CN114324245B - Quantitative phase microscopic device and method based on partially coherent structured light illumination - Google Patents
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Abstract
The invention discloses a quantitative phase microscopic device and a quantitative phase microscopic method based on partial coherent structured light illumination, wherein the device comprises an annular light source, a structured light modulation module, an illumination objective lens, a detection objective lens, a spatial light modulation module and an image acquisition module which are sequentially arranged along the direction of a light path, wherein the annular light source is used for generating partial coherent annular light beams; the structural light modulation module is used for carrying out structural modulation on the annular light beam to obtain + -1-order diffraction light; the sample is arranged between the illumination objective and the detection objective, and generates non-scattered light and scattered light carrying sample information under irradiation of + -1-level diffraction light; the spatial light modulation module is used for carrying out phase modulation on the non-scattered light so as to obtain a plurality of structured interference phase shift diagrams of the sample, and the image acquisition module is used for acquiring the structured interference phase shift diagrams. The invention can carry out high-resolution label-free detection on various scattering samples, and has wide application prospect in the fields of biomedicine and the like.
Description
Technical Field
The invention belongs to the technical field of optical microscopic imaging, and particularly relates to a quantitative phase microscopic device and method based on partial coherent structured light illumination, which are used for carrying out high-resolution label-free detection on a thicker scattering sample.
Background
Optical microscopy has a great advantage as a non-invasive imaging technique for the detection of living samples, and therefore plays an important role in the field of life science research. Most biological samples are translucent or fully transparent and conventional optical bright field microscopes cannot detect their internal structure. The fluorescence microscope can carry out high-resolution and high-contrast selective imaging on a transparent sample, the spatial resolution also reaches tens of nanometers, and a powerful technical means is provided for the research of the life process. However, fluorescence microscopy has many application limitations. First, fluorescent markers can adversely affect organelles within living cells, and long-term or high-frequency fluorescence excitation can cause cell death; secondly, due to the limitation of fluorescent markers and optical devices, the number of organelles capable of being stably marked is three to four, and complex life processes in living cells cannot be comprehensively detected by using a fluorescent microscope; in addition, the fluorescent substance generates peroxy ions when excited, has phototoxicity to living cells, and has photobleaching property when excited. Therefore, it is difficult to perform long-term dynamic observation of a living sample in a natural state with a fluorescence microscope.
Compared with a fluorescence microscope, the quantitative phase microscopic imaging technology can perform high-contrast and quantitative phase imaging on transparent organelles and substances in living cells without fluorescent markers, and can obtain information such as the structure and refractive index distribution of a sample. The digital holographic microscopy technology is a classical label-free quantitative phase microscopy imaging method, and the amplitude and phase modulation function of the scattered sample to the illumination light wave can be quantitatively obtained only through single data acquisition. However, in order to improve the spatial resolution, the same sample needs to be subjected to annular scanning illumination, so that the imaging speed is greatly reduced, and the influence of environmental disturbance and laser speckles on the image quality can be reduced by relatively complicated image processing in the later period. Scientific researchers further put forward object-reference common-path digital holographic microscopy based on coaxial point diffraction and point diffraction digital holographic microscopy based on polarization diffraction grating, however, the illumination light sources in the devices are still lasers with very high coherence, the image quality is extremely easily adversely affected by laser speckles, and the spatial resolution and the time resolution are still mutually restricted.
Diffraction quantitative phase microscopy based on iterative computation has been developed rapidly due to low cost. The system structure of the technology is very simple, and the structural information of the transparent sample can be quantitatively obtained only by carrying out a series of iterative computation on the diffraction image obtained by recording. Stacked diffraction imaging (Ptychographic Iterative Engine, PIE) and fourier stacked microscopy (Fourier Ptychographic Microscope, FPM) are the most representative two diffraction quantitative phase microscopy techniques that integrate optical synthetic aperture and phase recovery, greatly increasing the spatial bandwidth product of optical microscopy imaging, allowing high spatial resolution over a large field of view. However, the diffraction quantitative phase microscopy technology based on iterative computation needs to collect tens of original diffraction images to realize single high-resolution phase reconstruction, and no report on the rapid biodynamic process research by using the technology exists at present. In addition, the spatial resolution and image contrast of this type of technique is not sufficient to detect fine structures within living cells, and no marker-free detection of organelles using this type of technique has been reported. Scientific researchers put forward a multi-layer diffraction model on the basis of the technology to obtain a three-dimensional structure of a sample, however, various approximations obtained in the modeling process greatly reduce the spatial resolution of an image, and a complex processing process also brings about a serious artifact structure. The gradient light interference quantitative phase microscopy is established on a differential interference microscope, has two structures of transmission type and reflection type, and can perform three-dimensional imaging on thicker biological tissues. However, when the phase recovery is performed by using this type of technique, the integration operation needs to be performed on the acquired original image, so that the technique is only suitable for samples that continuously change. In addition, the spatial resolution of this type of technique is not yet sufficient to detect the fine structure of subcellular organelles within living cells, etc.
Quantitative phase microscopy based on phase contrast has significant advantages in the coordination of spatial resolution and temporal resolution. Taewoo et al first proposed a spatial light interference microscope that combines a fresnel phase contrast microscope and a phase shift algorithm, and achieved a lateral resolution of 350 nanometers at a time resolution of 16 frames per second. The technology has an optical structure of common-path interference, so that external disturbance does not influence the measurement process. In addition, the light source used in the technology is an extended light source with a certain spectral range, so that speckle noise caused by a laser and other related light sources is avoided, and the image quality is greatly improved. Further, researchers have proposed super-oblique illumination quantitative phase microscopy based on annular led illumination, which achieves a lateral resolution of 270 nm at an imaging speed of 250 frames per second. However, this type of technique is only suitable for detecting relatively thin weakly scattering samples, and cannot perform high-resolution three-dimensional imaging on thicker tissues, and even cannot perform high-contrast imaging on living cells with poor adhesion.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a quantitative phase microscopic device and a quantitative phase microscopic method based on partial coherent structured light illumination. The technical problems to be solved by the invention are realized by the following technical scheme:
The invention provides a quantitative phase microscopic device based on partial coherent structured light illumination, which comprises an annular light source, a structured light modulation module, an illumination objective lens, a detection objective lens, a spatial light modulation module and an image acquisition module which are sequentially arranged along the direction of a light path,
the ring light source is used for generating a partially coherent ring light beam;
the structural light modulation module is used for carrying out structural modulation on the annular light beam to obtain + -1-level diffraction light;
the sample is irradiated by the + -1-level diffraction light to generate non-scattered light and scattered light carrying sample information;
the spatial light modulation module is used for carrying out phase modulation on the non-scattered light so as to obtain a plurality of structured interference phase shift diagrams of the sample, and the image acquisition module is used for acquiring the structured interference phase shift diagrams.
