CN101609207A - Lienable fluorescence microscopy endoscopic imaging system and formation method - Google Patents
Lienable fluorescence microscopy endoscopic imaging system and formation method Download PDFInfo
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- CN101609207A CN101609207A CNA2009100889950A CN200910088995A CN101609207A CN 101609207 A CN101609207 A CN 101609207A CN A2009100889950 A CNA2009100889950 A CN A2009100889950A CN 200910088995 A CN200910088995 A CN 200910088995A CN 101609207 A CN101609207 A CN 101609207A
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- 238000003384 imaging method Methods 0.000 title claims abstract description 24
- 238000000799 fluorescence microscopy Methods 0.000 title claims abstract description 23
- 238000000034 method Methods 0.000 title claims description 9
- 230000015572 biosynthetic process Effects 0.000 title description 4
- 239000000835 fiber Substances 0.000 claims abstract description 73
- 230000003287 optical effect Effects 0.000 claims abstract description 63
- 241001465754 Metazoa Species 0.000 claims abstract description 27
- 230000005284 excitation Effects 0.000 claims abstract description 17
- 230000021615 conjugation Effects 0.000 claims abstract description 8
- 238000009434 installation Methods 0.000 claims abstract description 5
- 210000001519 tissue Anatomy 0.000 claims description 24
- 230000005540 biological transmission Effects 0.000 claims description 13
- 239000002639 bone cement Substances 0.000 claims description 4
- 230000000295 complement effect Effects 0.000 claims description 4
- 238000005057 refrigeration Methods 0.000 claims description 4
- 229920001285 xanthan gum Polymers 0.000 claims description 4
- 229940082509 xanthan gum Drugs 0.000 claims description 4
- 235000010493 xanthan gum Nutrition 0.000 claims description 4
- 239000000230 xanthan gum Substances 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 238000000695 excitation spectrum Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000002189 fluorescence spectrum Methods 0.000 claims description 3
- 238000005498 polishing Methods 0.000 claims description 3
- 230000007306 turnover Effects 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 2
- 239000007850 fluorescent dye Substances 0.000 abstract description 5
- 239000013307 optical fiber Substances 0.000 abstract description 4
- 238000001215 fluorescent labelling Methods 0.000 abstract description 3
- 230000008859 change Effects 0.000 abstract description 2
- 238000002591 computed tomography Methods 0.000 description 4
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 description 2
- 230000008676 import Effects 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 238000012857 repacking Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
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Abstract
Lienable fluorescence microscopy endoscopic imaging system belongs to the fluorescence microscopy field, gets involved and indwelling portion, and be a GRIN Lens, insert in Wicresoft's mouth of living animal; Exciting light and fluorescence transfer part are connected in sequence by optical fiber splicing device, image-carrying fiber bundle, fibre bundle stationary installation, and this image-carrying fiber bundle front end face and the interior observed zone of tissue are with respect to the GRIN Lens conjugation; The fluorescence shoot part, successively by along the horizontal microcobjective of fluorescent emission main shaft, dichroic mirror, fluorescent optical filter, and the CCD device form, CCD device and image-carrying fiber bundle rear end face are with respect to the microcobjective conjugation; Excitation light generation part is made of successively light source, exciting light optical filter, lens combination, exciting light main shaft and fluorescence emission main shaft quadrature, and all become miter angle with dichroic mirror.The present invention has: be easy to realize the high-speed video collection, to observe the dynamic change of fluorescence labeling target, also can observe continuously the living animal same position for a long time.
Description
Technical field:
The present invention relates to fluorescence microscopy and based endoscopic imaging field.Be specifically related to obtain the method for the image of Dynamic Fluorescence target-marking in the human or animal body by lienable fluorescence microscopy endoscopic imaging system.
Background technology:
Usually, use fluorescently-labeled biological tissue or cell, need to use fluorescent microscope, photofulorography or fluorescence computed tomography (SPECT) system in order to observe.To observe body internal cavity tissue in order getting involved, to need to use medical endoscope.
