CN110141182A - A kind of microscopy endoscopic imaging method and system based on Structured Illumination - Google Patents
A kind of microscopy endoscopic imaging method and system based on Structured Illumination Download PDFInfo
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- A61B1/00—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor
- A61B1/04—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances
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- A61B1/05—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor combined with photographic or television appliances characterised by the image sensor, e.g. camera, being in the distal end portion
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- A61B1/07—Instruments for performing medical examinations of the interior of cavities or tubes of the body by visual or photographical inspection, e.g. endoscopes; Illuminating arrangements therefor with illuminating arrangements using light-conductive means, e.g. optical fibres
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Abstract
A kind of microscopy endoscopic imaging method and system based on Structured Illumination, this method include that the first lighting source of control and the second lighting source successively emit exciting light, and generate fluorescence signal respectively；Control double-shutter camera is acquired the fluorescence signal generated respectively and obtains two images；Collected two images are handled, a frame image is obtained, first lighting source is Structured Illumination light source, and second lighting source is Both wide field illumination light source.The system includes the first illumination path, the second illumination path, combined beam light road and imaging optical path.The present invention is not necessarily to scanning means, in such a way that Structured Illumination and Both wide field illumination imaging combine, successively acquires fluorescence signal, only needs two images to synthesize the fluorescence that can obtain high quality in burnt image, image taking speed is fast；And light-blocking without pin hole, the efficiency of light energy utilization is high, and the fluorescence signal of generation is more, fluorescence imaging high sensitivity, the fluorescence organization chart picture abundant information got.
Description
Technical field
The invention belongs to microscopy endoscopic imaging field more particularly to a kind of microscopy endoscopic imaging sides based on Structured Illumination
Method and system.
Background technique
In microscopy endoscopic imaging field, optical microscopy imaging has many advantages, such as lossless, quick, inexpensive, always by
It is held in both hands to people's heat.Optical microscopy imaging technology is mainly based upon optical microscopy principle, by utilizing optical lens to object
Amplify imaging, it can be observed that the histiocytic morphology of microbiological and physiological feature, convenient for physiological tissue into
The screening of row lesion and diagnosis.
High-resolution fluorescence microscopy scope (High Resolution Microendoscopy, HRME) is a kind of novel
Microscopy endoscopic formula imaging system enters human body by a soft fiber optic bundle, and fiber optic bundle can be with simultaneous transmission illumination light and figure
As signal, realize to the lesion inspection of human body inner tissue.High-resolution fluorescence microscopy scope uses LED as light source, by proprietary
Light channel structure and fiber optic bundle, optical signal reach irradiation tissue surface fluorescent dye, the fluorescence signal inspired pass through optical fiber
Beam returns, and is amplified into ccd image detection device by imaging optical path, to generate image or video output.High-resolution
Fluorescence microscopy scope internal structure is simple, and image taking speed is fast, easy to operate, it is only necessary to by fiber optic bundle close to tissue surface, energy
Get tissue surface image.However, due to lacking the inhibition to background light signal, causing a large amount of out of focus in imaging process
Signal may also appear in display image, for severe jamming in the identification and observation of burnt signal, picture contrast is poor, unfavorable
Precisely judge to examine in lesion tissue cell.
Laser co-focusing microendoscopic (Laser Confocal Endomicroscopy, CLE) is at present clinically
A kind of advanced based endoscopic imaging technology to come into operation similarly enters human body also with a soft fiber optic bundle, shines simultaneously
Penetrate and transmit fluorescence signal.The feature of Laser scanning confocal microscopy technology maximum is that pin hole dress is added before signal sensor
It sets, can effectively stop the transmission of signal out of focus, inhibit background light signal, substantially increase image resolution ratio and contrast.Laser
It is copolymerized burnt microendoscopic accuracy rate with higher and susceptibility in terms of early-stage cancer screening, is disadvantageous in that and joined needle
Aperture apparatus, to the more demanding of light source, while Laser Scanning Confocal needs are scanned, and image taking speed is slow, and interior lights
Line structure is complicated, and higher cost has aggravated medical institutions and the financial burden of patient.
Therefore, it is necessary to a kind of new microscopy endoscopic imaging method and system based on Structured Illumination be provided, to adapt to
The demand in market.
Summary of the invention
In view of this, the microscopy endoscopic imaging method that the purpose of the present invention is to provide a kind of based on Structured Illumination and being
System improves the inhibition to background light signal in the case where guaranteeing image taking speed and image resolution ratio, enhances picture contrast.