In one embodiment of the invention, the annular light source is composed of a plurality of light emitting diodes which are uniformly distributed on an annular light source framework.
In one embodiment of the present invention, the structural light modulation module includes an industrial lens, a first thin lens, a first beam splitter, an optical diffraction unit, a first barrel lens, and a spatial filter mask sequentially disposed in an optical path direction, wherein,
The industrial lens and the first thin lens are used for zooming and converging the annular light beam;
the first beam splitter is obliquely arranged between the first thin lens and the optical diffraction unit, illumination light from the first thin lens is transmitted to the optical diffraction unit through the first beam splitter, and the optical diffraction unit can load a series of binarized fringe patterns with different direction angles to perform structural modulation on the illumination light to form 0-order diffraction light and +/-1-order diffraction light; the first beam splitter is further used for reflecting the 0 th-order diffraction light and the +/-1 st-order diffraction light to a first lens barrel lens, and the spatial filtering mask is arranged at a confocal surface of the first lens barrel lens and the illumination objective lens and used for shielding the 0 th-order diffraction light.
In one embodiment of the present invention, the working surface of the optical diffraction unit is located at a confocal surface of the first tube lens and the first thin lens.
In one embodiment of the invention, the optical diffraction unit is any one of a digital micromirror array, a reflective spatial light modulator, a transmissive spatial light modulator, a reflective diffraction grating, or a transmissive diffraction grating.
In one embodiment of the present invention, the spatial light modulation module includes a linear polarizer, a second tube lens, a dichroic mirror, a second thin lens, a second beam splitter, a spatial light modulator, and a third thin lens sequentially disposed in a light traveling direction, wherein,
the linear polarizer is used for adjusting the scattered light and the non-scattered light into linearly polarized light;
the second lens barrel lens and the second thin lens form a confocal system, and the dichroic mirror is used for reflecting light rays from the second lens barrel lens to the second thin lens;
the second beam splitter is obliquely arranged between the second thin lens and the spatial light modulator, light rays from the second thin lens are transmitted to the spatial light modulator through the second beam splitter, the spatial light modulator can perform phase modulation on the non-scattered light, and the modulated non-scattered light is reflected to the third thin lens through the second beam splitter;
the second thin lens and the third thin lens form a confocal system, the spatial light modulator is positioned at the confocal surface of the second thin lens and the confocal surface of the third thin lens, and the image acquisition module is arranged at the back focal surface of the third thin lens.
In one embodiment of the present invention, the quantitative phase microscopy apparatus further comprises a fluorescence excitation detection module disposed on a side of the dichroic mirror remote from the second tube lens.
In another aspect, the invention provides a quantitative phase microscopy method based on partially coherent structured light illumination, comprising:
s1: obtaining a plurality of structured interferometric phase-shift maps of the sample using the quantitative phase microscopy device based on partially coherent structured light illumination of any of the above embodiments;
s2: and calculating and obtaining phase information of the sample according to a plurality of the structured interference phase shift maps.
In one embodiment of the present invention, the S1 includes:
s11: turning on the annular light source to generate annular illumination light;
s12: loading a series of binarized fringe patterns with different direction angles through an optical diffraction unit to perform structured modulation on the annular illumination light;
s13: performing phase modulation of 0, 0.5 pi, pi and 1.5 pi on non-scattered light of the sample through a spatial light modulator;
s14: and recording a structured interference phase shift diagram corresponding to the sample under the phase modulation by using a phase image acquisition module.
In one embodiment of the present invention, the S2 includes:
For samples with a thickness of 10 μm or less, phase information of the samples was obtained using formula (a):
wherein θ represents the direction angle of the fringe diffraction pattern loaded on the digital micromirror array, I θ,0 、I θ,1 、I θ,2 、I θ,3 An interference phase shift diagram when the direction angle is theta and the modulation phase is 0, 0.5 pi, pi and 1.5 pi respectively;
for samples with a thickness above 10 μm, phase information of the samples was obtained using formula (b):
wherein,
represents the interference phase shift diagram acquired by the image acquisition module, m represents the phase sequence number of the fringe diffraction pattern, and n represents the non-scattering property of the spatial light modulatorThe phase modulation sequence number of the light.
Compared with the prior art, the invention has the beneficial effects that:
the quantitative phase microscopic device and the quantitative phase microscopic method based on the partial coherent structured light illumination combine the light cutting effect of the partial coherent structured light illumination with the quantitative phase microscopic imaging, and have the advantages of high measurement precision and: firstly, the device has an optical structure of common-path interference, so that the anti-interference capability of the device is greatly improved; secondly, the device uses a plurality of light emitting diodes which are uniformly distributed in a ring shape as a basic light source, and the quality of the image is greatly improved through an average effect; in addition, the device utilizes the optical diffraction device to carry out structured modulation on the partially coherent illumination light emitted by the annular light source, greatly improves the light cutting capacity and the transverse spatial resolution of the system, and is very suitable for three-dimensional high-resolution label-free detection of thick scattering samples; finally, the device can not only carry out high-resolution quantitative phase microscopic imaging on a thin weak scattering sample, but also carry out high-resolution light-cut quantitative phase microscopic imaging on a thick strong scattering sample, and can be coupled with a fluorescence microscopic technology to form a dual-mode microscopic imaging system. Therefore, the quantitative phase microscopic device and the quantitative phase microscopic method can carry out high-resolution label-free detection on various scattering samples, and have wide application prospects in the fields of biomedicine and the like.
The present invention will be described in further detail with reference to the accompanying drawings and examples.
Drawings
FIG. 1 is a schematic diagram of a quantitative phase microscope device based on partially coherent structured light illumination according to an embodiment of the present invention;
FIG. 2 is a diagram of a binarized fringe pattern distributed in a vertical direction loaded on an optical diffraction unit according to an embodiment of the present invention;
FIG. 3 is a schematic illustration of three demagnified version of an annular light source produced at the back focal plane of the first tube lens when loading the fringe pattern of FIG. 2 on a digital micromirror array;
FIG. 4 is a schematic illustration of a reduced version of an annular light source blocked by a spatially filtered mask;
FIG. 5 is a schematic diagram of a spatial light modulator that continuously phase modulates only non-scattered light regions (0, 0.5 pi, and 1.5 pi) exhibited by a + -1-level annular light source without modulating the scattered light;
FIG. 6 is a schematic diagram of another quantitative phase microscopy and fluorescence microscopy dual-mode system based on partially coherent structured light illumination according to an embodiment of the present invention;
FIG. 7A is a plot of the change in illumination optical transfer function at different defocus distances;
FIG. 7B is a plot of the intensity distribution of stripe structured light in the xz plane of the sample for the stripe frequency selected in FIG. 7A;
FIG. 8 is a comparison of four phase shift maps and a sample phase map obtained when the fringe diffraction pattern is unloaded and loaded;
FIG. 9 is a quantitative phase imaging contrast result for 200 nm polystyrene beads when no fringe diffraction pattern is loaded and loaded;
fig. 10 is a quantitative phase imaging contrast result of african green monkey kidney cells when the fringe diffraction pattern is unloaded and loaded.