Fluorescent microscope: can observe biological tissue's micromechanism and cell, but be confined to observe the stripped biological tissue section or the cell of cultured in vitro, can't carry out fluorescence imaging at body or live body.
Photofulorography and fluorescence computed tomography (SPECT) system: can carry out fluorescence imaging, but be merely able to observe the macroscopic view distribution of fluorescence, can't observe biological tissue's micromechanism and cell at body or live body.
Medical endoscope: can insert in human or animal's the cavity organ and observe, but can not observe biological tissue's micromechanism and cell, can not carry out fluorescence imaging.
Microscopy endoscopic imaging system based on laser confocal scanning microscope: have foreign periodical report to use the microscopy endoscopic imaging system of laser confocal scanning microscope repacking in recent years, realize under body or live body situation to the fluorescence imaging of biological tissue's micromechanism or cell.But cost is higher, and technical sophistication is difficult to quick adjustment, is difficult to promote; And can only once observe, be difficult to repeatedly same ad-hoc location be observed for a long time, therefore can't carry out continuous observation and the comparative study of long period.
Therefore as seen, in the practical application, have for carrying out at body or somatoscopy biological tissue micromechanism and cell fluorescence marking image, and for a long time, the actual needs of observation repeatedly.Therefore novel lienable fluorescence microscopy endoscopic imaging system and formation method have practical meaning and demand in actual applications.
Summary of the invention:
The objective of the invention is to, propose a kind of new fluorescence imaging system.
The invention is characterized in, contain: excitation light generation part, main optical path and fluorescence shoot part, exciting light and fluorescence transfer part and intervention and indwelling portion, wherein:
Excitation light generation part, contain: light source, exciting light optical filter and lens combination, wherein:
Light source is great power LED,
The exciting light optical filter is bandpass filter, centre wavelength and bandwidth and wait that the excitation spectrum of observing fluorescent target is complementary,
Lens combination is sent by described great power LED in order to realization, and the collimation of the light beam behind the described exciting light optical filter of process,
Main optical path and fluorescence shoot part, contain: successively horizontal positioned and and described image-carrying fiber bundle rear end face form CCD device, fluorescent optical filter, dichroic mirror and the microcobjective of main optical path together, wherein:
The CCD device for cryogenic refrigeration CCD device, is used for the detection to fluorescent signals,
Fluorescent optical filter is bandpass filter, and centre wavelength, bandwidth and living animal wait to observe the fluorescence emission spectrum of fluorescent target to be complementary, and the free transmission range of its free transmission range and described exciting light optical filter do not overlap, so that the light of filtering exciting light wave band,
Dichroic mirror, becoming 45 degree with described main optical path optical axis when, also become miter angle with the narrow band exciting light optical axis that is used for the fluorescence excitation target-marking that described excitation light generation part produces, make described main optical path optical axis and described narrow band exciting light optical axis be orthogonal, the turnover wavelength of this dichroic mirror is in the interval of the free transmission range of described exciting light optical filter and described fluorescent optical filter, so that reflect the photon of described exciting light wave band and the photon of the described fluorescence wave band of transmission
Exciting light and fluorescence transfer part, contain: snap ring in image-carrying fiber bundle, image-carrying fiber bundle stationary installation, image-carrying fiber bundle web member overcoat and the image-carrying fiber bundle web member, wherein:
Image-carrying fiber bundle is the flexible image-carrying fiber bundle that 20cm～100cm is long, and respectively there is the long hard sheath of 3cm～5cm at two ends, and external diameter is less than 1mm, two ends optical flat polishing, and inner fibril spacing is less than 10um,
The image-carrying fiber bundle stationary installation has center pit, is used for fixing the rear end face of described image-carrying fiber bundle,
Be connected by accurate thread between the snap ring in fiber optic connector overcoat and the described image-carrying fiber bundle, with observed position that guarantees living animal and the front end face conjugation of inserting the described image-carrying fiber bundle within the described image-carrying fiber bundle web member overcoat, make the fluoroscopic image of observed position in the tissue of living animal can blur-free imaging in the front end face of described image-carrying fiber bundle
Get involved and indwelling section; It is a GRIN Lens; External diameter is less than 1mm; Insert in the hole of described fiber optic connector overcoat; Get involved in the living animal body; Directly contact with the observed position of described tissue; Exciting light is converged to the observed position of described tissue by described image-carrying fiber bundle front end face; Simultaneously; The described fluorescence imaging at observed position of organizing in described image-carrying fiber bundle front end face; This GRIN Lens is to place observed position in the body of living animal by the Wicresoft's mouth less than 1mm that a puncture produces
Described CCD device, image-carrying fiber bundle rear end face are with respect to described microcobjective conjugation.
