CN108121059A - A kind of parallel micro imaging systems of STED based on Structured Illumination - Google Patents
A kind of parallel micro imaging systems of STED based on Structured Illumination Download PDFInfo
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- CN108121059A CN108121059A CN201711150961.0A CN201711150961A CN108121059A CN 108121059 A CN108121059 A CN 108121059A CN 201711150961 A CN201711150961 A CN 201711150961A CN 108121059 A CN108121059 A CN 108121059A
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/0004—Microscopes specially adapted for specific applications
- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
- G02B21/0032—Optical details of illumination, e.g. light-sources, pinholes, beam splitters, slits, fibers
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/06—Means for illuminating specimens
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- Optics & Photonics (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract
The parallel micro imaging systems of STED provided by the invention based on Structured Illumination, by being designed to lighting module, a branch of exciting light of exciting light laser emitting is divided into two beam coherent lights, a branch of loss light that light laser outgoing is lost is divided into two beam coherent lights, equally distributed excitation structure light and loss structure light are respectively obtained by interference, excitation structure light and loss structure light simultaneously excite sample and are lost, carry out the parallel micro-imagings of STED, image reconstruction is carried out using the coordinate location method and SIM frequency-domain spectrum figures fusion method of STED again, realize super-resolution imaging, contribute to the field range and image taking speed of expansion STED microscopic systems.
Description
Technical field
The present invention relates to microscopic detection instrument design and manufacturing field, more particularly, to a kind of based on Structured Illumination
The parallel micro imaging systems of STED.
Background technology
Super-resolution micro-imaging technique is a kind of cutting edge technology in the research fields such as biology, is broadly divided into coordinate and determines
Position class and the random two major class micro imaging method of class of coordinate.Typical Representative wherein in coordinate setting class micro imaging method be by
Swash radiation loss (STED) micro-imaging, STED micro-imagings directly using optical means, carry out point by point scanning to sample, realize
Super-resolution imaging.But it is limited by simple scan mode, the image taking speed of STED micro-imagings is slow.
In recent years, to improve the image taking speed of STED micro-imaging techniques, the parallel micro-imagings of STED are suggested.Wherein imitate
Fruit preferably uses two-dimensional structure optical illumination pattern, hence it is evident that improves image taking speed.But due to the addition of two-dimensional structure light,
Imaging system complexity is caused to become higher, and exciting light does not use wide field to excite using structure optical mode in the technology, makes
Obtaining the signal-to-noise ratio of effective fluorescence significantly reduces, and affects imaging resolution.
The content of the invention
The purpose of the present invention is：
The parallel micro-imaging systems of the STED based on Structured Illumination that a kind of imaging resolution is high and image taking speed is fast are provided
System.
To achieve the above object, the present invention uses following technical proposals：
The parallel micro imaging systems of a kind of STED based on Structured Illumination, including lighting module, detecting module, control mould
Block and image reconstruction module；
The lighting module includes：Excite light laser, loss light laser, LCD space light modulator, Wollaston
Prism, the first lens, vias masks plate, the second lens, the first half-wave plate, the second half-wave plate, speculum, the first dichroic mirror,
Two dichroic mirrors, cylinder mirror, object lens and three-dimensional manometer displacement platform, the three-dimensional manometer displacement platform can be described in XYZ three-dimensional direction movings
Three-dimensional manometer displacement platform carries detected sample；
The detecting module includes optical filter, finder lens and planar array detector, the pixel in the planar array detector
Dummy pinhole can be formed；
The control module is electrically connected at the three-dimensional manometer displacement platform, planar array detector and liquid crystal spatial light modulation
Device；Described image rebuilds module and is electrically connected at the control module；Wherein：
For the Gaussian beam that the excitation light laser is sent as exciting light, the exciting light incides into the liquid crystal spatial
Optical modulator, the LCD space light modulator are modulated the exciting light generation diffraction light, and the diffraction light includes ± 1
Order diffraction light and 0 order diffraction light；The diffraction light is incident into the vias masks plate after first lens focus, described
Vias masks plate blocks the 0 order diffraction light, only allows ± 1 order diffraction light by, ± 1 order diffraction light through described the
Again successively through first half-wave plate, the speculum, the dichroic mirror, second dichroic mirror and cylinder mirror after two collimateds
Afterwards into the object lens, and interfered at the object lens front focal plane, generate the excitation structure light of interference fringe shape, it is described to swash
Hair structure light is illuminated sample excitation；
The Gaussian beam that the loss light laser is sent is as loss light, and the loss light is through the Wollaston prism
Loss light is divided into the mutually perpendicular first loss light in two beam polarization directions and the second loss light, the first loss light and the second damage
Deplete, first loss light and second loss light through second half-wave plate after identical through the second half-wave plate rear polarizer direction
Again successively after second dichroic mirror and the cylinder mirror into the object lens, and done at the front focal plane of the object lens
It relates to, generates the loss structure light of interference fringe shape, sample is lost in the loss structure light；
The loss of excitation and loss structure light of the sample through the excitation structure light, forms structure light light field, the structure
Light light field is successively through the object lens, the cylinder mirror, second dichroic mirror, first dichroic mirror, the optical filter and described
Finder lens, then the image of the photosurface formation uniform stripe distribution in the planar array detector, the planar array detector detection
Described image is simultaneously converted into electric signal after carrying out space filtering to described image by the dummy pinhole by described image；
The control module gathers the electric signal, and described image rebuilds module and uses coordinate setting and SIM frequency-domain spectrums
Figure melts carries out image reconstruction to the electric signal, realizes the parallel micro-imagings of STED based on Structured Illumination.