Reference numerals illustrate:
1-a ring light source; 2-an industrial lens; 3-a first thin lens; 4-a first beam splitter; a 5-optical diffraction unit; 6-a first tube lens; 7-spatial filtering mask; 8-illumination objective; 9-sample; 10-detecting an objective lens; 11-linear polarizer; 12-a second tube lens; 13-dichroic mirrors; 14-a second thin lens; 15-a second beam splitter; a 16-spatial light modulator; 17-a third thin lens; 18-an image acquisition module; 19-fluorescence excitation detection module.
Detailed Description
In order to further illustrate the technical means and effects adopted by the invention to achieve the preset aim, the following describes in detail a quantitative phase microscopic device based on partial coherent structured light illumination according to the invention with reference to the attached drawings and the detailed description.
The foregoing and other features, aspects, and advantages of the present invention will become more apparent from the following detailed description of the preferred embodiments when taken in conjunction with the accompanying drawings. The technical means and effects adopted by the present invention to achieve the intended purpose can be more deeply and specifically understood through the description of the specific embodiments, however, the attached drawings are provided for reference and description only, and are not intended to limit the technical scheme of the present invention.
It should be noted that in this document relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that an article or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in an article or apparatus that comprises the element.
Example 1
Referring to fig. 1, fig. 1 is a schematic structural diagram of a quantitative phase microscope device based on partially coherent structured light illumination according to an embodiment of the present invention. The quantitative phase microscopic device comprises an annular light source 1, and a structural light modulation module, an illumination objective 8, a detection objective 10, a spatial light modulation module and an image acquisition module which are sequentially arranged along the light path direction, wherein the annular light source 1 is used for generating a partially coherent annular light beam; the structural light modulation module is used for carrying out structural modulation on the annular light beam to obtain + -1-level diffraction light; the sample 9 is arranged between the illumination objective 8 and the detection objective 10, and the sample 9 generates non-scattered light and scattered light carrying sample information under the irradiation of the + -1-order diffraction light; the spatial light modulation module is used for carrying out phase modulation on the non-scattered light so as to obtain a plurality of interference phase shift images of the sample 9, and the image acquisition module is used for acquiring the interference phase shift images.
Specifically, the ring-shaped light source 1 is composed of a plurality of identical light emitting diodes uniformly distributed on the ring-shaped light source skeleton. In actual operation, all the leds on the ring light source 1 are lit simultaneously, and fig. 1 only shows the propagation of light when a single led is lit, in order to clearly reveal the system light path. The more LEDs distributed on the annular light source 1, the more light energy the device can collect, and the greater the imaging speed. In the present embodiment, the ring-shaped light source 1 is uniformly distributed in a ring shape by 24 light emitting diodes having identical parameters, and emits white light. All light emitting diodes are connected in parallel and are simultaneously lighted when the phase device is in operation.
Further, the structural light modulation module of the present embodiment includes an industrial lens 2, a first thin lens 3, a first beam splitter 4, an optical diffraction unit 5, a first barrel lens 6, and a spatial filter mask 7, which are sequentially disposed in the light path direction. The number of leds on the annular light source 1 is proportional to the diameter of the annular light source, and therefore a zoom system is required to zoom the annular light beam generated by the annular light source 1, the industrial lens 2 and the first thin lens 3 being used to zoom and converge the annular light beam. The industrial lens 2 can collect as many light beams as possible emitted from the ring light source 1, and can reduce the light beams emitted from the ring light source 1. In order to maximize the light emitted by the leds, each led on the ring light source 1 needs to have a small divergence angle, and each led needs to be directed to the focal point of the industrial lens 2, as shown in fig. 1. The industrial lens 2 of the present embodiment is a small focal length industrial lens, the focal length is 12 mm, and the ultra wide angle is provided.
The image of the annular light source 1 reduced by the industrial lens 2 is located exactly at the front focal plane of the first thin lens 3. Each of the leds emits a large angular spread of approximately spherical waves after the annular light source 1 is contracted, and these approximately spherical waves converge through the first thin lens 3. Preferably, the first thin lens 3 is a two inch double cemented achromat with a focal length of 100 mm.
Further, the first beam splitter 4 is obliquely disposed between the first thin lens 3 and the optical diffraction unit 5, the illumination light from the first thin lens 3 is transmitted onto the optical diffraction unit 5 through the first beam splitter 4, the optical diffraction unit 5 is capable of loading a series of binarized stripe patterns having different direction angles to structurally modulate the illumination light, forming 0 th order diffraction light and ±1 st order diffraction light, and then the 0 th order diffraction light and ±1 st order diffraction light are reflected to the first barrel lens 6 through the first beam splitter 4.
Specifically, a part of the illumination light converged by the first thin lens 3 passes through the first beam splitter 4 and obliquely irradiates on the working surface of the optical diffraction unit 5 in the form of plane waves, and the other part of the light energy is far away from the system after being reflected by the first beam splitter 4. The beam splitting ratio of the first beam splitter 4 selected in this embodiment is 45:55, that is, 45% of the illumination light passes through the first beam splitter 4 and irradiates onto the working surface of the optical diffraction unit 5, and 55% of the illumination light is far away from the system after being reflected by the first beam splitter 4.
In operation of the microscopic device, the optical diffraction unit 5 can be loaded with a series of binarized stripe patterns with different direction angles to perform structural modulation on illumination light, and referring to fig. 2, fig. 2 is a schematic diagram of a binarized stripe pattern which is loaded on the optical diffraction unit and distributed along a vertical direction according to an embodiment of the present invention. The optical diffraction unit 5 may be a digital micromirror array, a reflective spatial light modulator, a transmissive spatial light modulator, a reflective diffraction grating, a transmissive diffraction grating, or the like, and is not limited to the specific type of the optical diffraction unit, but only a digital micromirror array is exemplified in the present embodiment. Preferably, the resolution of the digital micromirror array is 1920×1080, and the size of each pixel is 7.56 micrometers×7.56 micrometers.
Specifically, after the diffraction action of the dmd array 5, the oblique plane wave incident on the surface thereof generates multiple orders of diffraction light (0 order diffraction light, ±1 order diffraction light and ±2 order diffraction light, wherein±2 order diffraction light is far away from the system), fig. 1 only shows the propagation of 0 order and ±1 order diffraction light, and the exit angle of the ±1 order diffraction light depends on the fringe frequency loaded by the dmd array 5. The 0 th and ±1 th diffraction light is condensed on the rear focal plane thereof by the first barrel lens 6 after being reflected by the first beam splitter 4.
