CN107907981A - A kind of three-dimensional structure optical illumination super-resolution microscopic imaging device based on double galvanometers - Google Patents
A kind of three-dimensional structure optical illumination super-resolution microscopic imaging device based on double galvanometers Download PDFInfo
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- CN107907981A CN107907981A CN201711025464.8A CN201711025464A CN107907981A CN 107907981 A CN107907981 A CN 107907981A CN 201711025464 A CN201711025464 A CN 201711025464A CN 107907981 A CN107907981 A CN 107907981A
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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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B27/00—Optical systems or apparatus not provided for by any of the groups G02B1/00 - G02B26/00, G02B30/00
- G02B27/58—Optics for apodization or superresolution; Optical synthetic aperture systems
Abstract
The present invention discloses a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device based on double galvanometers, including laser and is sequentially arranged along the light path of the laser：The laser beam, is divided into the incident light being concerned with three tunnels by beam splitting component；Galvanometer scanning system, is separately mounted in two input paths, and a wherein input path is equipped with by mobile to change the speculum of optical path difference；Microcobjective, interference generation Structured Illumination pattern is carried out for three-beam to be incided on sample, and collects the fluorescence intensity signals that sample is sent；Further include a computer, by controlling, speculum is mobile and galvanometer scanning system is scanned, the multiple phase shift of Structured Illumination pattern progress and direction are rotated, and several postrotational fluorescence intensity images of phase shift and direction are carried out with data processing, reconstruct obtains super resolution image.Image taking speed of the present invention is fast, and incident laser energy utilization rate is high, and interference pattern contrast is high, and image quality is high, suitable for carrying out three-dimensional super-resolution imaging to vivo biological tissue.
Description
Technical field
The invention belongs to optical ultra-discrimination micro-imaging field, more particularly to a kind of three-dimensional structure illumination based on double galvanometers
Bright super-resolution microscopic imaging device.
Background technology
Light microscope, particularly fluorescence microscope, since it can study the three-dimensional internal structure of cell and organism
Ability and the advantage of specific observation can carry out molecule by fluorochrome label, and be widely used in life section
In the research in field.But the spatial resolution of conventional optical microscope is limited by lambda1-wavelength, diffraction can not be broken through
The limit, can only achieve 200nm or so.
As a kind of powerful super-resolution micro-imaging technique, Structured Illumination microscope can break through optical diffraction
The limit, the lateral resolution of traditional fluorescence microscope is improved twice.Its basic principle is using structure light pattern illumination sample
Product, are usually sinusoidal pattern, are moved into the radio-frequency component that can not be usually observed in sample using frequency alias effect
As system low frequency pass band within the scope of, so as to obtain the more detail of the high frequency of sample, improve resolution ratio.With it
He compares various imaging techniques, and Structured Illumination microscope has lot of advantages, is such as swept compared to stimulated emission depletion microscope point
Retouch needs acquisition great amount of images to be reconstructed with unimolecule position finding microscope, and structure light microscope need to only obtain small number of width
Field picture can recover super resolution image, and image taking speed is high, available for real-time monitored and living imaging；Positioned compared to unimolecule
Microscope needs specific fluorescent dye, and structure light microscope greatly expands its application range without specific dye.
In life science, the technology of another active demand is exactly that live body three-dimensional can be carried out to sample at present
Imaging, this contributes to researchers to be better understood from function and the mutual contact that organism is respectively organized.Due to two
Dimension Structured Illumination microscope is interfered using dual-beam, its optical transfer function occurs that middle absence of heart shows in the axial direction
As causing axial resolution too low, three-dimensional imaging can not be applied to.If interfered using three beams coherent light, what it was produced
Structured Illumination pattern can include more high fdrequency components, so as to eliminate the center deficient phenomena of optical transfer function, ensure
It can obtain the resolution ratio of twice conventional optical microscope on laterally and axially, available for three-dimensional super-resolution imaging.
The content of the invention
The present invention provides a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device based on double galvanometers, two are used
Beam splitter produces three beams coherent light, and wherein two-beam is scanned by galvanometer control respectively, and three beams is collected finally by object lens
Light shows that carrying out interference produces three-dimensional structure optical pattern in sample.Present apparatus image taking speed is fast；Experimental error is small；Incident laser energy
Utilization rate is high；Interference pattern contrast is high, and image quality is high；Three-dimensional super-resolution can be realized, especially suitable for fluorescent samples
Carry out live body three-dimensional imaging.
