstract
The invention discloses a multi-photon excited multidirectional lighting microscopy imaging system. From front to back, the microscopy imaging system comprises a multi-photon laser unit, a lighting unit and an imaging detection unit which are sequentially arranged on a fluorescent light path of the imaging system, wherein the multi-photon laser unit is used for generating and modulating laser and for transmitting the modulated laser to the lighting unit; the lighting unit is used for receiving the modulated laser, generating multidirectional light sheet illumination and exciting a to-be-detected sample to generate fluorescent light; and the imaging detection unit is used for detecting the fluorescent light generated from the to-be-detected sample and converting the fluorescent light into a digital image. The multi-photon excited multidirectional lighting microscopy imaging system disclosed by the invention can image rapidly, so as to image for the dynamic development of living organisms or record neuronal activities.
Description
Technical field
The invention belongs to optical field, particularly relate to Induced Fluorescence Microscopy.
Background technology
Existing fluorescent microscopic imaging can be divided into by the scanning microscopy imaging of spot scan mode imaging according to imaging mode and re-use the mating plate illumination micro-imaging that CCD etc. carries out detection imaging after irradiating fluorescence excitation by side thin-beam, in addition, existing fluorescent microscopic imaging can be divided into one-photon excitation and two-photon excitation by fluorescence excitation conditions, two-photon excitation is also known as multiphoton excitation, two-photon excitation refers under the sub-density of high light, fluorescence molecule can simultaneously stability two long wavelength photons, the photon that wavelength is shorter is launched after exciting the life-span, thus two-photon excitation can utilize the light wave longer compared with one-photon excitation wavelength to carry out fluorescence excitation, also near infrared light namely can be utilized to carry out fluorescence excitation, namely and near infrared light possesses two advantages: first is that higher also imaging depth is high thus be applicable to observe thick sample for penetration depth, second is little to biopsy sample toxicity to be detected thus is applicable to observe biological living sample.
In prior art, there is the multiphoton excitation scanning imaging technology utilizing Mapping mode, although this technology can detect living body biological sample, owing to being obtain fluoroscopic image by point by point scanning, use this technology for detection and the speed of synthetic image is slow.
Summary of the invention
The invention provides the multiphoton excitation multidirectional illumination micro imaging system that a kind of image taking speed is fast, for solving the technological deficiency that in existing micro-imaging technique, image taking speed is slow.
The invention provides a kind of multiphoton excitation multidirectional illumination micro imaging system, comprise be positioned at described imaging system from front to back successively fluorescence light path on: multi-photon laser cell, lighting unit, image checking unit;
Described multi-photon laser cell, for generation of with modulated laser and by modulation after laser send into lighting unit; Described lighting unit is located at the side of described sample to be detected, produces multidirectional mating plate again throw light on and excite sample to be detected to produce fluorescence for the laser after receiving described modulation; Described image checking unit is positioned at above sample to be detected, for detecting the fluorescence of sample to be detected generation and transferring digital picture to, at least comprises and detects described fluorescence and the photodetection camera transferring electric signal to.
Especially, described lighting unit be included in described imaging system fluorescence light path on position successively multi-photon mating plate rearward produce light path and multidirectional mating plate illumination objective lens group; Described multidirectional mating plate illumination objective lens group comprises at least two illumination objective lens be arranged circumferentially;
Described multi-photon mating plate produces light path and more described laser is divided into multidirectional mating plate for the laser after receiving described modulation and each illumination objective lens of corresponding incident described multidirectional mating plate illumination objective lens group respectively; Any described illumination objective lens throws light on for the mating plate receiving a direction in multidirectional mating plate again and excites sample to be detected to produce fluorescence.
Especially, all vertical with the axle of described image checking unit and the described mating plate of mating plate that each illumination objective lens of described multidirectional mating plate illumination objective lens group receives impinges upon and sample to be detected excites position in the focal plane of described image checking unit.
Especially, described multi-photon laser cell be included in described imaging system fluorescence light path on position LASER Light Source rearward and multiphoton excitation light path successively;
Described LASER Light Source for generation of high photon density laser and send into described multiphoton excitation light path, described multiphoton excitation light path be used for receive and modulate described laser and send into described lighting unit.
Especially, described image checking unit be included in described imaging system fluorescence light path on position objective table, image-forming objective lens and filter plate rearward successively;
Described objective table be used for fixing and change sample to be detected excite position, be excited the fluorescence produced in multiple angle or the degree of depth to make sample to be detected; Described image-forming objective lens is positioned at above sample to be detected, for focusing on described fluorescence and sending into described filter plate; Described filter plate is fixedly installed on the described light path front of described photodetection camera, for the veiling glare in fluorescence described in filtering.