To achieve the above object, the invention provides the following technical scheme:
A kind of microscopy endoscopic imaging method based on Structured Illumination comprising step:
The second lighting source controlled in the first lighting source and the second illumination path in the first illumination path is successively sent out
Exciting light is penetrated, and generates fluorescence signal respectively；
Control double-shutter camera is acquired the fluorescence signal generated respectively and obtains two images；
Collected two images are handled, a frame image is obtained.
Further, second in the first lighting source and the second illumination path in the first illumination path of the control is shone
Mingguang City source successively emit exciting light comprising steps of
It controls the first lighting source and emits exciting light in first time, only by the first lighting source within the first time
Generate the optical signal of excitation fluorescence；
Control the second lighting source after the first time the second time transmitting exciting light, second time only by
Second lighting source generates the optical signal of excitation fluorescence.
Further, collected two images are handled, obtaining a frame image further comprises:
Using composition algorithms, every collected two images of a pair are all handled, signal out of focus therein is restrained,
It extracts in burnt fluorescence signal, thus a frame image required for obtaining.
Further, first lighting source and the second lighting source are LED light source.
To achieve the above object, the present invention also provides following technical solutions:
A kind of microscopy endoscopic imaging system based on Structured Illumination comprising:
First illumination path, for generating the illumination light of first direction；
Second illumination path, for generating the illumination light of second direction；
Combined beam light road carries out conjunction beam for the illumination light to the first illumination path and the second illumination path；
Imaging optical path is imaged for collecting fluorescence signal, and to fluorescence signal.
Further, first illumination path successively includes the first lighting source, to the light of the first lighting source sending
The lens of the first collimation lens, grating and the diffraction light for collecting grating generation that are collimated.
Further, first lighting source is LED light source.
Further, the grating includes several light along the periodic groove of same direction, after optical grating diffraction
Striated structure is presented and realizes striped and illuminates.
Further, the grating is located at the front focal plane position of lens, and the lens joint imaging optical path is to grating surface
Projection imaging is carried out, so that the periodic stripe of grating is projected sample surfaces.
Further, second illumination path includes the second lighting source and carries out to the light that the second lighting source issues
Second collimation lens of collimation.
Further, second lighting source is LED light source.
Further, the combined beam light road successively includes the first optical filter, for reflecting after the filtering of the first optical filter
The dichroscope of spectrum, the object lens for collecting the optical signal that dichroscope reflects back and it is used for transmission illumination light light wave
Fiber optic bundle, first optical filter are used to carry out spectrum filter to from the illumination light of the first illumination path and the second illumination path
Wave stops the spectrum invalid to fluorescent dye excitation process.
Further, the object lens include object space and Image space, and the fiber optic bundle is located at the object space of object lens, described
The grating face of first lighting source, the second lighting source and grating is respectively positioned on Image space.
Further, the imaging optical path successively includes the fiber optic bundle for collecting transmission fluorescence signal, carries out to fluorescence signal
Amplify transmission object lens, to object lens transmission fluorescence signal handled dichroscope, to fluorescence signal carry out spectral filtering
The second optical filter.
Further, the imaging optical path further includes imaging lens and double-shutter camera, the imaging lens and double-shutter
Camera is used cooperatively, and the filtered fluorescence signal collection of the second optical filter is imaged, and projects the fluorescence letter on double-shutter camera
Number after photoelectric conversion, the image with tissue information is shown.
Further, the microscopy endoscopic imaging system based on Structured Illumination further includes beam splitter, the beam splitter
Positioned at the position that crosses of the first illumination path direction of propagation and the second illumination path direction of propagation, first illumination path is come from
Enter same light path system after beam splitter reflects with the light of the second illumination path.
It can be seen from the above technical proposal that the present invention on the basis of optical path of traditional wide field fluorescent microscopic imaging, is added
Structured Illumination optical path, while incoming fiber optic beam construct microscopy endoscopic imaging system, which is to be based on fluorescence imaging principle,
Corresponding to specific fluorescent dye, corresponding excitation light source and light path system are designed, and smoothly acquires fluorescence signal and shielding
The interference of stray light of its all band finally gets the fluorescent image that can reflect organizational information.
Compared with prior art, the beneficial effects of the present invention are:
1, using LED as light source, economic and environment-friendly, light energy conversion efficiency is high, avoids the potentially hazardous property of laser light source,
It is safe and efficient, long service life.
2, it is based on Induced Fluorescence Microscopy, cooperates corresponding fluorochrome label, available high-resolution tissue
Cellular Pathology Image, convenient for carrying out special Journal of Sex Research to cellular elements.