For achieving the above object, concrete technical scheme of the present invention is as follows：
A kind of three-dimensional structure optical illumination super-resolution microscopic imaging device based on double galvanometers, including excitation light path module, are used
In incident laser is divided into three tunnels, carries out interference in sample interior and produce three-dimensional structure optical illumination pattern；With imaging optical path module,
The fluorescence signal sent for collecting sample to be tested, data processing and image reconstruction are carried out using computer；
The excitation light path module includes what is be sequentially arranged：
Laser, sends laser beam；
Beam splitting component, including two beam splitters are sequentially arranged, for producing three road incident lights；
Galvanometer system, for controlling light beam scanning direction, realizes the rotation of structure light pattern；
Microcobjective, interference generation Structured Illumination figure is carried out for the tangential linearly polarized light of three beams to be focused on sample
Sample, and collect the fluorescence intensity signals that sample is sent；
The imaging optical path module includes what is be sequentially arranged：
Dichroscope, for the fluorescence for transmiting incident light, reflected sample is sent；
Industrial camera, for collecting the fluorescence intensity signals；
Computer, for controlling galvanometer system scanning and piezoelectric ceramics movement to change the direction of structure light pattern and phase
Position, control half-wave plate rotation ensure that normal incident light polarization direction remains tangential, and control sample stage carries out axial scan acquisition
Three-dimensional information, control industrial camera obtain the fluorescence signal that sends of sample, and to the fluorescence intensity signals of camera collection into line number
According to processing and phase reconstruction, two and three dimensions super resolution image is obtained.
Preferably, it has been sequentially placed between the laser and two beam splitters：Single mode optical fiber, for laser beam into
Row filtering；The polarizer, for laser beam to be changed into linearly polarized light；And half-wave plate, for controlling the line incided on beam splitter
Direction of polarized light.
Optionally, two sets of galvanometer systems use transmission-type or reflective galvanometer system.
Preferably, tangential light polarization plate converter is respectively placed with the lens focal plane position of two sets of galvanometer systems, is used
In the polarization direction of rotation two-way linearly polarized light, the incident light for ensureing to interfere on sample is tangential linearly polarized light, with
Produce the Structured Illumination pattern of contrast maximum.
Preferably, it has been sequentially placed between galvanometer system and microcobjective for described two sets：Plane mirror, shakes for changing
The direction of propagation for the light beam that mirror comes out, makes its oblique be mapped on the scanning lens of rear；And scanning lens, for making three road incident lights
Focus on after microcobjective at pupil face.
It is arranged on the plane mirror all the way in light path to be moved by Piezoelectric Ceramic, changes light path official post structure light
Illumination pattern produces phase shift.
Preferably, from the normal incidence of light all the way that beam splitter transmits to scanning lens middle position, the normal incidence light path
Beam splitter and scanning lens between be sequentially placed：Half-wave plate, for rotating the polarization direction of the route polarised light, ensures phase
It is tangential linearly polarized light to other two-way incident lights, the rotation of half-wave plate is controlled by computer.
Preferably, it has been sequentially placed between the dichroscope and industrial camera：Filter plate, for filtering off sample to be tested hair
Veiling glare in the fluorescence signal gone out；And plus lens, the fluorescence signal for sample to be tested to be sent are imaged onto industrial camera
On.
It is as follows using a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device operating process based on double galvanometers：
1) laser beam is divided into the linearly polarized light of three curb different directions propagation, wherein light is directly incident on sample all the way
On, in addition two-way light is incided on sample by galvanometer, and three-beam interferes in sample interior and produces three-dimensional structure optical illumination
Pattern；
2) structure light pattern excitation sample sends fluorescence, and the fluorescence signal sent by industrial camera collection sample, obtains
Fluorescence intensity information；
3) rotation structured light illumination pattern direction successively, while the phase of structure light pattern is varied multiple times under all directions,
Obtain several fluorescence intensity images under the corresponding phase of all directions；
4) mobile example platform carries out sample the information at axial scan acquisition sample different depth；
5) data processing is carried out to several obtained fluorescence intensity images, reconstructs the two dimension obtained at sample different depth and surpass
Resolution image.
6) several two-dimensional super-resolution images are reconstructed to obtain the three-dimensional super-resolution image of object.
In step 3), the direction of rotational structure optical pattern at least under three angles, changes at least five times under all directions
The phase of structure changes optical pattern.
