CN110989313B - Holographic microscopic imaging device - Google Patents
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- CN110989313B CN110989313B CN201911131752.0A CN201911131752A CN110989313B CN 110989313 B CN110989313 B CN 110989313B CN 201911131752 A CN201911131752 A CN 201911131752A CN 110989313 B CN110989313 B CN 110989313B
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- 238000003384 imaging method Methods 0.000 title claims abstract description 49
- 230000010287 polarization Effects 0.000 claims abstract description 60
- 238000005259 measurement Methods 0.000 claims abstract description 25
- 230000003287 optical effect Effects 0.000 claims abstract description 18
- 230000008878 coupling Effects 0.000 claims abstract description 9
- 238000010168 coupling process Methods 0.000 claims abstract description 9
- 238000005859 coupling reaction Methods 0.000 claims abstract description 9
- 239000002131 composite material Substances 0.000 claims abstract description 6
- 230000010365 information processing Effects 0.000 claims description 3
- 238000000386 microscopy Methods 0.000 claims 2
- 230000003321 amplification Effects 0.000 claims 1
- 238000003199 nucleic acid amplification method Methods 0.000 claims 1
- 238000000799 fluorescence microscopy Methods 0.000 abstract description 2
- 230000031700 light absorption Effects 0.000 abstract description 2
- 230000001988 toxicity Effects 0.000 abstract description 2
- 231100000419 toxicity Toxicity 0.000 abstract description 2
- 239000007844 bleaching agent Substances 0.000 abstract 1
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- 239000004973 liquid crystal related substance Substances 0.000 description 1
- 229910044991 metal oxide Inorganic materials 0.000 description 1
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/0005—Adaptation of holography to specific applications
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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
- G02—OPTICS
- G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
- G02B21/00—Microscopes
- G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
- G02B21/361—Optical details, e.g. image relay to the camera or image sensor
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/04—Processes or apparatus for producing holograms
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/0005—Adaptation of holography to specific applications
- G03H2001/005—Adaptation of holography to specific applications in microscopy, e.g. digital holographic microscope [DHM]
Abstract
An apparatus for realizing holographic microscopic imaging, which comprises an inverted microscope and a holographic imaging device, wherein: the inverted microscope is provided with a light path inlet and a light path outlet, an object to be observed is illuminated by a linear polarized laser bright field, light carrying information of the object to be observed enters the inverted microscope through the light path inlet, a holographic image of the object after being amplified is obtained in the inverted microscope, and the light path outlet of the inverted microscope is connected with the light path inlet of the holographic imaging device; the holographic imaging device is provided with an optical path inlet and an optical path outlet and is used for coupling the transverse mode and the polarization of an optical field and carrying out composite measurement. The quantitative phase imaging realizing device provided by the invention uses an imaging method of a non-labeled specimen, does not generate light absorption caused by fluorescence imaging to bleach the light of a molecular structure, overcomes the problem of toxicity of exciting light to biological cells, and can be used for observing the evolution of living cells.
Description
Technical Field
The invention relates to the technical field of microscopic imaging technology, interference imaging and phase imaging, in particular to a holographic microscopic imaging device.
Background
The optical field interacts with the non-homogeneous medium by modulating the wavefront and amplitude of the light, a process known as scattering of the light. Generally only elastic scattering is considered, i.e. the wavelength of the light after scattering is unchanged. Because the amplitude and phase of the scattered light carry the internal structure information of the scattering object, the structure of the object can be recovered by measuring the scattered field, and the process is called as the solution of the backscattering problem. This is the basis for tomographic imaging of transparent biological tissues or cells. The long-felt need for tomographic imaging of unlabeled three-dimensional living specimens has led to new phase imaging techniques, since intracellular tissue has many highly dynamic structures that are difficult to study using labeling methods.
Imaging of thin transparent multiple-scattered samples has been a difficult problem in optical imaging, in that multiple scattering creates an incoherent background that ultimately results in reduced image contrast, even for conventional quantitative phase imaging.
Disclosure of Invention
Technical problem to be solved
The invention mainly aims to provide a holographic microscopic imaging device for realizing holographic microscopic imaging of a phase-type object.