In wherein some embodiments, the photosurface of the LCD space light modulator, the front focal plane, described of the object lens
The exit plane of Wollaston prism and the planar array detector photosurface are in conjugate planes.
In wherein some embodiments, the planar array detector is the magazine one kind of CCD or CMOS.
In wherein some embodiments, pass through phase of the control module adjustment loading on LCD space light modulator
Distribution can change direction, cycle and the initial phase of exciting light light field so that the exciting light structure light light field and loss photo structure
Light light field has identical direction and cycle, initial phase difference half period.
It is of the invention to be using the advantages of above-mentioned technical proposal：
The parallel micro imaging systems of STED provided by the invention based on Structured Illumination, by being set to lighting module
Meter, is divided into two beam coherent lights by a branch of exciting light of exciting light laser emitting, a branch of loss of light laser outgoing will be lost
Light is divided into two beam coherent lights, and equally distributed excitation structure light and loss structure light, excitation structure light are respectively obtained by interference
Structure light and sample is excited and is lost with loss, carry out the parallel micro-imagings of STED, then the coordinate setting using STED
Method and SIM frequency-domain spectrum figures fusion method carry out image reconstruction, realize super-resolution imaging, help to expand STED microscopic systems
Field range and image taking speed.
Description of the drawings
Fig. 1 is the parallel micro imaging system structural representations of the STED provided in an embodiment of the present invention based on Structured Illumination
Figure.
Fig. 2 (a) is the structure diagram of excitation structure light light field provided in an embodiment of the present invention；
Fig. 2 (b) is the structure diagram of loss structure light light field provided in an embodiment of the present invention；
Fig. 2 (c) is the structure diagram of resulting structure light light field provided in an embodiment of the present invention；
Fig. 3 (a) is the striated image of structure light on planar array detector provided in an embodiment of the present invention；
Fig. 3 (b) is the structure diagram of dummy pinhole provided in an embodiment of the present invention；
Fig. 4 (a) is provided in an embodiment of the present invention in frequency domain to merge the frequency domain figure of every width two dimensional image
Structure diagram；
Fig. 4 (b) is transformed into spatial domain by inverse Fourier transform and obtains final oversubscription to be provided in an embodiment of the present invention
Distinguish the structure diagram of image.
Wherein：Lighting module 110, detecting module 120, control module 130, image reconstruction module 140, excitation light laser
111st, light laser 112, LCD space light modulator 113, Wollaston prism 114, the first lens 115, vias masks are lost
Plate 116, the second lens 117, the first half-wave plate 118, the second half-wave plate 119, speculum 1110, the first dichroic mirror 1111, second
Dichroic mirror 1112, cylinder mirror 1113, object lens 1114, three-dimensional manometer displacement platform 1115, optical filter 121, finder lens 122, face battle array are visited
Survey device 123.
Specific embodiment
It please refers to Fig.1, is a kind of parallel micro-imaging systems of STED based on Structured Illumination provided in an embodiment of the present invention
System 100, lighting module 110, detecting module 120, control module 130 and image reconstruction module 140.Wherein：
The lighting module 110 includes：Excite light laser 111, loss light laser 112, LCD space light modulator
113rd, Wollaston prism 114, the first lens 115, vias masks plate 116, the second lens 117, the first half-wave plate 118, second
Half-wave plate 119, speculum 1110, the first dichroic mirror 1111, the second dichroic mirror 1112, cylinder mirror 1113,1114 and three wiener of object lens
Rice displacement platform 1115, the three-dimensional manometer displacement platform 1115 can be in XYZ three-dimensional direction movings, the three-dimensional manometer displacement platform carryings
There is detected sample.