3, it introduces Structured Illumination optical path and there is Structured Illumination imaging system, illuminate mark using the striped that grating generates
Remember out in burnt signal, inhibits signal out of focus, effectively improve picture contrast.
4, it is not necessarily to scanning means, in such a way that Structured Illumination and Both wide field illumination imaging combine, successively acquires fluorescence
Signal only needs two images to synthesize the fluorescence that can obtain high quality in burnt image, and image taking speed is fast.
5, light-blocking without pin hole, the efficiency of light energy utilization is high, and the fluorescence signal of generation is more, and fluorescence imaging high sensitivity is got
Fluorescence organization chart picture abundant information.
6, using double-shutter camera, fluorescent image is acquired in pairs, and the fluorescence signal of two width different structure optical illumination excitation is adopted
Collection interval is shorter, reduces motion artifacts, stronger to histokinesis's information obtaining ability.
7, modularized design, illumination path and imaging optical path separation, Structured Illumination optical path and width in microscopy endoscopic system
Field illumination path separation, structure is simple, and stability is good.
Detailed description of the invention
Fig. 1 is the flow chart of the microscopy endoscopic imaging method the present invention is based on Structured Illumination.
Fig. 2 is the structural schematic diagram of the microscopy endoscopic imaging system the present invention is based on Structured Illumination.
Fig. 3 is the schematic diagram of the two images obtained the present invention is based on the microscopy endoscopic imaging system of Structured Illumination,
In, (a) figure is the fluorescence imaging schematic diagram that Structured Illumination generates, and (b) figure is the fluorescence imaging schematic diagram that Both wide field illumination generates.
Wherein: 100- beam splitter, the first lighting source of 11-, the first collimation lens of 12-, 13- grating, 14- lens, 21-
Two lighting sources, the second collimation lens of 22-, the first optical filter of 31-, the second optical filter of 32-, 33- object lens, 34- fiber optic bundle, 35-
Dichroscope, 36- imaging lens, 37- double-shutter camera.
Specific embodiment
The microscopy endoscopic imaging method and system based on Structured Illumination that the invention discloses a kind of is guaranteeing image taking speed
In the case where image resolution ratio, the inhibition to background light signal is improved, enhances picture contrast.
As shown in Figure 1, Fig. 1 is the flow chart of the microscopy endoscopic imaging method the present invention is based on Structured Illumination.This is based on
The microscopy endoscopic imaging method of Structured Illumination includes the following steps:
S101: control the first illumination path in the first lighting source and the second illumination path in the second lighting source according to
Secondary transmitting exciting light, and fluorescence signal is generated respectively.
Specifically, the second illumination in first lighting source and the second illumination path controlled in the first illumination path
Light source successively emit exciting light comprising steps of
It controls the first lighting source and emits exciting light in first time, only by the first lighting source within the first time
Generate the optical signal of excitation fluorescence；
Control the second lighting source after the first time the second time transmitting exciting light, second time only by
Second lighting source generates the optical signal of excitation fluorescence.
In one embodiment, first illumination path is Structured Illumination optical path, and second illumination path is width
Field illumination path.
In one embodiment, first lighting source and the second lighting source are LED light source, using LED conduct
Light source, economic and environment-friendly, light energy conversion efficiency is high, avoids the potentially hazardous property of laser light source, safe and efficient, long service life.
S102: control double-shutter camera is acquired the fluorescence signal generated respectively and obtains two images.
Wherein, it is different successively to emit two images caused by exciting light for first lighting source and the second lighting source
It causes.
S103: handling collected two images, obtains a frame image.
Wherein, collected two images are handled, obtaining a frame image further comprises:
Using composition algorithms, every collected two images of a pair are all handled, signal out of focus therein is restrained,
It extracts in burnt fluorescence signal, thus a frame image required for obtaining.
This method is not necessarily to scanning means, in such a way that Structured Illumination and Both wide field illumination imaging combine, successively acquires
Fluorescence signal only needs two images to synthesize the fluorescence that can obtain high quality in burnt image, and image taking speed is fast.
As shown in Fig. 2, Fig. 2 is the structural schematic diagram of the microscopy endoscopic imaging system the present invention is based on Structured Illumination.It should
Microscopy endoscopic imaging system based on Structured Illumination includes the first illumination path, the second illumination path, combined beam light road and imaging
Optical path.First illumination path is used to generate the illumination light of first direction.Second illumination path is for generating second party
To illumination light.The combined beam light road is for carrying out conjunction beam to the illumination light of the first illumination path and the second illumination path.It is described
Imaging optical path is used to collect the fluorescence signal generated, and fluorescence signal is imaged.The system uses modularized design, system
Middle illumination path and imaging optical path separation, structure is simple, and stability is good.The present invention is based on the imagings of the microscopy endoscopic of Structured Illumination
System is to cooperate corresponding fluorochrome label, available high-resolution histocyte based on Induced Fluorescence Microscopy
Pathological image, convenient for carrying out special Journal of Sex Research to cellular elements.