In the present invention, single width structure light pattern is projected in the fluorescence intensity information obtained on sample comprising five frequencies point
Amount, in order to separate this five frequency components, it is necessary to obtain five equations.Common Structured Illumination microtechnic passes through mobile light
Grid obtain five equations, and the present invention controls interfere arm speculum repeatedly to be moved by piezoelectric ceramics, and change projects sample
On structure light pattern phase, several dephased fluoroscopic images are obtained, so that each frequency component is extracted and moved.
Further, in order to all realize super-resolution imaging in each direction, it is also necessary to structure light pattern is carried out multiple
Rotation.Common Structured Illumination microtechnic realizes the rotation of structure light pattern by rotating grating, and the present invention passes through galvanometer
Control interfering beam is scanned, so as to change the direction of the structure light pattern projected on sample, obtains postrotational fluorescence
Image, realizes the super-resolution imaging of all directions.
Preferably, respectively in the direction of 0 °, 60 ° and 120 ° rotational structure optical pattern, and control structure optical pattern phase respectively
Move 0 °, 72 °, 144 °, 216 °, 288 °.It is only limitted to herein as optimal example, in theory, direction of rotation and phase shift
Angle can be arbitrary numerical value, meet each direction of rotation and phase shift angle difference；Alternatively, it is also possible to increase rotation side
To the number with phase shift angle, it can also realize the technique effect of the invention to be reached and solve proposed technical problem.
Preferably, when axial scan acquisition sample three-dimensional information is carried out to sample, its axial scan spacing and number need
Determined according to specific sample characteristics of for example and combination nyquist sampling theorem, can generally choose axial scan spacing 100nm or so,
Scanning times 10 times or so.
Compared with prior art, the present invention has technique effect beneficial below：
(1) grating mechanical scanning and movement are replaced using vibration mirror scanning and piezoelectric ceramics movement, realizes Structured Illumination figure
The rotation and phase shift of sample, reduce experimental error, the microscopical image taking speed of Structured Illumination are improved, suitable for three-dimensional live body
Imaging；
(2) mechanical rotation for replacing linear polarizer using tangential light polarization converter ensures structure light pattern contrast most
Height, on the one hand further increases image taking speed, on the other hand avoids error caused by mechanical rotation possibility；
(3) replace optical grating diffraction to obtain three beams coherent light using beam splitter, improve incident laser energy utilization rate；
(4) three-dimensional super-resolution is realized, especially suitable for carrying out live body three-dimensional imaging to fluorescent samples.
Brief description of the drawings
Fig. 1 is a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device schematic diagram based on double galvanometers of the present invention；
Fig. 2 is the tangential light polarization converter structure schematic diagram that the present apparatus uses；
Fig. 3 is the three-dimensional structure optical pattern schematic diagram that the present apparatus is produced in sample interior, wherein, (a) figure is three-dimensional structure
Optical pattern transverse direction schematic diagram, (b) figure are three-dimensional structure optical pattern axial direction schematic diagram.
Fig. 4 shows for a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device operating process based on double galvanometers of the present invention
It is intended to.
Embodiment
With reference to embodiment and attached drawing, the present invention will be described in detail, but the present invention is not limited to this.
Three-dimensional structure optical illumination super-resolution microscopic imaging device as shown in Figure 1, including：Laser 1, single mode optical fiber 2, rise
Inclined device 3, the first half-wave plate 4, the first beam splitter 5, the second beam splitter 6, the second half-wave plate 7, the first convex lens 8, the second convex lens
9th, the first galvanometer system 10, the first tangential light polarization converter 11, the first plane mirror 12, the second galvanometer system 13, second
Tangential light polarization converter 14, the second plane mirror 15, piezoelectric ceramics 16, scanning lens 17, microcobjective 18, fluorescence to be measured
Sample 19, dichroscope 20, filter plate 21, plus lens 22, industrial camera 23 and computer 24.
Laser 1 sends laser beam, single mode optical fiber 2, the polarizer 3, the first half-wave plate 4,5, second points of the first beam splitter
Beam mirror 6, the second half-wave plate 7, the first convex lens 8 and the second convex lens 9 are placed sequentially on the optical axis of input path.Single mode optical fiber
2 are used to be filtered laser beam.The polarizer 3 is used to emergent light being converted into linearly polarized light.First half-wave plate 4 is used to control
Linearly polarized light incides the direction on beam splitter.First beam splitter 5 and the second beam splitter 6 are used to incident light being divided into three tunnels.The
Two half-wave plates 7 are used to ensure that the light beam of normal incidence light path to be always tangential linearly polarized light.First convex lens 8 and the second convex lens 9
Focal plane overlaps but focal length is different, and image planes can be shifted, while carries out shrink beam to light beam.