It should be noted that the working surface of the dmd 5 is at the confocal plane of the first tube lens 6 and the first thin lens 3, and at this time, three decentered reduced-version annular light sources are generated at the back focal plane of the first tube lens 6 from the 0 th and ±1 st diffracted light, and the connecting lines of their centers are parallel to the diffraction direction of the pattern loaded by the dmd 5, as shown in fig. 3.
The quantitative phase microscope apparatus of the present embodiment requires only ±1-order diffraction terms to illuminate the sample simultaneously, and therefore a spatial filter mask 7 for blocking 0-order diffracted light and transmitting only ±1-order diffracted light is provided at the confocal plane of the first tube lens 6 and the illumination objective lens 8, as shown in fig. 4.
Further, the ±1-level annular light source at the back focal plane of the illumination objective 8 is converged by the illumination objective 8, and then obliquely irradiated onto the sample 9 placed at the common focal plane of the illumination objective 8 and the detection objective 10 at the same time in the form of plane waves, and the sample is subjected to structured illumination by the stripe structure light. The stripe structure light at this time is actually an image of a stripe diffraction pattern loaded on the digital micromirror array 5 at the front focal plane of the illumination objective lens 8 through a confocal system composed of the first tube lens 6 and the illumination objective lens 8. Irradiation of the sample 9 with the + -1 st order diffracted light produces non-scattered light that is not affected by the sample and scattered light that carries information about the structure and refractive index profile of the sample. Preferably, the illumination objective 8 of the present embodiment is a immersion objective, the magnification is 25 times, and the numerical aperture na=1.1; the probe objective 10 is an immersion objective with a magnification of 60 times and a numerical aperture na=1.4.
The spatial light modulation module of the present embodiment includes a linear polarizer 11, a second barrel lens 12, a dichroic mirror 13, a second thin lens 14, a second beam splitter 15, a spatial light modulator 16, and a third thin lens 17, which are disposed in this order in the light propagation direction. The linear polarizer 11 is used to adjust scattered light and non-scattered light of the sample to linearly polarized light. Since the spatial light modulator 16 has polarization direction selectivity when performing phase modulation on the light field, and the light emitted by all the light emitting diodes on the annular light source is unpolarized, the linear polarizer 11 is installed in front of the spatial light modulator 16, and the linear polarizer 11 adjusts the scattered light and the non-scattered light into linear polarized light, so that the scattered light and the non-scattered light irradiated onto the spatial light modulator 16 only comprise polarization components along the acting direction of the spatial light modulator 16, and accurate phase modulation on the light field by the spatial light modulator 16 is ensured.
The first barrel lens 12 and the second thin lens 14 constitute a confocal system, and the dichroic mirror 13 is used to emit light from the second barrel lens 12 to the second thin lens 14. The second beam splitter 15 is obliquely disposed between the second thin lens 14 and the spatial light modulator 16, and the light from the second thin lens 14 is transmitted to the spatial light modulator 16 through the second beam splitter 15, and the spatial light modulator 16 can perform phase modulation on the non-scattered light, and the modulated non-scattered light is reflected to the third thin lens 17 through the second beam splitter 15. After passing through the linear polarizer 11, the linearly polarized light containing the scattered light component and the non-scattered light component is magnified and imaged to the back focal plane of the second barrel lens 12 by the confocal system composed of the detection objective lens 10 and the second barrel lens 12, and then imaged to the working plane of the image pickup module 18 by the confocal system composed of the second thin lens 14 and the third thin lens 17. In the present embodiment, the dichroic mirror 13 only plays a role of reflection, in order to make the system more compact, so the dichroic mirror 13 may also be replaced with a planar mirror.
Specifically, for the non-scattered light of the + -1 level, they are imaged in the form of + -1 level annular light source after being converged by the detection objective lens 10 to the confocal surface of the detection objective lens 10 and the second tube lens 12, and then imaged to the working surface of the spatial light modulator 16 at the back focal surface of the second thin lens 14 through the confocal system composed of the first tube lens 12 and the second thin lens 14. For scattered light, it is mainly composed of first-order spherical waves, and passes through a confocal system composed of the detection objective lens 10 and the first tube lens 12, a confocal system composed of the first tube lens 12 and the second thin lens 14, and a confocal system composed of the second thin lens 14 and the third thin lens 17, and finally converges on the working surface of the image acquisition module 18 at the back focal surface of the third thin lens 17. During this time, the scattered light propagates onto the active surface of spatial light modulator 16 as a uniform plane wave. Only a part of the energy of the non-scattered light and the scattered light enters the system after passing through the second beam splitter 15, and the energy enters the system after being modulated by the spatial light modulator 16 and then being reflected by the second beam splitter 15.
It should be noted that, in the present embodiment, spatial light modulator 16 is capable of performing phase modulation of 0, 0.5 pi, and 1.5 pi on the non-scattered light of the sample, as shown in fig. 5, by performing continuous phase modulation (0, 0.5 pi, and 1.5 pi) only on the non-scattered light region exhibited by the ±1-order ring light source, and not modulating the scattered light in the form of a plane wave. The working surface of the spatial light modulator 16 is at the front focal plane of the third thin lens 17, so that the modulated non-scattered light of + -1 order is obliquely incident on the working surface of the image acquisition module 18 in the form of a plane wave after the convergence of the third thin lens 17. Finally, the scattered light and the + -1 order non-scattered light produce a structured phase-shifted interferogram carrying sample information at the back focal plane of the third thin lens 17 and are recorded by the image acquisition module 18.
In the present embodiment, it is preferable that the average wavefront deformation of the linear polarizer 11 caused by light transmission is less than 1/4 wavelength; the second barrel lens 12 is a barrel lens having a focal length of 200 mm; the dichroic mirror 13 has a thickness of 3 mm, the wavefront distortion caused by light reflected from its surface is less than 1/5 wavelength, and the second thin lens 14 is a double cemented achromat with a focal length of 150 mm; the beam splitting ratio of the second beam splitter 15 is 45:55; the phase modulation resolution of the spatial light modulator 16 is 8 bits; the third thin lens 17 is a double cemented achromat with a focal length of 200 mm; the image acquisition module 18 is a high sensitivity sCMOS camera with individual pixels of 6.5 microns by 6.5 microns in size.
Further, the quantitative phase microscopy device further comprises a data processing module for calculating phase information of the sample according to a plurality of the interference phase shift maps, and a specific data processing process will be described in detail below.