In one embodiment, the microscopy endoscopic imaging system based on Structured Illumination further includes beam splitter, described
Beam splitter is located at the position that crosses of the first illumination path direction of propagation and the second illumination path direction of propagation, shines from described first
The light of Mingguang City road and the second illumination path enters same light path system after beam splitter reflects.The combined beam light road is located at beam splitter
In optical path later.Modularized design, illumination path and imaging optical path separation in microscopy endoscopic system, Structured Illumination optical path and
The separation of Both wide field illumination optical path, structure is simple, and stability is good；Also, light-blocking without pin hole, the efficiency of light energy utilization is high, the fluorescence of generation
Signal is more, fluorescence imaging high sensitivity, the fluorescence organization chart picture abundant information got.
In one embodiment, first illumination path is Structured Illumination optical path, and second illumination path is width
Field illumination path.
In one embodiment, first lighting source and the second lighting source are LED light source, using LED conduct
Light source, economic and environment-friendly, light energy conversion efficiency is high, avoids the potentially hazardous property of laser light source, safe and efficient, long service life.
In one embodiment, due to excite wave band, first lighting source and the second illumination light corresponding to special fluorescent dye
Source is all the LED illumination using specific band, economical cheap.
As shown in Fig. 2, in one embodiment, in Structured Illumination optical path (i.e. the first illumination path), with spectroscope
For point, first illumination path successively includes the first lighting source 11, carries out to the light that the first lighting source 11 issues
The first collimation lens 12, grating 13 and the lens 14 for collecting the diffraction light that grating 13 generates of collimation.First lighting source
11 specifically include LED lamp bead.Grating 13 includes several light along the periodic groove of same direction, after 13 diffraction of grating
Striated structure is presented and realizes striped and illuminates.Grating 13 is located at the front focal plane position of lens, and 14 joint imaging optical path of lens is to light
Grid surface carries out projection imaging, so that the periodic stripe of grating is projected sample surfaces.Present invention introduces structure light imagings
System, the striped illuminated sign generated using grating 13 are gone out in burnt signal, inhibit signal out of focus, effectively improve picture contrast.
As shown in Fig. 2, the LED lamp bead due to use has the very big angle of divergence, in order to improve the utilization rate of luminous energy,
The light that the first collimation lens 12 issues LED lamp bead is added before LED to collimate, collects more illumination lights and enters key light
Road.Illumination light after collimation is incident on 13 surface of grating, diffraction light is generated by grating 13, for realizing Structured Illumination, light
Grid 13 are at least to be made of several along the periodic groove of same direction, therefore the light appearance after 13 diffraction of grating is specific
Striated structure, thus realize striped illuminate.It is collected by the lens 14 that the diffraction light that grating 13 generates is placed behind, light
Grid 13 are located at the front focal plane position of lens 14, and the design of part in 14 joint imaging optical path of lens can carry out 13 surface of grating
Projection imaging, so that the periodic stripe of grating 13 is projected sample surfaces.The fringe structure light that structure optical path generates reaches
One beam splitter 10 enters same light path system with the light from Both wide field illumination after the reflection of beam splitter 10.What beam splitter 10 played
Effect is light wave to be separated or closed beam.Beam splitter 10 is located at the structure optical path direction of propagation and wide field optical path illumination direction
Cross position.
In one embodiment, second illumination path includes the second lighting source 21 and sends out the second lighting source 21
The second collimation lens 22 that light out is collimated.Second lighting source 21 specifically includes LED lamp bead.Both wide field illumination optical path is (i.e.
Second illumination path) the second collimation lens 22 lighting source 21 and matched comprising second, the light incidence side launched
It is spatially mutually perpendicular to structure light sense, in this way in the case where not interfereing with each other, light wave after collimation is from another
A direction also can smoothly enter into beam splitter.
It is evidenced from the above discussion that should microscopy endoscopic imaging system emphasis based on Structured Illumination be divided into illumination path and at
As optical path, wherein illumination path matches that there are two lighting sources, is respectively at different positions, by beam splitter by two not Tongfangs
To illumination light be introduced into the same light path system.