(II) technical scheme
An apparatus for realizing holographic microscopic imaging, comprising: inverted microscope and holographic imaging apparatus, wherein:
the inverted microscope is provided with a light path inlet and a light path outlet, light rays emitted by the linear polarization laser bright field enter the light path inlet, the light path outlet of the inverted microscope is connected with the light path inlet of the holographic imaging device, and the inverted microscope is used for obtaining a holographic image of an object after being amplified;
the holographic imaging device is provided with an optical path inlet and an optical path outlet and is used for coupling the transverse mode and the polarization of an optical field and carrying out composite measurement.
In the above aspect, the inverted microscope includes: the device comprises a linear polaroid, a first beam expanding lens, a second beam expanding lens and a reflector, wherein the linear polaroid, the first beam expanding lens, the second beam expanding lens and the reflector are sequentially arranged along the direction of a light path.
In the above aspect, the hologram imaging apparatus includes: a polarization evolution module and a polarization measurement module, wherein:
the polarization evolution module is provided with a light path inlet and a light path outlet and is used for coupling the light field containing the information of the object to be detected with polarization;
and the polarization measurement module is provided with a light path inlet, and the light path inlet of the polarization measurement module is connected with the light path outlet of the polarization evolution module and is used for measuring the light field containing the information of the object to be measured.
In the above scheme, the polarization evolution module comprises a first fourier lens, a second fourier lens and a spatial light modulator, the first fourier lens and the second fourier lens form a linear optical information processing system, the spatial light modulator is located on a confocal plane of the first fourier lens and the second fourier lens, and the first fourier lens, the spatial light modulator and the second fourier lens are sequentially arranged in the device sequence according to the light path direction.
In the above solution, the first fourier lens is used to transform the light beam from x, y spatial distribution to momentum space;
the spatial light modulator is used for rotating the light polarization of a p-0 component in a momentum space by any angle;
the second fourier lens is used to transform the beam from the momentum space to the x, y space.
In the above scheme, the polarization measurement module comprises a half-wave plate, a quarter-wave plate, a polarization beam splitter and a photosensitive device, and the quarter-wave plate, the half-wave plate and the polarization beam splitter are arranged in sequence according to the direction of the light path.
In the scheme, the half-wave plate and the quarter-wave plate are used for obtaining intensity images of light beams under six groups of measurement base diagonal angles, reverse diagonal angles, left-handed rotation, right-handed rotation, horizontal rotation and vertical rotation;
the polarization beam splitter is used for changing the phase of the light beam to realize the polarization rotation of the zero momentum component;
the photosensitive device is used for acquiring images.
The light sensing device may be a camera such as a CCD or a CMOS.
(III) advantageous effects
1. The quantitative phase imaging realizing device provided by the invention uses an imaging method of a non-labeled specimen, so that the photobleaching of a molecular structure due to light absorption caused by fluorescence imaging is avoided, the problem of toxicity of exciting light to biological cells is solved, and the quantitative phase imaging realizing device can be used for observing the evolution of living cells.
2. The quantitative phase imaging realizing device provided by the invention can effectively inhibit background scattering, so that the imaging result has high contrast, and the device can be used for observing biological structures such as a small amount of or a single unmarked cell.
3. The quantitative phase imaging realization device provided by the invention has the advantage of common light path design, so that the imaging has extremely high spatial and temporal coherence stability.
4. The quantitative phase imaging device provided by the invention uses a low-coherence light source as the illumination light source, and can effectively inhibit laser speckles.
5. The quantitative phase imaging realizing device provided by the invention can carry out real-time measurement and monitor the dynamic change of a target object.
Drawings
Fig. 1 is a schematic structural diagram of a quantitative phase imaging apparatus according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to specific embodiments and the accompanying drawings.