Than the practical application of existing fluorescence imaging system, new fluorescence microscopy endoscopic imaging system has remarkable advantages.The same with fluorescent microscope, new system can tissues observed micromechanism and cell.The same with the fluorescence computed tomography (SPECT) system with the photofulorography of routine, new system can carry out the observation at body or live body.And new system unit area exciting light energy is higher, therefore is easy to realize the high-speed video collection, to observe the dynamic change of fluorescence labeling target.The kept somewhere design of new system can be observed human or animal's same position in long-time.Than the photofulorography and the fluorescence computed tomography (SPECT) system of routine, tangible saving is being arranged aspect excitation source and the fluorescent probe.Than existing sorts of systems, the present invention has some advantages.This system can also adjust enlargement factor and spatial resolution by changing grin lens, image-carrying fiber bundle and object lens; Can support the imaging applications of fluorescent markers such as various fluorescent dyes, quantum dot, fluorescin by switching excitation source, exciting light optical filter, dichroic mirror and fluorescent optical filter.
Description of drawings:
Fig. 1 shows block diagram of the present invention, and wherein each part description is as follows:
1, grin lens (or claiming GRIN Lens, gradient-index lens)
2, fiber optic connector overcoat,
3, snap ring in the fiber optic connector,
4, image-carrying fiber bundle,
5, optic fibre fixing device,
6, microcobjective,
7, dichroic mirror (or claiming color separation film) or beam split plain film (or claiming the half-transmitting and half-reflecting mirror),
8, fluorescent optical filter,
9, lens or lens combination,
10, light source,
11, exciting light optical filter,
12, CCD (charge-coupled image sensor).
Embodiment:
This system comprises at least one or a plurality of light source, and (light source is laser instrument, great power LED (light emitting diode), laser diode or other light fixtures, the light that also can be optical fiber import from outside other light sources, preferably great power LED), exciting light optical filter, fluorescent optical filter and dichroic mirror group.Described light source, exciting light optical filter, fluorescent optical filter, cooperate jointly with dichroic mirror, will with the excitation wavelength of corresponding fluorescent marker under work.
This system by the working chamber of operating theater instruments or the inner chamber of syringe needle, inserts grin lens in the human or animal tissues to be observed when using.When needing the extended immobilization observation place, the grin lens insertion end can be kept somewhere in human or animal tissues, the other end is exposed to outside the human or animal tissues, and passes through surgical attachment between the human or animal tissues (using methods such as bone cement, xanthan gum or stitching).
This system converges the importing image-carrying fiber bundle with exciting light, and enters grin lens by the image-carrying fiber bundle transmission, and then shine biological tissue surface, fluorescence excitation by the lens in light source the place ahead and the cooperation of microcobjective.Fluorescence amplifies via microcobjective, and is finally surveyed by CCD (charge-coupled image sensor) again by grin lens and image-carrying fiber bundle.
This system is by the fixing and governor motion between grin lens and the image-carrying fiber bundle, finish the adjusting of grin lens and image-carrying fiber bundle end face distance, to realize adaptation for imaging object distance (human or animal tissues and grin lens insert the gap that end face may exist).And can be by should fixing and governor motion, realize separating fast and assembling of grin lens and system other parts, make things convenient for the indwelling of grin lens and observe repeatedly.