As shown in Fig. 2, including combined beam light road in closing the light path system after beam, the combined beam light road successively includes first
It is optical filter 31, the dichroscope 35 for reflecting the spectrum after the filtering of the first optical filter 31, anti-for collecting dichroscope 35
The object lens 33 of incoming optical signal and the fiber optic bundle 34 for being used for transmission illumination light light wave.First optical filter 31 is used for coming from
The illumination light of first illumination path and the second illumination path carries out spectral filtering, stops the light invalid to fluorescent dye excitation process
Spectrum.In the illumination optical system, an optical filter is only used, i.e. an optical filter and combined beam light road position are only used in combined beam light road
In optical path after beam splitter 10, spectral filtering, resistance can be carried out to from the light of structure optical path and Both wide field illumination optical path simultaneously
Keep off the spectrum invalid to fluorescent dye excitation process.Dichroscope 35 cooperates the first optical filter 31 to use, by the first optical filter
Spectrum after 31 filtering, all can be reflected into object lens by dichroscope 35, after being collected through object lens 33 to optical signal, two kinds
The illuminating bundle of type is smoothly coupled into fiber optic bundle 34.Object lens are the key elements during light processing.
In one embodiment, object lens 33 include object space and Image space, and fiber optic bundle 34 is located at the object space of object lens, the
The grating face of one lighting source 11, the second lighting source 21 and grating 13 is respectively positioned on Image space.Specifically, in this illumination light
Lu Zhong, optical fiber are located at the object space of object lens, and the light source of two kinds of structures and the grating face in structure optical path are all located at the picture of object lens
Space of planes, for Both wide field illumination, object lens 33, which mainly collect optical signal, to be transmitted, and for Structured Illumination, object lens 33 will contain
The optical signal diminution of grating fringe structure is imaged onto optical fiber target surface, and optical fiber target surface and grating surface are in optical conjugate position, because
This object lens has good aberration correction and flat field imaging function.Fiber optic bundle mainly plays the role of transmitting lighting light wave,
Optical excitation signal is transferred to human tissue surface.
As shown in Fig. 2, in one embodiment, the imaging optical path successively includes the fiber optic bundle for collecting transmission fluorescence signal
34, the object lens 33 of transmission, the dichroscope 35 handled the fluorescence signal that object lens 33 transmit are amplified to fluorescence signal
And the second optical filter 32 of spectral filtering is carried out to fluorescence signal.Wherein, fiber optic bundle 34, object lens 33 and dichroscope 35 can be
Combined beam light road and imaging optical path share.
As shown in Fig. 2, the imaging optical path further includes imaging lens 36 and double-shutter camera 37, imaging lens 36 and double quick
Door camera 37 is used cooperatively, and is imaged, is projected on double-shutter camera 37 to the filtered fluorescence signal collection of the second optical filter 32
Fluorescence signal after photoelectric conversion, show the image with tissue information.Using double-shutter camera 37, adopt in pairs
Collect fluorescent image, the fluorescence signal acquisition interval of two width different structures illumination excitation is shorter, reduces motion artifacts, transports to tissue
Dynamic information obtaining ability is stronger.Specifically, imaging optical path is to generate designed by position to phosphor collection position to build from fluorescence
A series of optical transmission systems successively include fiber optic bundle 34, object lens 33, dichroscope 35, the second optical filter 32,36 and of imaging lens
Double-shutter camera 37.Fluorescence signal is mainly by illumination light excitation fluorescent dye generation, therefore the fluorescence signal inspired
It is collected and transmits by fiber optic bundle 34 simultaneously, and from the other end of fiber optic bundle 34, that is, the port outgoing being located at 33 front focal plane of object lens.Object
Mirror 33 plays the role of amplification transmission to fluorescence signal, and the fluorescence signal generated at this time may include the illumination light from exciting light
Spectrum, therefore the optical signal after amplification also needs to filter out invalid spectrum by dichroscope 35 and the second optical filter 32.Due to
Excitation spectrum and fluorescence spectrum have certain SPECTRAL DIVERSITY, by using the dichroscope 35, Ke Yiyou of corresponding SPECTRAL DIVERSITY
Effect ground excitation spectrum and fluorescence spectrum are separated, here the function of dichroscope 35 be optical excitation signal is reflected and
Fluorescence signal is transmitted, so fluorescence signal can pass through dichroscope 35, while in dichroscope in imaging optical path
Another piece of the second optical filter 32 is set after 35 and guarantees the pure of fluorescence signal for further carrying out blocking rejecting to stray light
It is net interference-free.Imaging lens 36 and double-shutter camera 37 are used cooperatively, and are that filtered fluorescence signal collection is imaged, projection
Fluorescence signal on to camera CCD target surface shows the image with tissue information after photoelectric conversion.It is above to complete
The transmission imaging process of entire fluorescence light path.