As shown in fig. 1, fig. 1 is a schematic structural diagram of a holographic microscopic imaging apparatus provided in an embodiment of the present invention, and the apparatus includes: an inverted microscope a and a holographic imaging device b. The inverted microscope a is provided with a light path inlet and a light path outlet, light rays emitted by a linear polarization laser bright field enter the light path inlet and irradiate an object 2 to be detected through a linear polaroid 1, a holographic image of the object after being amplified is obtained on the inverted microscope a, and the light path outlet of the inverted microscope a is connected with the light path inlet of the holographic imaging device b. The inverted microscope a includes: the device comprises a linear polaroid 1, a first beam expanding lens 3, a second beam expanding lens 4 and a reflecting mirror 5, wherein the linear polaroid 1, the first beam expanding lens 3, the second beam expanding lens 4 and the reflecting mirror 5 are arranged in sequence along the direction of a light path.
The linear polarizer 1, the first beam expanding lens 3 and the second beam expanding lens 4 are used for expanding incident light;
the reflector 5 is used for reflecting the expanded light.
The holographic imaging device b is provided with an optical path inlet and an optical path outlet and is used for coupling the transverse mode and the polarization of an optical field and carrying out composite measurement, and the holographic imaging device b comprises: the polarization evolution module and the polarization measurement module.
The polarization evolution module is provided with a light path inlet and a light path outlet and is used for coupling a light field containing information of an object to be detected and polarization, the polarization evolution module comprises a first Fourier lens 6, a second Fourier lens 8, a spatial light modulator 7 or a liquid crystal phase plate and other polarization devices with controllable pixels, the first Fourier lens and the second Fourier lens form a linear optical information processing system, the spatial light modulator 7 is positioned on a confocal plane of the first Fourier lens 6 and the second Fourier lens 8, and the first Fourier lens 6, the spatial light modulator 7 and the second Fourier lens 8 are sequentially arranged according to the direction of the light path. Wherein the first Fourier lens is used for transforming the light beam from an x, y spatial distribution to a momentum space; the spatial light modulator is used for rotating the light polarization of a p-0 component in the momentum space by any angle; the second fourier lens is used to transform the beam from the momentum space to the x, y space.
The polarization measurement module is provided with a light path inlet, the light path inlet of the polarization measurement module is connected with a light path outlet of the polarization evolution module and used for measuring a light field containing information of an object to be measured, and the polarization measurement module comprises a half-wave plate 10, a quarter-wave plate 9, a polarization beam splitter 11 and a photosensitive device 12, wherein the photosensitive device can be a camera such as a CCD (charge coupled device) or a CMOS (complementary metal oxide semiconductor) and the like. The device is sequentially a quarter wave plate 9, a half wave plate 10, a polarizing beam splitter 11 and a photosensitive device 12 according to the direction of the optical path. The imaging light path firstly passes through the polarization evolution module and then enters the polarization measurement module. The half-wave plate and the quarter-wave plate are used for obtaining intensity images of light beams under six groups of measurement base diagonal angles, reverse diagonal angles, left-handed rotation, right-handed rotation, horizontal rotation and vertical rotation; the polarization beam splitter is used for changing the phase of the light beam to realize the polarization rotation of the zero momentum component; the photosensitive device is used for acquiring images.
After the sample is imaged by the microscope, the x, y spatial distribution of the light beam is transformed into a distribution of momentum space by a first Fourier lens. On the back focal plane of the first fourier lens, the spatial light modulator polarizes light of a portion where the momentum p is 0 by the rotation angle θ. The specific implementation method is to load proper gray scale images on a plurality of pixels at the central position of a light beam on the spatial light modulator, and change the phase of the light beam to realize the polarization rotation of the zero momentum component. After passing through a linear polarizer, the light beam is transformed to x and y space through a first Fourier lens, enters a polarization measurement module, the angle of a half-wave plate and a quarter-wave plate is appropriately changed, the measurement of base diagonal, left-handed, right-handed, horizontal and vertical measurement bases is carried out, the image result obtained on a CCD is extracted, the real part and the imaginary part of a wave function can be calculated, and the phase distribution diagram of an observed object is calculated according to theta (x, y) ═ arctan (Re psi/Im psi).
The principle of the present device is described below in a quantitative manner.