Fig. 1 has schematically shown fluorescence microscopy endoscopic imaging system of the present invention, and concrete each several part is described as follows:
The excitation light generation module
This module comprises light source 10, exciting light optical filter 11, lens or lens combination 9, generation is used for the narrow band exciting light of fluorescence excitation target-marking, be shaped as suitable beam shape and send into system's main optical path (hereinafter introducing), send into the excitation beam and the main optical path light shaft positive cross of system's main optical path.
Wherein light source 10 can be laser instrument, great power LED (light emitting diode), laser diode or other light fixtures, also can be the light that optical fiber imports from outside other light sources, preferably great power LED.
Wherein exciting light optical filter 11 is a bandpass filter, the selection of its centre wavelength and bandwidth should with wait to observe the excitation spectrum of fluorescent target adapt.
Wherein lens or lens combination 9 when using laser instrument as light source, need to realize the expansion bundle of light beam, are the laser beam expanding lens of lens combination formation; When using other light sources, need to realize the collimation of light beam, be simple lens or lens combination.
Main optical path and fluorescence taking module
This module comprises CCD (charge-coupled image sensor) 12, fluorescent optical filter 8, dichroic mirror 7, microcobjective 6, is used for exciting light is converged importing image-carrying fiber bundle 4, and fluoroscopic image amplification imaging that simultaneously image-carrying fiber bundle is spread out of and collection.
Can be science cryogenic refrigeration CCD or civilian CCD comprising CCD (charge-coupled image sensor) 12, science cryogenic refrigeration CCD preferably, so that wideer dynamic range, higher signal to noise ratio (S/N ratio) and higher light receiving efficiency to be provided, help detection more for fluorescent signals.
Wherein fluorescent optical filter 8 is a bandpass filter, the selection of its centre wavelength and bandwidth should with wait to observe the fluorescence emission spectrum of fluorescent target adapt, and the free transmission range of its free transmission range and exciting light optical filter can not overlap, in order to the light of filtering exciting light wave band.
Wherein dichroic mirror (perhaps beam split plain film) 7 becomes 45 degree to place (all becoming miter angle with the optical axis of main optical path optical axis and excitation light generation module) in light path, in order to the photon of reflection exciting light wave band and the photon of transmission fluorescence wave band, when using dichroic mirror, its turnover wavelength should be between the free transmission range of exciting light optical filter and fluorescent optical filter, preferably dichroic mirror.
Exciting light and fluorescence transfer module
This module comprises snap ring 3 in image-carrying fiber bundle 4, optic fibre fixing device 5, fiber optic connector overcoat 2 and the fiber optic connector.Below in the explanation, with the end end face of " front end face " expression image-carrying fiber bundle, with the end end face of " rear end face " expression image-carrying fiber bundle near microcobjective near grin lens 1.
Image-carrying fiber bundle 4 is the long flexible imaging fibre bundle of 20cm～100cm, it is the hard sheath of 3cm～5cm that respectively there is length at two ends, so that clamping and fixing, external diameter is less than 1mm, two ends optical flat polishing, inner fibril spacing is preferably 2 μ m or following less than 10 μ m, to realize higher spatial resolution.Under particular application image-carrying fiber bundle length can be for 100cm more than, but long fiber lengths will cause more light intensity attenuation and add higher wire broken rate in man-hour.
Snap ring 3 is supporting structure in fiber optic connector overcoat 2 and the fiber optic connector, overcoat can be realized fixing simply fast with grin lens 1 or remove, interior snap ring and image-carrying fiber bundle 4 are fixing, be connected by accurate thread between overcoat and the interior snap ring, guarantee simultaneously the front end face conjugation of the observed position of human or animal tissues and image-carrying fiber bundle 4 by this accurate thread, also i.e. assurance organize the fluoroscopic image of observed position can blur-free imaging in the image-carrying fiber bundle front end face.