In the technical scheme, Structured Illumination light source and Both wide field illumination light source are successively to light excitation, i.e., in structure light
In the case that lighting source is lighted, a structure light excitation fluorescent image is acquired, Structured Illumination light source is then shut off and lights
Both wide field illumination light source, then a Both wide field illumination excitation fluorescent image is acquired, collection process is realized by double-shutter camera, that is, is existed
In the case where single exposure, fluorescent image is acquired in couples, and acquisition speed is fast, ensure that imaging frame rate.Since Both wide field illumination produces
It is included in burnt and out of focus organizational information in raw fluorescent image simultaneously, and striated structure in the light activated fluorescent image of structure
Being come out in burnt information flag, by carrying out algorithm synthesis processing to this two images, can extract wherein in coke
Signal abandons signal out of focus simultaneously, to realize the inhibition of background light signal, improves the contrast of image, while entire imaging
It is light-blocking without pin hole in the process without scanning means, therefore image taking speed is very fast.
It is described below with reference to technical solution of the specific embodiment to this law,
As shown in figure 3, the blue led that the light source of two Structured Illumination optical paths is all 450nm using central wavelength
Light source, power are consistent.It is Structured Illumination optical path first, the light that LED light source 11 (i.e. the first lighting source 11) is launched has
The very big angle of divergence is successively converged by the first collimation lens 12, after grating 13, lens 14 and beam splitter 10 reflect, is reached
First optical filter 31.First collimation lens 12 is located at before the LED lamp bead of LED light source 11, and the LED lamp bead of LED light source 11 is in
The front focal plane position of first collimation lens 12, transmitting illumination light are propagated in parallel from 12 rear space of the first collimation lens.Transmission-type
The grating 13 of diffraction is between the first collimation lens 12 and lens 14 and is located at the front focal plane of lens 14, for being directed at direct light
Shu Jinhang shaping simultaneously generates the illuminating bundle with specific structure, and 14 lens that structure light beam is placed below are collected and from beam splitting
Reflective surface in mirror 10 is to the corresponding direction of propagation.Beam splitter 10 have two to close Shu Gongneng, can be by the light beam of different directions
It is transmitted in the same optical path, beam splitter 10 is located at the structure optical path direction of propagation and wide field optical path illumination direction crosses position, with
The wide field that structure optical path direction upright position is made of LED light source 21 (i.e. the second lighting source 21) and the second collimation lens 22
Illumination path, similarly, for the lamp bead of the LED of LED light source 21 also in the front focal plane position of the second collimation lens 22, second is quasi-
Straight lens 22 are closely located to beam splitter 10.Structured Illumination light and Both wide field illumination light by this joint of beam splitter 10 it
Afterwards, the first optical filter 31 can be first passed around and specific band spectrum can smoothly penetrate the first optical filter 31, put by 45 degree of angles
After the dichroscope 35 set reflects, object lens 33 are entered.Pair dichroscope 35 is to reflect long wave transmission function with shortwave, i.e.,
Light wave around 450nm is reflected, and zero deflection is collimatedly incident on object lens 33, while also can spectrum wave to 500nm or more
Section is lossless to be passed through.Object lens 33 are imaged between fiber optic bundle 34 and dichroscope 35, to light beam and are conducted compression, are made
Flat field achromatic micro objective usually has 10 or 20 amplification factor, in this way to from its image space aperture illumination and
The beam and focus come is compressed, its beam radius is narrowed down to the one fixed width smooth coupled into optical fibres of low-loss to the maximum extent
Beam.One end of fiber optic bundle 34 is located at the front focal plane position of object lens 33, and the other end is contacted by channel with tissue.Illuminating bundle
It reaches by this fiber optic bundle by the tissue surface of fluorescent dyeing, excitation generates the fluorescence of corresponding wave band, while fluorescence can edge
Fiber optic bundle backtracking.
The exciting light of 450nm as used in the present embodiment, the fluorescence bands generally generated 500nm it
Afterwards, fluorescence passes through dichroscope after microcobjective amplification in this way.Second optical filter 32 is located at 35 He of dichroscope
Between imaging lens 36, the second optical filter 32 only can smoothly let pass to the light wave of central wavelength 510nm and mask its all band
Light, especially 500nm spectrum below.Double-shutter camera 37 generally uses CCD or CMOS, makes with imaging lens cooperation
With by filtered fluorescence signal collection and by being imaged on display after photoelectric conversion.