The other two-way incident light that first beam splitter 5 and the second beam splitter 6 separate is respectively enterd by the first galvanometer system 10
With the second galvanometer system 13.Two sets of galvanometer systems are made of two galvanometers and two convex lenses, the placement position of four optical elements
Put needs and ensure object-image relation with subsequent optical path, two convex lens focal planes overlap but focal length is different, can carry out shrink beam to light beam.Two
A galvanometer system controls it to be scanned by computer 24.Galvanometer system can also use other structures, such as reflective four mirrors system
System or reflective three-mirror system etc..The focal plane of two galvanometer systems is placed with tangential light polarization converter 11 and tangential respectively
Light polarization converter 14, its structure is as shown in Fig. 2, linear polarization for incident ray polarized light to be converted into two beam tangential polarizations
Light, ensures the Structured Illumination pattern contrast highest to be formed.
The tangential linearly polarized light of two beams come out from the first galvanometer system 10 and the second galvanometer system 13 passes through plane mirror
Tangential linearly polarized light behind 12 and 15 change paths directions with central normal incidence light path passes through sweeping for coaxial placement successively again
Lens 17 and microcobjective 18 are retouched, are incided on fluorescent samples 19 to be measured.The side focal plane of scanning lens 17 and lens 9 and shake
The focal plane of mirror system overlaps, and opposite side focal plane is overlapped with the entrance pupil face of microcobjective 18, for shifting object-image relation, improves
Vibration mirror scanning edge light beam incides the ability in object lens entrance pupil face.Plane mirror 15 passes through its position of 16 drive control of piezoelectric ceramics
Move, change the optical path difference of incident light, realize the phase shift of Structured Illumination pattern.Microcobjective 18 is used to focus on three beams incident light
Interfered at 19 same position of sample to be tested and produce three-dimensional structure light optical illumination pattern, as shown in figure 3, excitation sample is sent
Fluorescence.Sample to be tested 19 is placed on sample stage, is controlled it to move axially by computer 24, is obtained sample different depth
The information at place is used to reconstruct three-dimensional super-resolution image.
The fluorescence signal that sample to be tested 19 is sent passes sequentially through microcobjective 18, dichroscope 20, filter plate 21 and convergence
Lens 22, into industrial camera 23.Dichroscope 20 is used to transmit incident light, reflected fluorescent light.Filter plate 21 is used to filter off to be measured
Veiling glare in the fluorescence signal that sample 19 is sent.Plus lens 22 is used to fluorescence signal focusing on industrial camera 23.
Computer 24 is used to control the rotation of half-wave plate 7 to ensure that normal incident light polarization direction remains tangential, control galvanometer
The scanning of system 10 and 14 changes the direction of Structured Illumination pattern, 16 displacement of control piezoelectric ceramics changes Structured Illumination pattern
Phase, control sample stage 19 carry out sample axial scan and obtain three-dimensional information and control industrial camera 23 to gather what sample was sent
Fluorescence intensity signals, while data processing and phase reconstruction are carried out by algorithm, obtain the two and three dimensions super-resolution figure of sample
Picture.
As shown in figure 4, a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device workflow based on double galvanometers is such as
Under：
The laser beam that laser 1 is sent is incided on the first beam splitter 5 and the second beam splitter 6, resolves into three beams vibration
The orthogonal linearly polarized light in direction enters, and is imaged onto pupil face after microcobjective 18, focuses on the sample to be tested 19 with fluorescent marker
On interfere produce striated structure optical pattern.Structured Illumination pattern excitation sample to be tested 19 sends fluorescence signal by micro- thing
Mirror 18 collects, and filtered wave plate 21 filters off the veiling glare in the fluorescence being collected into, and is then imaged onto CMOS industrial cameras 23
On, obtain the fluorescence intensity distributed intelligence inside sample to be tested 19.