Get involved and the indwelling module
This module includes only grin lens 1, external diameter is less than 1mm, length is decided according to the degree of depth of observed tissue, in order to get involved in the human or animal body, directly contact with organizing observed position, from image-carrying fiber bundle 4 front end faces exciting light is converged to and to organize observed position, the fluorescence imaging that to organize observed position simultaneously is in the front end face of image-carrying fiber bundle 4, the Wicresoft's mouth less than 1mm that this module is produced by a puncture places the observed position in the human or animal body, keep somewhere so that observe repeatedly for a long time as needs, then use bone cement, mode such as xanthan gum or operation stitching is fixed in Wicresoft's mouth, keep an end to contact with observed position, the other end is exposed to outside Wicresoft's mouth, is connected with it to make things convenient for optical fiber to connect overcoat 2.
The imaging operation method
1, according to imaging at fluorescence labeling target light spectroscopy feature, switch to corresponding light source, exciting light optical filter, fluorescent optical filter and dichroic mirror.
2, use puncture needle or syringe that the human or animal is treated that look-out station punctures,, need carry out the operation of opening cranium operation if carry out the animal brain observations.Inner chamber by puncture needle or syringe is sent to position to be observed with grin lens, takes out puncture needle or syringe, and grin lens is kept somewhere in treating look-out station.
3, observe this privileged site if desired for a long time repeatedly, then use methods such as bone cement, xanthan gum or operation stitching that grin lens is fixed in the Kou Chu of Wicresoft; Observe if only need carry out single, then can skip this step.
4, the fiber optic connector overcoat is installed on grin lens and is exposed to the outer end of Wicresoft's mouth, by the focusing of the minute adjustment screw thread between fiber optic connector overcoat and the interior snap ring (regulate the human or animal and be observed the front end face of tissue and image-carrying fiber bundle) with respect to the grin lens conjugation.
5, use CCD (charge-coupled image sensor) to carry out the shooting of single-frame images or continuous video acquisition.
Claims (5)
1, lienable fluorescence microscopy endoscopic imaging system is characterized in that, contains: excitation light generation part, main optical path and fluorescence shoot part, exciting light and fluorescence transfer part and intervention and indwelling portion, wherein:
Excitation light generation part, contain: light source, exciting light optical filter and lens combination, wherein:
Light source is great power LED,
The exciting light optical filter is bandpass filter, centre wavelength and bandwidth and wait that the excitation spectrum of observing fluorescent target is complementary,
Lens combination is sent by described great power LED in order to realization, and the collimation of the light beam behind the described exciting light optical filter of process,
Main optical path and fluorescence shoot part, contain: successively horizontal positioned and and described image-carrying fiber bundle rear end face form CCD device, fluorescent optical filter, dichroic mirror and the microcobjective of main optical path together, wherein:
The CCD device for cryogenic refrigeration CCD device, is used for the detection to fluorescent signals,
Fluorescent optical filter is bandpass filter, and centre wavelength, bandwidth and living animal wait to observe the fluorescence emission spectrum of fluorescent target to be complementary, and the free transmission range of its free transmission range and described exciting light optical filter do not overlap, so that the light of filtering exciting light wave band,
Dichroic mirror, becoming 45 degree with described main optical path optical axis when, also become miter angle with the narrow band exciting light optical axis that is used for the fluorescence excitation target-marking that described excitation light generation part produces, make described main optical path optical axis and described narrow band exciting light optical axis be orthogonal, the turnover wavelength of this dichroic mirror is in the interval of the free transmission range of described exciting light optical filter and described fluorescent optical filter, so that reflect the photon of described exciting light wave band and the photon of the described fluorescence wave band of transmission
Exciting light and fluorescence transfer part, contain: snap ring in image-carrying fiber bundle, image-carrying fiber bundle stationary installation, image-carrying fiber bundle web member overcoat and the image-carrying fiber bundle web member, wherein:
Image-carrying fiber bundle is the flexible image-carrying fiber bundle that 20cm～100cm is long, and respectively there is the long hard sheath of 3cm～5cm at two ends, and external diameter is less than 1mm, two ends optical flat polishing, and inner fibril spacing is less than 10um,
The image-carrying fiber bundle stationary installation has center pit, is used for fixing the rear end face of described image-carrying fiber bundle,
Be connected by accurate thread between the snap ring in fiber optic connector overcoat and the described image-carrying fiber bundle, with observed position that guarantees living animal and the front end face conjugation of inserting the described image-carrying fiber bundle within the described image-carrying fiber bundle web member overcoat, make the fluoroscopic image of observed position in the tissue of living animal can blur-free imaging in the front end face of described image-carrying fiber bundle
Get involved and indwelling section; It is a GRIN Lens; External diameter is less than 1mm; Insert in the hole of described fiber optic connector overcoat; Get involved in the living animal body; Directly contact with the observed position of described tissue; Exciting light is converged to the observed position of described tissue by described image-carrying fiber bundle front end face; Simultaneously; The described fluorescence imaging at observed position of organizing in described image-carrying fiber bundle front end face; This GRIN Lens is to place observed position in the body of living animal by the Wicresoft's mouth less than 1mm that a puncture produces
Described CCD device, image-carrying fiber bundle rear end face are with respect to described microcobjective conjugation.