Especially, described objective table at least comprises microoperation controller, and described microoperation controller is used for the operation that sample to be detected is performed to translation or rotates around vertical axes, to adjust the relative position of described sample to be detected and described imaging system light path.
Especially, described image checking unit also comprises electrical signal collection control module and central processing module, described electrical signal collection control module is specifically for receiving the electric signal of described photodetection camera output and being converted to digital signal, and described central processing module is specifically for receiving described digital signal and generating digital picture.
Beneficial effect of the present invention is:
Multiphoton excitation multidirectional illumination micro imaging system of the present invention carries out detection imaging by adopting mating plate illumination micro-imaging technique to the fluorescence that multiphoton excitation sample to be detected produces, corresponding image checking unit is made to carry out parallel image collection, thus improve image taking speed compared to multi-photon laser dot scans micro-imaging of the prior art, and multiphoton excitation improves the detection degree of depth owing to make use of near-infrared excitation light, that is the higher detection degree of depth can be reached under the condition ensureing image taking speed, therefore the dynamic imaging to thicker biological living can be realized, in addition multiphoton excitation multidirectional illumination micro imaging system of the present invention carries out imaging by adopting the mating plate of multiple directions to carry out the illumination biosome that can realize having opaque section to sample to be detected, owing to combining the advantage that can realize many mating plate illumination two aspects such as the image taking speed of micro-imaging and the imaging depth of two-photon excitation that parallel image gathers, at medical imaging field, the present invention may be used for carrying out dynamic development imaging and neuron activity record to the living body biological such as zebra fish, fruit bat.
Accompanying drawing explanation
Fig. 1 is the structured flowchart that the present invention's real multiphoton excitation multidirectional illumination micro imaging system executes example one;
Fig. 2 is the Organization Chart that the present invention's real multiphoton excitation multidirectional illumination micro imaging system executes example one;
In figure: 1-multi-photon laser cell, 11-LASER Light Source, 12-multiphoton excitation light path, 2-lighting unit, 21-multi-photon mating plate produces light path, 22-multidirectional mating plate illumination objective lens group, 3-image checking unit, 31-objective table, 32-image-forming objective lens, 33-filter plate, 34-photodetection camera, 35-electrical signal collection control module, 36-central processing module.
Embodiment
Fig. 1 is the structured flowchart that the present invention's real multiphoton excitation multidirectional illumination micro imaging system executes example one, Fig. 2 is the Organization Chart of the embodiment of the present invention one multiphoton excitation multidirectional illumination micro imaging system, as depicted in figs. 1 and 2, multiphoton excitation multidirectional illumination micro imaging system of the present invention, comprise be positioned at described imaging system from front to back successively fluorescence light path on: multi-photon laser cell 1, lighting unit 2, image checking unit 3;
Described multi-photon laser cell 1, for generation of with modulated laser and by modulation after laser send into lighting unit; Described lighting unit 2 is located at the side of described sample to be detected, produces multidirectional mating plate again throw light on and excite sample to be detected to produce fluorescence for the laser after receiving described modulation; Described image checking unit 3 is positioned at above sample to be detected, for detecting the fluorescence of sample to be detected generation and transferring digital picture to, at least comprises and detects described fluorescence and the photodetection camera 34 transferring electric signal to; Preferably, described photodetection camera can be high speed SCOMS chip camera, can the fluorescence that produces to sample to be detected of quick detection.
Preferably, described lighting unit 2 be included in described imaging system fluorescence light path on position successively multi-photon mating plate rearward produce light path 21 and multidirectional mating plate illumination objective lens group 22; Described multidirectional mating plate illumination objective lens group 22 comprises at least two illumination objective lens be arranged circumferentially;
Described multi-photon mating plate produces light path 21 and more described laser is divided into multidirectional mating plate for the laser after receiving described modulation and each illumination objective lens of corresponding incident described multidirectional mating plate illumination objective lens group 22 respectively; Any described illumination objective lens throws light on for the mating plate receiving a direction in multidirectional mating plate again and excites sample to be detected to produce fluorescence.
Preferably, the mating plate that each illumination objective lens of described multidirectional mating plate illumination objective lens group 22 receives also is all that the vertical and described mating plate of the axle of image-forming objective lens 32 impinges upon sample to be detected excites position in the focal plane of described image checking unit also namely in the focal plane of image-forming objective lens with the axle of described image checking unit 3.