The quantitative phase microscopic device based on the partial coherent structured light illumination combines the light cutting effect of the partial coherent structured light illumination with quantitative phase microscopic imaging, and has the advantages of high measurement precision and: firstly, the device has an optical structure of common-path interference, so that the anti-interference capability of the device is greatly improved; secondly, the device uses a plurality of light emitting diodes which are uniformly distributed in a ring shape as a basic light source, and the quality of the image is greatly improved through an average effect; in addition, the device utilizes the optical diffraction device to carry out structured modulation on the partially coherent illumination light emitted by the annular light source, greatly improves the light cutting capacity and the transverse spatial resolution of the system, and is very suitable for three-dimensional high-resolution label-free detection of thick scattering samples.
Example two
The present embodiment provides another quantitative phase microscopic device based on partially coherent structured light illumination, as shown in fig. 6, wherein a fluorescence excitation detection module 19 is added on the basis of the structure of the first embodiment, the fluorescence excitation detection module 19 includes a fluorescence light source and a fluorescence image acquisition unit, parallel excitation light can be generated by the fluorescence light source, the excitation light excites the sample 9 after passing through the second lens barrel lens 12 and the detection objective lens 10, and then the generated fluorescence is received by the fluorescence image acquisition unit in the fluorescence excitation detection module 19 after passing through the detection objective lens 10 and the second lens barrel lens 12. Other structures of the quantitative phase microscopy apparatus are identical to those of the first embodiment, and will not be described again here.
Note that the dichroic mirror 13 in the quantitative phase microscopy apparatus is different in function, and in the structure shown in the first embodiment, the dichroic mirror 13 is used to make the system more compact, whereas in the structure shown in the present embodiment, the dichroic mirror 13 is used to couple the fluorescence excitation detecting module 19 and the structure in the first embodiment into a dual-mode microscopy system. In this embodiment, the specific optical path of the fluorescence excitation detection module is not limited, and the fluorescence excitation detection module is applicable to all fluorescence-related microscopic imaging systems. The quantitative phase microscopic device of the embodiment of the invention can be combined with fluorescence microscopic imaging and other technologies to form a dual-mode imaging system, and the two modes can be used simultaneously.
Example III
On the basis of the first embodiment, the present embodiment provides a quantitative phase microscopy method based on partially coherent structured light illumination, the method comprising:
s1: a plurality of interferometric phase-shift patterns of a sample were obtained using the ring light illumination-based epi-quantitative phase microscopy apparatus described in embodiment one.
Specifically, turning on the annular light source to generate annular illumination light; loading a series of binarized fringe patterns with different direction angles through an optical diffraction unit to perform structured modulation on the annular illumination light; performing phase modulation of 0, 0.5 pi, pi and 1.5 pi on non-scattered light of the sample through a spatial light modulator; and recording a structured interference phase shift diagram corresponding to the sample under the phase modulation by using a phase image acquisition module.
S2: and calculating and obtaining phase information of the sample according to a plurality of interference phase shift maps.
Specifically, during imaging, the leds in the ring light source 1 are mounted on a ring frame processed by 3D printing, and each led has a certain inclination so as to ensure that as much light as possible emitted by them is collected by the industrial lens 2, and all leds are lit simultaneously. Each light emitting diode has a relatively wide spectral bandwidth, the light emitted by different light emitting diodes is incoherent, and the light emitted by each light emitting diode itself is partially coherent. Considering now the illumination light with wavelength lambda emitted by a certain LED, after the zooming action of the industrial lens 2 and the converging action of the first thin lens 3, the illumination light irradiates the working surface of the digital micro-mirror array 5 in the form of plane wave, and the wave vector can be expressed as Wherein (1)>Is the unit vector of the illumination direction).
The digital micromirror array 5 is loaded with a spatial frequency vector of the sampleIs +.>Is diffracted when it impinges on the surface of the dmd array, thereby producing a series of diffracted light. After being blocked by the spatial filtering mask 7, only the + -1-order diffraction item passes through the system and illuminates the sample to generate a new light field, the light field is detected by the image acquisition module 18 in the form of intensity after reaching the imaging surface by the imaging system, and the light intensity detected by the image acquisition module 18 can be expressed as:
wherein,is the amplitude of plane wave irradiated onto the working surface of the digital micro-mirror array, < >>Spatial position vector representing sample,/->Representing the initial phase of the fringe pattern loaded on the digital micromirror array,/->Representing the complex amplitude modulation function of the sample to be measured on the light field, < >>Representing a frequency vector at the pupil plane; />A spatial position vector representing the position of the imaging plane (the working plane of the image acquisition module 18); />Representing the pupil function of the quantitative phase microscopy apparatus, which is continuous only for non-scattered light areas exhibited by a class + -1 annular light sourcePhase modulation (0, 0.5 pi, and 1.5 pi). It should be noted that the pair ++in the formula (1) >Including the integral of the illumination wavelength (each led having a broad spectral bandwidth) and the integral of the illumination light direction vector (the different leds). Further, the spatial fourier transform is performed on the left and right ends of the formula (1):
that is:
wherein-the spatial fourier transform of the representation variable,coordinate vector representing spectrum domain, "-four" represents spatial cross correlation operation, ">Formula (3) contains three parts, the first part +.>Representing the spatial Fourier transform of intensity signals recorded by an image acquisition module when the diffraction light of the + -1 order generated by digital micromirror array modulation is used as two incoherent annular light sources to illuminate a sample; the two latter parts->And->Representing the diffracted light of the + -1 order produced by digital micromirror array modulation asAnd the two coherent light sources are used for carrying out space Fourier transform on the mutual dry intensity signals recorded by the image acquisition module when the sample is illuminated. Changing the phase of the fringe diffraction pattern loaded on the dmd array (>Representing the initial phase of the fringes, ψ representing the current phase of the fringes, m=0, 1, 2) and collecting the corresponding raw data, three parts in equation (3) can be obtained by solving the system of equations.