In the specific implementation process, the light source of two different light structures is successively to light sequentially in time, that is, works as LED
When light source 11 is lighted, what it is by fiber optic bundle arrival biological tissue surface excitation fluorescence can only be structure optical signal, similarly, when
When LED light source 21 is lighted, exciting the optical signal of fluorescence is exactly Both wide field illumination light beam.When the two light sources successively light generation excitation
Light time, double-shutter camera is all acquired the fluorescence signal generated each time, and exports image.Using composition algorithm, to every
A pair of collected two images are all handled, and signal out of focus therein is restrained, and are extracted in burnt fluorescence signal, will be adopted
The two width fluorescent images collected do algorithm pretreatment and seek equal Fang Yinzi, processing result are based on, respectively to Structured Illumination fluorogram
Picture and Both wide field illumination fluorescent image carry out low frequency and High frequency filter, last high and low frequency information reconfigure, thus required for obtaining
A frame image.Two light sources successively light illumination caused by fluorescent image it is inconsistent, as shown in figure 3, (a) figure is structure light
The fluorescence imaging schematic diagram generated is illuminated, wherein striped is the light structures that structure light generates, and each Structured Illumination is come
It says, the frequency of the striped and position are all constant, and (b) figure is the fluorescence imaging schematic diagram that Both wide field illumination generates, and no structure light is shone
Bright fringe structure, this two images are acquired by double-shutter camera single exposure, are carried out algorithm process to this two images, are synthesized
With the fluorescent image for effectively inhibiting signal out of focus.
From above-mentioned technical proposal description as can be seen that the present invention is on the basis of the optical path of traditional wide field fluorescent microscopic imaging,
Structured Illumination optical path, while incoming fiber optic beam is added, constructs microscopy endoscopic imaging system, which is former based on fluorescence imaging
Reason corresponds to specific fluorescent dye, designs corresponding excitation light source and light path system, and smoothly acquires fluorescence signal and screen
The interference of stray light of its all band is covered, the fluorescent image that can reflect organizational information is finally got.
Compared with prior art, the beneficial effects of the present invention are:
1, using LED as light source, economic and environment-friendly, light energy conversion efficiency is high, avoids the potentially hazardous property of laser light source,
It is safe and efficient, long service life.
2, it is based on Induced Fluorescence Microscopy, cooperates corresponding fluorochrome label, available high-resolution tissue
Cellular Pathology Image, convenient for carrying out special Journal of Sex Research to cellular elements.
3, it introduces Structured Illumination optical path and there is Structured Illumination imaging system, illuminate mark using the striped that grating generates
Remember out in burnt signal, inhibits signal out of focus, effectively improve picture contrast.
4, it is not necessarily to scanning means, in such a way that Structured Illumination and Both wide field illumination imaging combine, successively acquires fluorescence
Signal only needs two images to synthesize the fluorescence that can obtain high quality in burnt image, and image taking speed is fast.
5, light-blocking without pin hole, the efficiency of light energy utilization is high, and the fluorescence signal of generation is more, and fluorescence imaging high sensitivity is got
Fluorescence organization chart picture abundant information.
6, using double-shutter camera, fluorescent image is acquired in pairs, and the fluorescence signal of two width different structure optical illumination excitation is adopted
Collection interval is shorter, reduces motion artifacts, stronger to histokinesis's information obtaining ability.
7, modularized design, illumination path and imaging optical path separation, Structured Illumination optical path and width in microscopy endoscopic system
Field illumination path separation, structure is simple, and stability is good.
The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill
For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and
Modification, these improvements and modifications also should be regarded as protection scope of the present invention.
Claims (16)
1. a kind of microscopy endoscopic imaging method based on Structured Illumination, which is characterized in that itself comprising steps of
Control the second lighting source in the first lighting source and the second illumination path in the first illumination path successively emit swash
It shines, and generates fluorescence signal respectively；
Control double-shutter camera is acquired the fluorescence signal generated respectively and obtains two images；
Collected two images are handled, a frame image is obtained.
2. the microscopy endoscopic imaging method according to claim 1 based on Structured Illumination, which is characterized in that the control
The second lighting source in the first lighting source and the second illumination path in first illumination path successively emits exciting light and includes
Step:
It controls the first lighting source and emits exciting light in first time, only generated by the first lighting source within the first time
Excite the optical signal of fluorescence；
The the second time transmitting exciting light of the second lighting source after the first time is controlled, in second time only by second
Lighting source generates the optical signal of excitation fluorescence.
3. the microscopy endoscopic imaging method according to claim 1 based on Structured Illumination, which is characterized in that collecting
Two images handled, obtain a frame image further comprise:
Using composition algorithm, every collected two images of a pair are all handled, restrain signal out of focus therein, are extracted
Out in burnt fluorescence signal, thus a frame image required for obtaining.