Preferably, described multi-photon laser cell 1 be included in described imaging system fluorescence light path on position LASER Light Source 11 rearward and multiphoton excitation light path 12 successively;
Described LASER Light Source 11 for generation of high photon density laser and send into described multiphoton excitation light path 12, described multiphoton excitation light path 12 is for receiving and modulating described laser and send into described lighting unit 2; Preferably, described LASER Light Source 11 is ultrafast femtosecond pulse laser, can produce the exciting light of high photon density; Described multiphoton excitation light path 12 comprises the optical elements such as laser beam expander, catoptron and scanning galvanometer, and the laser specifically for producing the described LASER Light Source 11 received is modulated and the described multi-photon mating plate sent in described lighting unit 2 produces light path 21.
Preferably, described image checking unit 3 be included in described imaging system fluorescence light path on position objective table 31, image-forming objective lens 32 and filter plate 33 rearward successively;
Described objective table 31 for fixing and change sample to be detected excite position, be excited the fluorescence produced in multiple angle or the degree of depth to make sample to be detected, to produce single sectioning image; Described image-forming objective lens 32 is positioned at above sample to be detected, for focusing on described fluorescence and sending into described filter plate 33; Described filter plate 33 is fixedly installed on the described light path front of described photodetection camera 34, for the veiling glare in fluorescence described in filtering.
Preferably, described objective table 31 at least comprises microoperation controller, described microoperation controller is used for the operation that sample to be detected is performed to translation or rotates around vertical axes, to adjust the relative position of described sample to be detected and described imaging system light path, described sample to be detected can move at three dimensions and rotate at vertical direction, sample to be detected is excited to produce fluorescence to enable the described multidirectional mating plate illumination objective lens 22 groups of multi-angles in described lighting unit 2 and multilayer, to produce multiple sectioning images of sample to be detected, again the 3-D view that three-dimensionalreconstruction can obtain sample to be detected is carried out to sectioning image.
Preferably, described image checking unit 3 also comprises electrical signal collection control module 35 and central processing module 36, described electrical signal collection control module 35 is specifically for receiving the electric signal of described photodetection camera output and being converted to digital signal, described central processing module 36 is specifically for receiving described digital signal and generating digital picture, also namely described central processing module 36 specifically for receive described photodetection camera 34 according to detected fluorescence conversion and the electric signal exported and it is preserved and processes, thus finally obtain the high resolution digital image of sample to be detected, preferably, described central processing module 36 can be computing machine, described central processing module 36 is provided with the slot of graphics processing card and can supports figure parallel computation, the electric signal corresponding for the fluorescence continuously sent by the sample to be detected collected transfers digital picture to, now digital picture is the two-dimensional slice image of sample to be detected, again the process such as position registration, image co-registration and image reconstruction are carried out to the digital picture collected, finally image is carried out the 3-D view that three-dimensional reconstruction obtains sample to be detected.
Preferably, described electrical signal collection control module 35 also connects and the control signal sent by central processing module 36 to the microoperation controller input of described LASER Light Source 11 and described objective table 31 respectively; Preferably, to realize the electrical signal collection control module of central processing module to objective table, described central processing module 36 is provided with the communication control interface for connecting objective table, this interface can be USB interface or serial line interface, these interfaces are connected with objective table by USB line or string line, its parameter such as position and speed can be read from objective table 31, and the parameter of acquisition is returned described central processing module 36; Also namely described central processing module 36 specifically for sending to the microoperation controller of objective table 31 kinematic parameter of microoperation controller that three-dimensional that steering order controls sample to be detected moves and rotate and receive described objective table 31 by electrical signal collection control module; The control of central processing module 36 pairs of LASER Light Source 11 is similar, repeats no more.
Multiphoton excitation multidirectional illumination micro imaging system of the present invention carries out detection imaging by adopting mating plate illumination micro-imaging technique to the fluorescence that multiphoton excitation sample to be detected produces, corresponding image checking unit is made to carry out parallel image collection, thus improve image taking speed compared to multi-photon laser dot scans micro-imaging of the prior art, and multiphoton excitation improves the detection degree of depth owing to make use of near-infrared excitation light, that is the higher detection degree of depth can be reached under the condition ensureing image taking speed, therefore the dynamic imaging to thicker biological living can be realized, in addition multiphoton excitation multidirectional illumination micro imaging system of the present invention carries out imaging by adopting the mating plate of multiple directions to carry out the illumination biosome that can realize having opaque section to sample to be detected, owing to combining the advantage that can realize multidirectional mating plate illumination two aspects such as the image taking speed of micro-imaging and the imaging depth of two-photon excitation that parallel image gathers, at medical imaging field, the present invention may be used for carrying out dynamic development imaging and neuron activity record to the living body biological such as zebra fish, fruit bat
Last it is noted that above each embodiment is only in order to illustrate technical scheme of the present invention, be not intended to limit; Although with reference to foregoing embodiments to invention has been detailed description, those of ordinary skill in the art is to be understood that: it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein some or all of technical characteristic; And these amendments or replacement, do not make the essence of appropriate technical solution depart from the scope of various embodiments of the present invention technical scheme.