At the time of image reconstructionAs zero order remain unchanged, but +.>And->It is necessary to move in the frequency domain separately +.>And->When the array of digital micromirrors 5 is loaded with a series of fringe diffraction patterns with different direction angles and the spatial light modulator 16 performs a series of phase modulation on the non-scattered light, the phase shift intensity pattern acquired by the image acquisition module can be expressed as +.>(m is 0,1,2 represents the phase number of the fringe diffraction pattern, and the corresponding phase value is +.>n is 0,1,2,3, which represents the phase modulation sequence number of the spatial light modulator 16 for the non-scattered light, and the corresponding phase value is n pi/2; θ represents the angle of orientation of the striped diffraction pattern loaded on the dmd array, typically taken as 0,pi/3 and 2pi/3). Thus, when the dmd is loaded with a diffraction pattern in a certain direction, and the spatial light modulator performs a certain amount of phase modulation on the non-scattered light, three parts in equation (3) can be calculated as:
therefore, the corresponding high resolution phase shift intensity map can be reconstructed as:
where iFFT represents the inverse spatial Fourier transform. When the direction angle of the stripe diffraction pattern loaded on the digital micromirror array is theta, the spatial light modulator is utilized to carry out four-time phase modulation (n pi/2, n is 0,1,2, 3) on the non-scattered light, four high-resolution phase shift intensity images can be reconstructed and obtained, and the high-resolution phase distribution of the sample under the current unidirectional illumination can be obtained through a phase shift algorithm. In order to facilitate the phase modulation of non-scattered light, the diffraction terms of the 0 order and + -1 order generated by the annular light source after being modulated by the digital micromirror array are not intersected every two, so that the zero-order annular light source can be easily filtered out by using a spatial filter mask. Further, the sample can be illuminated at multiple angles by changing the direction angle of the fringe diffraction pattern loaded on the digital micromirror array. Finally, the high resolution phase information of the sample is calculated as:
Wherein tan is -1 Represents an arctangent function, θ represents the directional angle of a striped diffraction pattern loaded on a digital micromirror array, I θ,0 、I θ,1 、I θ,2 、I θ,3 Phase shift intensity diagrams when the direction angle is θ and the modulation phases are 0, 0.5 pi, and 1.5 pi, respectively, are shown. The sample phase distribution calculated by equation (6) has a very high lateral spatial resolution, which is determined by the numerical aperture of the detection objective, the wavelength of the illumination light and the angle of the illumination light, i.e. 1/(k) g +k s +NA det λ), where k g +k s ≤NA illu /λ，NA det Is the numerical aperture, NA, of the detection objective illu Is the numerical aperture of the illumination objective and λ is the wavelength of the illumination light. Therefore, the quantitative phase microscopic imaging with high resolution can be realized by the quantitative phase microscopic method based on the partial coherent structured light illumination.
It should be noted that in imaging thicker samples (typically more than 10 μm samples) using the above theory, out-of-focus signals outside the focal plane can severely impact the quality of the focused image. Thus for thick strongly scattering samples, further development of new reconstruction models is needed to achieve three-dimensional high resolution quantitative phase imaging. For simplicity, consider the extreme case where the light from each led on the ring light source is incoherent, and where the intensity distribution produced in the sample space after modulation of the digital micromirror array by the light from the ring light source can be expressed as:
Wherein m represents the phase number of the stripe structure light,is expressed in the sample space->Three-dimensional intensity distribution of stripe structured light, I 0 Representing the amplitude of the stripe structure light, +.>Frequency vector representing fringe pattern loaded on the digital micromirror array in sample space, +.>Representing the illumination optical transfer function +.>At frequency->Value of phi m Representing the phase value of the stripe structure light, the corresponding phase value is +.>As can be seen from equation (7), the frequency of the fringe structured light remains spatially unchanged, but the contrast of the fringe structured light at different axial locations is different, depending on the illumination optical transfer function and the fringe frequency. From the stoketh approximation, the illumination optical transfer functions at different axial positions can be approximately plotted as shown in fig. 7A. It can be seen that in the same axial plane, the amplitude of the illumination optical transfer function gradually decreases with increasing fringe frequency, which means that the contrast of the fringes gradually decreases with increasing fringe frequency in this axial plane. On the other hand, when the frequency of the fringes remains unchanged, the amplitude of the illumination optical transfer function gradually decreases as the axial position deviates (the absolute value of z gradually becomes larger), which means that the greater the degree of deviation from the focal plane, the lower the contrast of the fringe-structured light, with the fringe frequency remaining unchanged. Stripe frequency selected for FIG. 7A +. >FIG. 7B shows fringe structured light in the xz-plane of the sampleIntensity profile. It is clear from the figure that the contrast of the structured light of the fringes is relatively good in a small area near the focal plane, whereas the contrast of the structured light of the fringes is almost zero in an area far from the focal plane. Thus, the sample is illuminated in a structured manner in a very narrow region near the focal plane, while the sample is illuminated uniformly in a region away from the focal plane, a characteristic which is an important basis for achieving light-cut quantitative phase imaging with partially coherent structured light illumination.
Under the illumination of the structured light shown in the formula (7), the intensity signal recorded by the image acquisition module can be expressed as:
wherein,representing the three-dimensional structure distribution of the sample,/->Representing the point spread function of the sample signal at a distance z from the focal plane as it is imaged by the imaging system onto the working surface of the image acquisition module. When phi is m Respectively take outWhen the imaging device is used, the image acquisition module at the imaging surface acquires a series of intensity signals, and the signals are synthesized to obtain the result with the light cutting performance:
that is:
/>
to verify whether the imaging system is under incoherent structured light illuminationWith light cutting properties, this embodiment contemplates a special sample whose structural information can be expressed as Bringing this sample information into equation (10) yields:
further, only the cases where the sample is in the focus plane and far from the focus plane are considered. Z when the two-dimensional sample is in the focal plane s The value of (2) is 0, and the image acquisition module at the imaging surface acquires a clear image. And when the two-dimensional sample is far away from the focusing surface, for convenience, letC is a constant, that is, the sample is a uniform two-dimensional sample, and at this time, equation (9) can be calculated as:
wherein, OTF det Is PSF det Is a spatial fourier transform of (a). From equation (12), it can be seen that as structural information in the out-of-focus plane of the sample propagates through the system to the imaging plane, the signal decays to the third power of the out-of-focus distance. Compared with confocal microscopy (quadratic decay), the quantitative phase microscopy system device in the embodiment of the invention has better light cutting performance.
Equations (7) through (12) quantitatively demonstrate the ability of the quantitative phase microscopy system arrangement of the present invention to perform three-dimensional tomography under illumination of an annular light source and structured modulation of a digital micromirror array. Thus, the photoperiod quantitative phase image of the sample can be calculated as:
wherein,
Wherein,representing the phase shift intensity diagram acquired by the image acquisition module, wherein m is 0,1 and 2, representing the phase sequence number of the fringe diffraction pattern, and the corresponding phase value is +.>n is 0,1,2 and 3, and represents the phase modulation sequence number of the spatial light modulator on the non-scattered light, and the corresponding phase value is n pi/2; θ represents the directional angle of the fringe diffraction pattern loaded on the digital micromirror array, typically taking 0, pi/3 and 2 pi/3.