Single width three-dimensional structure optical pattern is projected in the fluorescence intensity information obtained on sample comprising five independent frequencies point
Amount, so needing to obtain five image equations to separate this five frequency components, passes through 16 control plane speculum of piezoelectric ceramics
15 are moved so that project 0 ° of Structured Illumination pattern phase shift on sample to be tested 19,72 °, 144 °, 216 °, 288 °, obtain
To five dephased fluoroscopic images, so that each frequency component is extracted and moved, the high-frequency information of sample is obtained.
The frequency component obtained after phase shift is only distributed in the horizontal direction, in order to all realize super-resolution in all directions, it is necessary to
Galvanometer system 10 and 13 is controlled to be scanned using computer 24 so as to project the direction rotation of the structure light pattern on sample 19
Turn 0 °, 60 °, 120 °, obtain the fluorescence intensity image under respective angles, obtain each side at 19 image space of sample at this time
Upward information.
In order to obtain the three-dimensional information of sample 19, it is also necessary to control sample stage movement to carry out axial scan to sample 19, its
Axial scan spacing and number need to be determined according to specific sample characteristics of for example, can generally choose axial scan spacing 100nm or so,
Scanning times 10 times or so.
Data processing is finally carried out to several obtained fluorescence intensity images by computer 24 again, reconstruct obtains sample 19
Two and three dimensions super resolution image.
The foregoing is merely the preferable implementation example of the present invention, be not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on, should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of three-dimensional structure optical illumination super-resolution microscopic imaging device based on double galvanometers, including send the laser of laser beam
Device, it is characterised in that：
Including what is be sequentially arranged along the light path of the laser：
The laser beam, is divided into the incident light being concerned with three tunnels by beam splitting component；
Galvanometer scanning system, is separately mounted in two input paths, and a wherein input path is equipped with by mobile to change light
The speculum of path difference；
Microcobjective, interference generation three-dimensional structure optical illumination pattern is carried out for three-beam to be incided on sample, and collects sample
The fluorescence intensity signals that product are sent；
A computer is further included, for controlling the speculum movement and galvanometer scanning system to be scanned, makes structure light
Illumination pattern carries out multiple phase shift and direction rotates, and carries out data to several postrotational fluorescence intensity images of phase shift and direction
Processing, reconstruct obtain super resolution image.
2. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that the laser
Device has been sequentially placed with beam splitting inter-module：
Single mode optical fiber, for being filtered to laser beam；
The polarizer, for laser beam to be changed into linearly polarized light；
With the first half-wave plate, the linearly polarized light direction on beam splitting component is incided for control.
3. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that the beam splitting
Component includes the first beam splitter and the second beam splitter on the transmitted light path of first beam splitter.
4. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that the galvanometer
Scanning system is transmission-type or reflective galvanometer system, and inside is placed with a tangential light polarization plate converter, for rotating two-way
The polarization direction of linearly polarized light so that the Structured Illumination pattern contrast of generation is maximum.
5. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that described three
In the incident light of road, installation is incident by the road by rotating second half-wave plate of computer control in remaining incident light all the way
Light rotates to be tangential linearly polarized light.
6. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 5, it is characterised in that described second
The first convex lens and the second convex lens that focal plane overlaps but focal length is different are disposed between half-wave plate and microcobjective, for turning
Move image planes and shrink beam.
7. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that the reflection
Mirror is moved by Piezoelectric Ceramic, the signal which sends according to computer, changes the optical path difference of input path
Structured Illumination pattern is set to produce phase shift.
8. such as claim 1~7 any one of them three-dimensional structure optical illumination super-resolution microscopic imaging device, it is characterised in that
The computer control speculum is at least five times mobile, at least five dephased fluorescence intensity images is obtained, to each fluorescence
The frequency component of intensity image is extracted and moved, and obtains the high-frequency information of sample；
Computer controls the galvanometer scanning system to be scanned sample so that projects structure light pattern on sample at least
Direction with three different angles rotates, and obtains the fluorescence intensity image under different angle, obtains each at sample image space
Information on a direction.
9. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 8, it is characterised in that dephased glimmering
Light intensity image is the image that gathers after 0 °, 72 °, 144 °, 216 ° and 288 ° of Structured Illumination pattern phase shift；
Fluorescence intensity image under different angle rotates the figure gathered after 0 °, 60 ° and 120 ° for the direction of Structured Illumination pattern
Picture.
10. three-dimensional structure optical illumination super-resolution microscopic imaging device as claimed in claim 1, it is characterised in that further include and hold
The sample stage of the sample is carried, the sample stage drives sample to be moved along optical axis direction, for obtaining the three-dimensional information of sample.
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