Claims (7)
1. a multiphoton excitation multidirectional illumination micro imaging system, is characterized in that, comprise be positioned at described imaging system from front to back successively fluorescence light path on: multi-photon laser cell, lighting unit, image checking unit;
Described multi-photon laser cell, for generation of with modulated laser and by modulation after laser send into lighting unit; Described lighting unit is located at the side of described sample to be detected, produces multidirectional mating plate again throw light on and excite sample to be detected to produce fluorescence for the laser after receiving described modulation; Described image checking unit is positioned at above sample to be detected, for detecting the fluorescence of sample to be detected generation and transferring digital picture to, at least comprises and detects described fluorescence and the photodetection camera transferring electric signal to.
2. multiphoton excitation multidirectional illumination micro imaging system according to claim 1, is characterized in that, on the fluorescence light path that described lighting unit is included in described imaging system position successively multi-photon mating plate rearward produce light path and multidirectional mating plate illumination objective lens group; Described multidirectional mating plate illumination objective lens group comprises at least two illumination objective lens be arranged circumferentially;
Described multi-photon mating plate produces light path and more described laser is divided into multidirectional mating plate for the laser after receiving described modulation and each illumination objective lens of corresponding incident described multidirectional mating plate illumination objective lens group respectively; Any described illumination objective lens throws light on for the mating plate receiving a direction in multidirectional mating plate again and excites sample to be detected to produce fluorescence.
3. multiphoton excitation multidirectional illumination micro imaging system according to claim 2, it is characterized in that, all vertical with the axle of described image checking unit and the described mating plate of the mating plate that each illumination objective lens of described multidirectional mating plate illumination objective lens group receives impinges upon and sample to be detected excites position in the focal plane of described image checking unit.
4. multiphoton excitation multidirectional illumination micro imaging system according to claim 1, is characterized in that, position LASER Light Source rearward and multiphoton excitation light path successively on the fluorescence light path that described multi-photon laser cell is included in described imaging system;
Described LASER Light Source for generation of high photon density laser and send into described multiphoton excitation light path, described multiphoton excitation light path be used for receive and modulate described laser and send into described lighting unit.
5. multiphoton excitation multidirectional illumination micro imaging system according to claim 1, is characterized in that, position objective table, image-forming objective lens and filter plate rearward successively on the fluorescence light path that described image checking unit is included in described imaging system;
Described objective table be used for fixing and change sample to be detected excite position, be excited the fluorescence produced in multiple angle or the degree of depth to make sample to be detected; Described image-forming objective lens is positioned at above sample to be detected, for focusing on described fluorescence and sending into described filter plate; Described filter plate is fixedly installed on the described light path front of described photodetection camera, for the veiling glare in fluorescence described in filtering.
6. multiphoton excitation multidirectional illumination micro imaging system according to claim 5, it is characterized in that, described objective table at least comprises microoperation controller, described microoperation controller is used for the operation that sample to be detected is performed to translation or rotates around vertical axes, to adjust the relative position of described sample to be detected and described imaging system light path.
7. multiphoton excitation multidirectional illumination micro imaging system according to claim 1, it is characterized in that, described image checking unit also comprises electrical signal collection control module and central processing module, described electrical signal collection control module is specifically for receiving the electric signal of described photodetection camera output and being converted to digital signal, and described central processing module is specifically for receiving described digital signal and generating digital picture.
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| CN106419889A (en) * | 2016-10-31 | 2017-02-22 | 佛山科学技术学院 | Device and method for three-dimensionally imaging blood flow based on lamella light |
| CN111352284A (en) * | 2020-04-30 | 2020-06-30 | 上海交通大学 | Photon neuron realization method and device based on passive nano beam resonant cavity |
| WO2021147198A1 (en) * | 2020-01-21 | 2021-07-29 | 广东湛江海洋医药研究院 | Multi-photon microscope transparent specimen adapter and imaging method thereof |
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