The ring light source of this embodiment is composed of a plurality of white light emitting diodes, and although the ring light source is partially coherent, the light cutting ability is not excessively lost. To sum up, in the quantitative phase microscopic method based on the partially coherent structured light illumination according to the embodiment of the present invention, the high-resolution phase image of the thin sample can be obtained by calculation using the formulas (4) to (6), and the high-resolution optical cut quantitative phase image of the thick sample can be obtained by calculation using the formulas (13) and (14). For high resolution quantitative phase imaging as described in equations (4) through (6), the fringe pattern frequency of the digital micromirror array should be as high as possible, as the illumination objective numerical aperture allows, in order to increase the lateral spatial resolution as much as possible. For the light-cut quantitative phase imaging described in formulas (13) and (14), the frequency of the fringe diffraction pattern needs to be adjusted by the digital micromirror array to increase the light-cut capability of the system as much as possible, and in general, the frequency of the fringe pattern loaded by the digital micromirror array on the sample surface should be half the cut-off frequency of the detection objective lens, namely 0.5NA det /λ。
To further reflect the feasibility of the quantitative phase microscopic imaging device based on partial coherent illumination of the present embodiment, the feasibility of the quantitative phase microscopic device shown in the first embodiment is experimentally verified as follows, and the model and parameters of the selected device are as follows.
The annular light source 1 is composed of 24 identical parameters of annular uniform distribution of light emitting diodes, and all of the 24 identical parameters emit white light. All the LEDs are connected in parallel and are lighted simultaneously when the system is in operation. The focal length of the small-focal-length industrial lens 2 is 12 mm, and the small-focal-length industrial lens has an ultra-large wide angle; the first thin lens 3 is a two inch double cemented achromat with a focal length of 100 mm; the beam splitting ratio of the first beam splitter 4 to the second beam splitter 15 is 45:55; the resolution of the digital micromirror array 5 is 1920×1080, and the size of each pixel is 7.56 micrometers×7.56 micrometers; the first barrel lens 6 and the second barrel lens 12 are both barrel lenses having a focal length of 200 mm; the illumination objective 8 is a immersion objective, the magnification is 25 times, and the numerical aperture na=1.1; the detection objective lens 10 is an immersion objective lens, the magnification is 60 times, and the numerical aperture NA=1.4; the linear polarizer 11 causes an average wavefront distortion of less than 1/4 wavelength for light transmission; the second thin lens 14 is a doublet acromatic lens with a focal length of 150 mm; the dichroic mirror 13 has a thickness of 3 mm and causes a wavefront distortion of less than 1/5 wavelength for light reflected from its surface; the phase modulation resolution of the spatial light modulator 16 is 8 bits; the third thin lens 17 is a double cemented achromat with a focal length of 200 mm; the image capture device 18 is a high sensitivity sCMOS camera with individual pixels of 6.5 microns by 6.5 microns in size.
Under this structure, the embodiment of the invention firstly verifies the high-resolution quantitative phase imaging described in formulas (4) to (6) through an analog simulation experiment. In the simulation experiment, the spectrum of each luminous point on the annular light source 1 adopts the actual spectrum value of the actually measured white light emitting diode. The simulation was performed in the absence of structured light and in the presence of structured light, respectively, during which the direction angle of the fringe diffraction pattern on the digital micromirror array 5 was changed only three times (θ was 0 °,60 °, and 120 °, respectively). In the simulation experiment, the numerical aperture of the detection objective lens is set to be 1.4, and the frequency value k of the stripe structure light at the sample collection surface is set g Is 2.16um -1 Annular light source illumination wave vector k s Is 0.72um in amplitude -1 。
Using the standard structure (siemens) as shown in fig. 8 (a) as a sample, when the digital micromirror array is not loaded with a striped diffraction pattern, the illumination light source is a conventional ring light source as shown in fig. 8 (B). At this time, the detector acquires four phase shift maps (0 pi, 0.5 pi, and 1.5 pi) as shown in fig. 8 (C) - (F), and a sample phase map calculated using a conventional four-step phase algorithm as shown in fig. 8 (G). When the digital micromirror array is loaded with stripe diffraction patterns having the direction angles θ of 0 °,60 °, and 120 °, respectively, the composite image of illumination light at the pupil plane of the illumination objective lens is as shown in fig. 8 (H). At this time, four high-resolution phase shift intensity maps (0 pi, 0.5 pi, and 1.5 pi) obtained by reconstruction are shown in fig. 8 (I) - (L), and a high-resolution sample phase map calculated using the formula (6) is shown in fig. 8 (M). As can be seen by comparing fig. 8 (D) and 8 (J), fig. 8 (F) and 8 (L), and fig. 8 (G) and 8 (M), the scalloped stripe structure is indistinguishable under ordinary annular illumination, but clearly identifiable under partially coherent structured light illumination in an embodiment of the present invention. Thus, the high resolution quantitative phase imaging based on partially coherent structured light illumination proposed by the present embodiment is feasible both in theory and in analog verification.
Further, the high-resolution quantitative phase imaging described in formulas (4) to (6) was verified experimentally. In the experiment, the 200 nm polystyrene pellets and living cells are respectively subjected to label-free quantitative phase imaging under the two conditions of no-structured light and structured light adopted by the embodiment of the invention. The result shown in fig. 9 (a) is a high resolution quantitative phase diagram reconstructed using formulas (4) to (6) when the digital micromirror array is loaded with a series of stripe patterns having different direction angles, and the result shown in fig. 9 (B) is a quantitative phase diagram calculated when the digital micromirror array is not loaded with a stripe pattern. Fig. 9 (C) depicts the contour lines of the individual polystyrene beads of fig. 9 (a) and (B), wherein the solid line represents the case without structured light, the dashed line represents the case with structured light, from which the lateral spatial resolution of the system under partially coherent structured light illumination is calculated to be 220 nanometers, and the lateral spatial resolution of the system without structured light modulation is 326 nanometers.
Further, in this example, the results shown in fig. 9 (D) are high-resolution quantitative phase maps reconstructed by using formulas (4) to (6) when the african green monkey kidney cells are loaded with a series of stripe patterns having different direction angles, and the results shown in fig. 9 (E) are quantitative phase maps calculated when the digital micromirror array is not loaded with a stripe pattern, and it can be seen that the resolution of the results shown in fig. 9 (D) is higher. Thus, the results shown in fig. 9 experimentally verify the high-resolution quantitative phase imaging described in formulas (4) to (6).
In addition, this example experimentally verified the high resolution photoperiod quantitative phase imaging described by equations (13) and (14). In the experimental process, three-dimensional scanning is carried out on a sample (living breast cancer cells cultured for 24 hours), the number of scanning layers is 80, and the axial distance between two adjacent layers is 200 nanometers. The structure shown in fig. 10 (a) is a high-resolution light-cut quantitative phase diagram reconstructed by the breast cancer cells using formulas (13) and (14) when the digital micromirror array is loaded with a series of stripe patterns having different direction angles, and the result shown in fig. 10 (B) is a quantitative phase diagram calculated when the digital micromirror array is not loaded with a stripe pattern. As can be seen from the results shown in fig. 10, for relatively thick breast cancer cells, conventional quantitative phase imaging is affected by structures outside the focal plane, and three-dimensional tomography cannot be achieved; the light cutting quantitative phase imaging based on the partial coherent structured light illumination provided by the embodiment of the invention, namely, the formulas (13) and (14) are utilized to carry out three-dimensional tomography on lipid droplets at different layers in breast cancer cells, and various organelles in living cells such as mitochondria, cell nucleus, pseudopodia and the like can be clearly identified.