2, lienable fluorescence microscopy endoscopic imaging system according to claim 1, it is characterized in that, described GRIN Lens use that bone cement is bonding, xanthan gum is bonding or the operation stitching mode in any mode be fixed in Wicresoft's mouth of living animal.
3, lienable fluorescence microscopy endoscopic imaging system according to claim 1 is characterized in that, each fibril spacing is less than 2um in the described image-carrying fiber bundle.
4, lienable fluorescence microscopy endoscopic imaging system according to claim 1 is characterized in that, described light source is any in laser instrument, laser diode, lamp and the outside lead-in light.
5, lienable fluorescence microscopy endoscopic imaging system according to claim 1 is characterized in that, described CCD device is civilian CCD device.
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| CN2009100889950A CN101609207B (en) | 2009-07-17 | 2009-07-17 | Indwellable fluorescence microscopy endoscopic imaging system and imaging method thereof |
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|---|---|---|---|
| CN2009100889950A CN101609207B (en) | 2009-07-17 | 2009-07-17 | Indwellable fluorescence microscopy endoscopic imaging system and imaging method thereof |
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| CN101609207A true CN101609207A (en) | 2009-12-23 |
| CN101609207B CN101609207B (en) | 2011-01-05 |
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| CN101904737A (en) * | 2010-08-09 | 2010-12-08 | 华中科技大学 | Living body fluorescent endoscopic spectrum imaging device |
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| CN108784629A (en) * | 2017-04-28 | 2018-11-13 | 凝辉(天津)科技有限责任公司 | A kind of distal end plug-in type MEMS based endoscopic imaging equipment |
| CN109833102A (en) * | 2017-11-29 | 2019-06-04 | 上海复拓知达医疗科技有限公司 | Position the marker and telltale mark system of the small space occupying lesion of intrapulmonary |
| CN108815588A (en) * | 2018-07-16 | 2018-11-16 | 中国科学技术大学 | A kind of self-focusing lens is used for the method for coating of brain area imaging |
| CN108732739A (en) * | 2018-07-25 | 2018-11-02 | 中国科学院苏州生物医学工程技术研究所 | The system being imaged in body multidigit point is realized using fiber optic bundle |
| CN113180598A (en) * | 2021-04-16 | 2021-07-30 | 中国科学院苏州生物医学工程技术研究所 | OCT and fluorescence composite microscopic endoscopic imaging system |
| CN115508857A (en) * | 2022-08-31 | 2022-12-23 | 核工业西南物理研究院 | Imaging device applied to fusion plasma |
| WO2024108517A1 (en) * | 2022-11-25 | 2024-05-30 | 中国科学院深圳理工大学(筹) | Method and system for fluorescence synchronous imaging in deep brain region and whole-brain cortex scale |
| CN117918770A (en) * | 2024-01-26 | 2024-04-26 | 杭州微新医疗科技有限公司 | Inspection device and endoscopic equipment |
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