The detecting module 120 includes optical filter 121, finder lens 122 and planar array detector 123, and the face battle array is visited
The pixel surveyed in device 123 can form dummy pinhole.
The control module 130 is electrically connected at the three-dimensional manometer displacement platform 1115, planar array detector 123 and liquid crystal
Spatial light modulator 113；Described image rebuilds module 140 and is electrically connected at the control module 130.
The work of the parallel micro imaging systems 100 of the following detailed description STED provided by the invention based on Structured Illumination
Principle：
For the Gaussian beam that the excitation light laser 111 is sent as exciting light, the exciting light 111 incides into the liquid
Brilliant spatial light modulator 113, the LCD space light modulator 113 is modulated the exciting light generation diffraction light, described
Diffraction light includes ± 1 order diffraction light and 0 order diffraction light；The diffraction light is incident after first lens 115 focusing to enter institute
State vias masks plate 116, the vias masks plate 116 blocks the 0 order diffraction light, only allow ± 1 order diffraction light by,
± 1 order diffraction light is after second lens 117 collimation again successively through first half-wave plate 118, the speculum
1110th, into the object lens 1114 after first dichroic mirror 1111, second dichroic mirror 1112 and cylinder mirror 1113, and in institute
State and interfered at 1114 front focal plane of object lens, generate interference fringe shape excitation structure light, the excitation structure light to sample into
Row illumination excitation；
The Gaussian beam that the loss light laser 112 is sent is as loss light, and the loss light is through the Wollaston
Prism 114 is lost light and is divided into the mutually perpendicular first loss light in two beam polarization directions and the second loss light, the first loss light
And second loss light it is identical through the 119 rear polarizer direction of the second half-wave plate, after second half-wave plate 119 first loss
Light and the second loss light again successively after second dichroic mirror 1112 and the cylinder mirror 1113 into the object lens 1114, and
Interfered at the front focal plane of the object lens 1114, generate the loss structure light of interference fringe shape, the loss structure light is to sample
Product are lost；
The loss of excitation and loss structure light of the sample through the excitation structure light, forms structure light light field, the structure
Light light field successively through the object lens 1114, the cylinder mirror 1113, second dichroic mirror 1112, first dichroic mirror 1111,
The optical filter 121 and the finder lens 122, then form uniform stripe distribution in the photosurface of the planar array detector 122
Image, the planar array detector 122 detects described image and simultaneously carries out space filtering to described image by the dummy pinhole
Described image is converted into electric signal afterwards；
The control module 130 gathers the electric signal, and described image rebuilds module 140 using coordinate setting and SIM frequencies
Domain spectrogram melts carries out image reconstruction to the electric signal, realizes the parallel micro-imagings of STED based on Structured Illumination.
Preferably, front focal plane P3, the Wollaston of the photosurface P1 of the LCD space light modulator 113, object lens 1114
114 exit plane P4 of prism and 123 photosurface P5 of planar array detector are in conjugate planes.
Further, planar array detector 123 can be CCD or CMOS cameras, can receive resulting structure in fluorescent samples
The fluorescence signal of light, and it is translated into electric signal；And the pixel in planar array detector 123 can form dummy pinhole, to visiting
The structure light measured carries out space filtering.
Further, since the control module 140 is electrically connected at the three-dimensional manometer displacement platform 1115, face battle array detection
Device 123 and LCD space light modulator 113 can be realized by the control module 140 to the three-dimensional manometer displacement platform
1115th, the scan control of planar array detector 123 and LCD space light modulator 113, and the control module 130 will gather electricity
Signal transmission to described image rebuilds module 140, and described image rebuilds module 140 according to SIM frequency-domain spectrum figure fusion methods to institute
It states electric signal and carries out image reconstruction, realize the parallel micro-imagings of STED based on Structured Illumination.
Image reconstruction module 140 described further below is melted using coordinate setting and SIM frequency-domain spectrum figures to the electric signal
Image reconstruction is carried out, realizes the course of work of the parallel micro-imagings of STED based on Structured Illumination：
First, parallel detecting resulting structure light light field
By phase distribution of the adjustment loading of control module 130 on LCD space light modulator 113, change excitation light
Direction, cycle and initial phase so that exciting light structure light light field and loss photo structure light light field have identical direction and
Cycle, initial phase difference half period；Sample is formed effective after the excitation of excitation structure light and the loss of loss structure light
Structure light light field, the resulting structure light light field is successively through the object lens 1114, the cylinder mirror 1113, second dichroic mirror
1112nd, first dichroic mirror 1111, the optical filter 121 and the finder lens 122, then in the planar array detector 123
Photosurface formed uniform stripe distribution image, so as to fulfill parallel optical signal detection collect, refer to Fig. 2 (a), Fig. 2
(b) and Fig. 2 (c) is expressed as excitation structure light light field, and structure light light field and resulting structure light light field is lost.