4. the microscopy endoscopic imaging method according to claim 1 based on Structured Illumination, which is characterized in that described first
Lighting source and the second lighting source are LED light source.
5. a kind of microscopy endoscopic imaging system based on Structured Illumination, characterized in that it comprises:
First illumination path, for generating the illumination light of first direction；
Second illumination path, for generating the illumination light of second direction；
Combined beam light road carries out conjunction beam for the illumination light to the first illumination path and the second illumination path；
Imaging optical path is imaged for collecting fluorescence signal, and to fluorescence signal.
6. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that described first
Light the first collimation lens, the light that are collimated that illumination path successively includes the first lighting source, is issued to the first lighting source
The lens of grid and the diffraction light for collecting grating generation.
7. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that described first
Lighting source is LED light source.
8. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the grating
Including several along the periodic groove of same direction, the light after optical grating diffraction is presented striated structure and realizes striped and shine
It is bright.
9. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the grating
Positioned at the front focal plane position of lens, the lens joint imaging optical path carries out projection imaging to grating surface, thus by grating
Periodic stripe projects sample surfaces.
10. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that described
Two illumination paths include the second collimation lens that the second lighting source and the light issued to the second lighting source are collimated.
11. the microscopy endoscopic imaging system according to claim 10 based on Structured Illumination, which is characterized in that described
Two lighting sources are LED light source.
12. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the conjunction
Beam optical path successively includes the first optical filter, the dichroscope for reflecting the spectrum after the first optical filter filters, for collecting
The object lens for the optical signal that dichroscope reflects back and the fiber optic bundle for being used for transmission illumination light light wave, first optical filter are used
In carrying out spectral filtering to from the illumination light of the first illumination path and the second illumination path, stop to fluorescent dye excitation process
Invalid spectrum.
13. the microscopy endoscopic imaging system according to claim 12 based on Structured Illumination, which is characterized in that the object
Mirror includes object space and Image space, and the fiber optic bundle is located at the object space of object lens, first lighting source, the second illumination light
Source and the grating face of grating are respectively positioned on Image space.
14. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that it is described at
As optical path successively includes collecting the fiber optic bundle of transmission fluorescence signal, the object lens that transmission is amplified to fluorescence signal, to object lens biography
The dichroscope that defeated fluorescence signal is handled and the second optical filter to fluorescence signal progress spectral filtering.
15. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that it is described at
As optical path further includes imaging lens and double-shutter camera, the imaging lens and double-shutter camera are used cooperatively, and are filtered to second
The filtered fluorescence signal collection imaging of piece, projects the fluorescence signal on double-shutter camera after photoelectric conversion, shows
Image with tissue information.
16. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the base
It further include beam splitter in the microscopy endoscopic imaging system of Structured Illumination, the beam splitter is located at the first illumination path direction of propagation
With the position that crosses of the second illumination path direction of propagation, the light from first illumination path and the second illumination path is through beam splitting
Enter same light path system after mirror reflection.
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| CN111580259A (en) * | 2020-04-29 | 2020-08-25 | 深圳大学 | Optical imaging system, imaging method and microscope |
| CN112075925A (en) * | 2020-09-21 | 2020-12-15 | 北京脑科学与类脑研究中心 | Fluorescent imaging illumination device, imaging system and imaging method based on speckle principle |
| CN112890736A (en) * | 2019-12-03 | 2021-06-04 | 精微视达医疗科技（武汉）有限公司 | Method and device for obtaining field mask of endoscopic imaging system |
| WO2021179127A1 (en) * | 2020-03-09 | 2021-09-16 | 深圳华大生命科学研究院 | Super-resolution imaging system and method, biological sample identification system and method, nucleic acid sequencing imaging system and method, and nucleic acid identification system and method |
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| CN112890736A (en) * | 2019-12-03 | 2021-06-04 | 精微视达医疗科技（武汉）有限公司 | Method and device for obtaining field mask of endoscopic imaging system |
| WO2021179127A1 (en) * | 2020-03-09 | 2021-09-16 | 深圳华大生命科学研究院 | Super-resolution imaging system and method, biological sample identification system and method, nucleic acid sequencing imaging system and method, and nucleic acid identification system and method |
| CN111580259A (en) * | 2020-04-29 | 2020-08-25 | 深圳大学 | Optical imaging system, imaging method and microscope |
| CN111580259B (en) * | 2020-04-29 | 2023-10-13 | 深圳大学 | Optical imaging system, imaging method and microscope |
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