Considering the distribution of the beam in the x-y plane as the system state | ψ, assuming that the illumination beam propagates along the z-axis>s=∫ψ(x,y)|x,y>dxdy, where ψ (x, y) is the wave function of the light field to be measured, and the index state is the polarization of the photon, where the initial state of the polarization is|V>Respectively representing horizontal polarization and vertical polarization. Performing unitary evolution after the system state and the pointer state are coupled, wherein the composite state is written as follows:
|Ψ>SP=U(θ)|ψ>|si>
wherein|si>Is the initial state of the pointer state, theta is the coupling strength,is { |0>,|1>And U (theta) represents the evolution operation of the composite state of the coupling of the polarization pointer and the two-dimensional space wave function. The position is measured next:
<x|ψ>|sf>=<x|U(θ)|ψ>|si>
< x | ψ > is the function to be measured, and the pointer state is:
is a weak value, and makes a polarization measurement on the end state, | sf>Representing the complex state | Ψ>SPPost-selection of the location space is performed and the resulting final state is normalized.
Can be selected by an appropriate initial pointer state (e.g. | s)i>=|H>,|H>Representing horizontal polarization) such that<si|σ1|si>=0,<si|σ2|si>0, so do1,σ2,σ3After the measurement
Obtaining a function to be measured
It can be seen that the real part of the function to be measured is inversely proportional to σ1,σ3The measured value, the imaginary part, is inversely proportional to σ2And (6) measuring the values.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention, and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (5)
1. An apparatus for realizing holographic microscopic imaging, which is characterized by comprising: inverted microscope (a) and holographic imaging device (b), wherein:
the inverted microscope (a) is provided with a light path inlet and a light path outlet, light rays emitted by a linear polarization laser bright field enter the light path inlet, the light path outlet of the inverted microscope (a) is connected with the light path inlet of the holographic imaging device (b), and the inverted microscope (a) is used for obtaining a holographic image of an object after amplification;
the holographic imaging device (b) is provided with an optical path inlet and an optical path outlet and is used for coupling the transverse mode and the polarization of an optical field and carrying out composite measurement;
wherein the holographic imaging device (b) comprises: a polarization evolution module and a polarization measurement module, wherein,
the polarization evolution module is provided with a light path inlet and a light path outlet and is used for coupling the light field containing the information of the object to be detected with polarization;
the polarization measurement module is provided with a light path inlet, and the light path inlet of the polarization measurement module is connected with the light path outlet of the polarization evolution module and is used for measuring a light field containing information of an object to be measured;
the polarization evolution module comprises a first Fourier lens, a second Fourier lens and a spatial light modulator, wherein the first Fourier lens and the second Fourier lens form a linear optical information processing system, the spatial light modulator is positioned on a confocal plane of the first Fourier lens and the second Fourier lens, and the first Fourier lens, the spatial light modulator and the second Fourier lens are sequentially arranged as devices according to the direction of a light path;
wherein the first Fourier lens is used to transform the beam from an x, y spatial distribution to a momentum space;
the spatial light modulator is used for rotating the light polarization of a p-0 component in a momentum space by any angle;
the second fourier lens is used to transform the beam from the momentum space to the x, y space.
2. The holographic microscopy imaging realisation device according to claim 1, characterised in that said inverted microscope (a) comprises: the device comprises a linear polaroid, a first beam expanding lens, a second beam expanding lens and a reflector, wherein the linear polaroid, the first beam expanding lens, the second beam expanding lens and the reflector are sequentially arranged along the direction of a light path.
3. The holographic microscopic imaging implementing apparatus of claim 1, wherein the polarization measuring module comprises a half-wave plate, a quarter-wave plate, a polarization beam splitter and a photosensitive device, and the devices are sequentially the quarter-wave plate, the half-wave plate and the polarization beam splitter according to the optical path direction.
4. The holographic microscopic imaging realization apparatus of claim 3, wherein the half wave plate and the quarter wave plate are used to obtain intensity images of light beams in six sets of measurement base diagonal, anti-diagonal, left-handed, right-handed, horizontal, and vertical;
the polarization beam splitter is used for changing the phase of the light beam to realize the polarization rotation of the zero momentum component;
the photosensitive device is used for acquiring images.
5. The holographic microscopy imaging realization apparatus as claimed in claim 3, wherein the light sensing device can be a CCD or CMOS camera.
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