Second, obtain given structure light direction and the STED images of given sample position
It is appreciated that when given structure light direction and given sample position, formed in the photosurface of planar array detector 123
Row's dummy pinhole is all corresponded to comprising many optical signal stripeds, every optical signal striped in the image of uniform stripe distribution, such as
Fig. 3 (a) and Fig. 3 (b) is respectively the striated image and dummy pinhole of structure light on planar array detector, wherein, strip rectangle
A stripe light distribution is represented, small square lattice represents the pixel of planar array detector, and larger square lattice represents
One dummy pinhole (including multiple pixels), positioned at the center of optical signal striped, inhibition light disturbance will for the arrangement of dummy pinhole
The light intensity that each pixel detects in dummy pinholeiSuperposition can obtain any corresponding STED image pixel value in sampleThe corresponding all dummy pinholes of each optical signal striped are similarly handled, a line in sample can be obtained
Dummy pinhole in all optical signal stripeds is all made identical processing, will obtain a width picture by corresponding STED image pixel values
The STED images of element value striped stride distribution.
3rd, obtain the STED images after scan sample under given structure light direction
Holding structure light (excitation structure light and loss structure light) direction is constant, is driven using three-dimensional manometer displacement platform 1115
Sample is moved along perpendicular to structural light stripes direction substep, and total mobile number is d under each structural light stripes directionp/ds-
1, dpFor fringe spatial cycle, dsFor single moving step pitch, displacement platform often stops a position, is recorded with planar array detector 123
Sample striated fluoroscopic image at the position using the processing method of above-mentioned second step, will be obtained between a width pixel value strip
Every the STED images of distribution, so, d will be obtained under each structural light stripes directionp/dsWidth pixel value striped stride point
The STED images of cloth, by these image superpositions, generation corresponds to the piece image in a structural light stripes direction, by this image
Method for reconstructing is denoted as coordinate positioning method, which realizes super-resolution imaging in the direction perpendicular with the structural light stripes.
4th, obtain the STED images after scan sample under all directions structure light
Wollaston prism 114 is rotated, and correspondingly rotates the phase diagram loaded on LCD space light modulator 113, is made
Different directions can be located at by obtaining the structure light light field striped of exciting light and loss light at 1114 focal plane of object lens, generate different directions
Effective fluorescence structure light light field, for all directions effective fluorescence structure light light field carry out the 3rd step it is identical with second step
Processing can obtain including the two dimensional image of one-dimensional super resolution information in light field all directions.
5th, it merges to obtain super resolution image by SIM frequency-domain spectrums
Image reconstruction is carried out using SIM frequency-domain spectrum figures fusion method, the two-dimensional images for first obtaining the 4th step carry out
Fourier transformation is transformed into frequency domain, as shown in Fig. 4 (a), merges the frequency domain figure of every width two dimensional image in frequency domain,
As shown in Fig. 4 (b), spatial domain finally is transformed into using inverse Fourier transform, obtains final super resolution image.
The parallel micro imaging systems of STED provided by the invention based on Structured Illumination, by lighting module 110 into
A branch of exciting light that light laser 111 is excited to be emitted is divided into two beam coherent lights by row design, and loss light laser 112 is emitted
A branch of loss light be divided into two beam coherent lights, by interference respectively obtain equally distributed excitation structure light and loss structure light,
Excitation structure light and loss structure light simultaneously excite sample and are lost, and carry out the parallel micro-imagings of STED, then using STED
Coordinate location method and SIM frequency-domain spectrum figures fusion method carry out image reconstruction, realize super-resolution imaging, contribute to expand STED
The field range and image taking speed of microscopic system.
Certainly the parallel micro imaging systems of the STED based on Structured Illumination of the present invention can also have a variety of conversion and change
Type, it is not limited to the concrete structure of the above embodiment.In short, protection scope of the present invention should include those for this field
It obviously converts or substitutes and retrofit for those of ordinary skill.