The simulation and experimental results show that the quantitative phase microscopic device provided by the embodiment of the invention can be used for carrying out high-resolution quantitative phase imaging on a thin sample and carrying out three-dimensional light-cut quantitative phase imaging on a thick sample by combining the light-cut effect of partial coherent structure light illumination and quantitative phase contrast microscopic imaging. The annular light source used by the device greatly improves the image quality and avoids coherent noise caused by coherent light sources such as laser and the like.
The foregoing is a further detailed description of the invention in connection with the preferred embodiments, and it is not intended that the invention be limited to the specific embodiments described. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Claims (10)
1. The quantitative phase microscopic device based on the partial coherent structured light illumination is characterized by comprising an annular light source (1), a structured light modulation module, an illumination objective lens (8), a detection objective lens (10), a spatial light modulation module and an image acquisition module which are sequentially arranged along the light path direction,
-said annular light source (1) for generating a partially coherent annular light beam;
The structural light modulation module is used for carrying out structural modulation on the annular light beam to obtain + -1-level diffraction light;
the illumination objective (8) and the detection objective (10) are used for arranging a sample (9), and the sample (9) generates non-scattered light and scattered light carrying sample information under irradiation of + -1-order diffraction light;
the spatial light modulation module is used for carrying out phase modulation on the non-scattered light so as to obtain a plurality of structured interference phase shift diagrams of the sample (9), and the image acquisition module is used for acquiring the structured interference phase shift diagrams.
2. Quantitative phase microscopy device based on partly coherent structured light illumination according to claim 1, characterized in that the ring light source (1) consists of a plurality of light emitting diodes uniformly distributed on the ring light source skeleton.
3. The quantitative phase microscopic device based on the illumination of the partially coherent structured light according to claim 2, wherein the structured light modulation module comprises an industrial lens (2), a first thin lens (3), a first beam splitter (4), an optical diffraction unit (5), a first tube lens (6) and a spatial filter mask (7) which are sequentially arranged along the direction of the light path,
-said industrial lens (2) and said first thin lens (3) are used for zooming and converging said annular light beam;
the first beam splitter (4) is obliquely arranged between the first thin lens (3) and the optical diffraction unit (5), illumination light from the first thin lens (3) is transmitted to the optical diffraction unit (5) through the first beam splitter (4), and the optical diffraction unit (5) can load a series of binarized stripe patterns with different direction angles to perform structural modulation on the illumination light to form 0-order diffraction light and +/-1-order diffraction light; the first beam splitter (4) is further used for reflecting the 0 th order diffraction light and the +/-1 st order diffraction light to the first tube lens (6), and the spatial filtering mask (7) is arranged at a confocal plane of the first tube lens (6) and the illumination objective lens (8) and used for shielding the 0 th order diffraction light.
4. A quantitative phase microscopy device based on partly coherent structured light illumination according to claim 3, characterized in that the working surface of the optical diffraction unit (5) is located at the confocal plane of the first tube lens (6) and the first thin lens (3).
5. The quantitative phase microscopy device based on partially coherent structured light illumination according to claim 4, characterized in that the optical diffraction unit (5) is any one of a digital micromirror array, a reflective spatial light modulator, a transmissive spatial light modulator, a reflective diffraction grating or a transmissive diffraction grating.
6. The quantitative phase microscopy device based on partially coherent structured light illumination according to claim 5, wherein the spatial light modulation module comprises a linear polarizer (11), a second tube lens (12), a dichroic mirror (13), a second thin lens (14), a second beam splitter (15), a spatial light modulator (16) and a third thin lens (17) sequentially arranged in a light propagation direction,
the linear polarizer (11) is used for adjusting the scattered light and the non-scattered light into linearly polarized light;
the second tube lens (12) and the second thin lens (14) form a confocal system, and the dichroic mirror (13) is used for reflecting light rays from the second tube lens (12) to the second thin lens (14);
the second beam splitting mirror (15) is obliquely arranged between the second thin lens (14) and the spatial light modulator (16), light rays from the second thin lens (14) are transmitted to the spatial light modulator (16) through the second beam splitting mirror (15), the spatial light modulator (16) can perform phase modulation on the non-scattered light, and the modulated non-scattered light is reflected to the third thin lens (17) through the second beam splitting mirror (15);
the second thin lens (14) and the third thin lens (17) form a confocal system, the spatial light modulator (16) is located at the confocal surface of the second thin lens (14) and the confocal surface of the third thin lens (17), and the image acquisition module (18) is arranged at the back focal surface of the third thin lens (17).
7. The quantitative phase microscopy device based on partially coherent structured light illumination according to claim 6, further comprising a fluorescence excitation detection module arranged at a side of the dichroic mirror (13) remote from the second tube lens (12).
8. A quantitative phase microscopy method based on partially coherent structured light illumination, comprising:
s1: obtaining a plurality of structured interferometric phase patterns of the sample using the quantitative phase microscopy device based on partially coherent structured light illumination of claim 6;
s2: and calculating and obtaining phase information of the sample according to a plurality of the structured interference phase shift maps.
9. The quantitative phase microscopy method based on partially coherent structured light illumination of claim 8, wherein S1 comprises:
s11: turning on the annular light source to generate annular illumination light;
s12: loading a series of binarized fringe patterns with different direction angles through an optical diffraction unit to perform structured modulation on the annular illumination light;
s13: performing phase modulation of 0, 0.5 pi, pi and 1.5 pi on non-scattered light of the sample through a spatial light modulator;
s14: and recording a structured interference phase shift diagram corresponding to the sample under the phase modulation by using a phase image acquisition module.
10. The quantitative phase microscopy method based on partially coherent structured light illumination of claim 8, wherein S2 comprises:
for samples with a thickness of 10 μm or less, phase information of the samples was obtained using formula (a):
wherein θ represents the direction angle of the fringe diffraction pattern loaded on the digital micromirror array, I θ,0 、I θ,1 、I θ,2 、I θ,3 An interference phase shift diagram when the direction angle is theta and the modulation phase is 0, 0.5 pi, pi and 1.5 pi respectively;
for samples with a thickness above 10 μm, phase information of the samples was obtained using formula (b):
wherein, the phase shift diagram of interference acquired by the image acquisition module is represented by m, the phase sequence number of the fringe diffraction pattern is represented by n, and the phase modulation sequence number of the spatial light modulator on the non-scattered light is represented by n.
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