Claims (4)
1. the parallel micro imaging systems of a kind of STED based on Structured Illumination, which is characterized in that including lighting module, detection mould
Block, control module and image reconstruction module；
The lighting module includes：Excite light laser, loss light laser, LCD space light modulator, Wollaston prism,
First lens, vias masks plate, the second lens, the first half-wave plate, the second half-wave plate, speculum, the first dichroic mirror, the two or two color
Mirror, cylinder mirror, object lens and three-dimensional manometer displacement platform, the three-dimensional manometer displacement platform can be in XYZ three-dimensional direction movings, three wieners
Rice displacement platform carries detected sample；
The detecting module includes optical filter, finder lens and planar array detector, and the pixel in the planar array detector can shape
Into dummy pinhole；
The control module is electrically connected at the three-dimensional manometer displacement platform, planar array detector and LCD space light modulator；
Described image rebuilds module and is electrically connected at the control module；Wherein：
For the Gaussian beam that the excitation light laser is sent as exciting light, the exciting light incides into the liquid crystal spatial light tune
Device processed, the LCD space light modulator are modulated the exciting light generation diffraction light, and the diffraction light spreads out including ± 1 grade
Penetrate light and 0 order diffraction light；The diffraction light is incident after first lens focus to enter the vias masks plate, the through hole
0 order diffraction light described in mask plate shielding only allows ± 1 order diffraction light by the way that ± 1 order diffraction light is saturating through described second
It is laggard through first half-wave plate, the speculum, the dichroic mirror, second dichroic mirror and cylinder mirror successively again after mirror collimation
Enter the object lens, and interfered at the object lens front focal plane, generate the excitation structure light of interference fringe shape, the excitation knot
Structure light is illuminated sample excitation；
The Gaussian beam that the loss light laser is sent is lost as loss light, the loss light through the Wollaston prism
Light is divided into the mutually perpendicular first loss light in two beam polarization directions and the second loss light, the first loss light and the second loss light
It is identical through the second half-wave plate rear polarizer direction, after second half-wave plate first loss light and second loss light again according to
It is secondary to enter the object lens after second dichroic mirror and the cylinder mirror, and interfered at the front focal plane of the object lens, it is raw
Into the loss structure light of interference fringe shape, sample is lost in the loss structure light；
The loss of excitation and loss structure light of the sample through the excitation structure light, forms structure light light field, the structure light light
Field is successively through the object lens, the cylinder mirror, second dichroic mirror, first dichroic mirror, the optical filter and the detection
Lens, then form the image of uniform stripe distribution in the photosurface of the planar array detector, described in planar array detector detection
Described image is simultaneously converted into electric signal after carrying out space filtering to described image by the dummy pinhole by image；
The control module gathers the electric signal, and described image rebuilds module and coordinate setting and SIM frequency-domain spectrum figures is used to melt
Image reconstruction is carried out to the electric signal, realizes the parallel micro-imagings of STED based on Structured Illumination.
2. the parallel micro imaging systems of the STED according to claim 1 based on Structured Illumination, which is characterized in that described
The photosurface of LCD space light modulator, the front focal plane of the object lens, the exit plane of the Wollaston prism and the face
Array detector photosurface is in conjugate planes.
3. the parallel micro imaging systems of the STED according to claim 1 based on Structured Illumination, which is characterized in that described
Planar array detector is the magazine one kind of CCD or CMOS.
4. the parallel micro imaging systems of the STED according to claim 1 based on Structured Illumination, which is characterized in that pass through
Phase distribution of the control module adjustment loading on LCD space light modulator can change direction, the week of exciting light light field
Phase and initial phase so that the exciting light structure light light field and loss photo structure light light field have identical direction and cycle, just
Phase differs half period.
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| CN108957720A (en) * | 2018-09-26 | 2018-12-07 | 中国科学院化学研究所 | Lighting system and STED optical microscopy for STED optical microscopy |
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| CN112326672A (en) * | 2020-11-06 | 2021-02-05 | 之江实验室 | Rapid imaging system based on multicolor parallel frequency shift illumination |
| CN117451683A (en) * | 2023-12-20 | 2024-01-26 | 深圳赛陆医疗科技有限公司 | Super-resolution microscopic imaging system, imaging method of sample to be detected and gene sequencer |
| WO2024144454A1 (en) * | 2022-12-27 | 2024-07-04 | National University Of Singapore | System and method for image reconstruction using structured illumination microscopy |
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| WO2024144454A1 (en) * | 2022-12-27 | 2024-07-04 | National University Of Singapore | System and method for image reconstruction using structured illumination microscopy |
| CN117451683A (en) * | 2023-12-20 | 2024-01-26 | 深圳赛陆医疗科技有限公司 | Super-resolution microscopic imaging system, imaging method of sample to be detected and gene sequencer |
| CN117451683B (en) * | 2023-12-20 | 2024-03-08 | 深圳赛陆医疗科技有限公司 | Super-resolution microscopic imaging system, imaging method of sample to be detected and gene sequencer |
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