CN114324245A - Quantitative phase microscope device and method based on partially coherent structured light illumination - Google Patents
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Abstract
The invention discloses a quantitative phase microscope device and a method based on partially coherent structured light illumination, wherein the device comprises an annular light source, and a structured light modulation module, an illumination objective, a detection objective, 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 a partially coherent annular light beam; the structured light modulation module is used for carrying out structured modulation on the annular light beam to obtain +/-1-order diffraction light; a sample is arranged between the illumination objective lens and the detection objective lens, and the sample generates non-scattered light and scattered light carrying sample information under the irradiation of +/-1 st-order diffracted light; the spatial light modulation module is used for carrying out phase modulation on the non-scattering light so as to obtain a plurality of structured interference phase shift images of the sample, and the image acquisition module is used for acquiring the plurality of structured interference phase shift images. 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 high-resolution unmarked detection of a relatively thick scattering sample.
Background
Optical microscopes have great advantages for the detection of living specimens as a non-invasive imaging technique, and thus play an important role in the field of life science research. Most of the biological samples are translucent or fully transparent and their internal structure cannot be probed by conventional optical bright field microscopes. The fluorescence microscope can carry out high-resolution and high-contrast selective imaging on a transparent sample, the spatial resolution also reaches dozens of nanometers, and a powerful technical means is provided for the research of the life process. There are many limitations to the application of fluorescence microscopy. First, fluorescent labels can adversely affect organelles within living cells, and long-term or high-frequency fluorescence excitation can cause cell death; secondly, limited by fluorescent markers and optical devices, only three to four organelles can be stably marked at the same time, and the complex life process in living cells cannot be detected in an all-around manner by using a fluorescent microscope; in addition, fluorescent substances generate peroxy ions when excited, and are phototoxic to living cells, while fluorescent substances are photobleachable when excited. Therefore, it is difficult to dynamically observe a living body sample in a natural state for a long time 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 marks, and can obtain information such as the structure, the refractive index distribution and the like of a sample. The digital holographic microscopy is a classical label-free quantitative phase microscopic imaging method, and can quantitatively obtain the amplitude and phase modulation function of a scattering sample on an illumination light wave only by single data acquisition. However, in order to improve the spatial resolution, the same sample needs to be illuminated by circular scanning, which greatly reduces the imaging speed, and a relatively complicated image processing is needed at the later stage to reduce the influence of environmental disturbance and laser speckle on the image quality. The scientific researchers further provide a parametric-to-object digital holographic microscopy based on coaxial point diffraction and a point diffraction digital holographic microscopy based on polarization diffraction grating, however, the illumination light sources in the devices are still lasers with high coherence, the image quality is extremely easily affected by laser speckles, and the spatial resolution and the time resolution are still mutually restricted.
The diffraction quantitative phase microscopy based on iterative computation is rapidly developed 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 calculations on the recorded diffraction image. The stacked diffraction imaging (PIE) and the Fourier stacked microscopy imaging (FPM) are two most representative diffraction quantitative phase microscopy techniques, which integrate the optical synthetic aperture and the phase recovery, greatly improve the spatial bandwidth product of the optical microscopy imaging, and can obtain high spatial resolution under a large field of view. However, the diffraction quantitative phase microscopy based on iterative computation needs to acquire tens of original diffraction images to realize single high-resolution phase reconstruction, and a report of performing rapid biodynamic process research by using the technology is not available at present. In addition, the spatial resolution and image contrast of such techniques are not sufficient to detect fine structures within living cells, and no label-free detection of organelles using such techniques has been reported. On the basis of the technology, researchers provide a multilayer diffraction model to obtain the three-dimensional structure of a sample, however, various approximations taken in the modeling process greatly reduce the spatial resolution of the image, and the complex processing process also brings about serious artifact structures. The gradient light interference quantitative phase microscope technology is established in a differential interference microscope, has two structures of a transmission type and a reflection type, and can carry out three-dimensional imaging on thicker biological tissues. However, when the technique is used for phase recovery, the integration operation needs to be performed on the acquired original image, so the technique is only suitable for continuously changing samples. In addition, the spatial resolution of such techniques is not sufficient to detect fine structures of subcellular organelles and the like within living cells.
The phase contrast-based quantitative phase microscopy has significant advantages in terms of coordination of spatial resolution and temporal resolution. Taewoo et al first proposed a spatial light interference microscope combining Zernike phase contrast microscopy and phase shift algorithms and achieved a lateral resolution of 350 nm at a temporal resolution of 16 frames per second. The technology has an optical structure with common-path interference, so that external disturbance cannot influence the measurement process. In addition, the light source used by the technology is an extended light source with a certain spectral range, so that speckle noise caused by coherent light sources such as laser is avoided, and the image quality is greatly improved. Further, researchers have proposed super-oblique illumination quantitative phase microscopy based on annular led illumination to achieve 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, or even high-contrast imaging on poorly adherent living cells.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a quantitative phase microscope device and a quantitative phase microscope method based on partially coherent structured light illumination. The technical problem to be solved by the invention is realized by the following technical scheme:
one aspect of the invention provides a quantitative phase microscope device based on partially coherent structured light illumination, which comprises an annular light source, and a structured light modulation module, an illumination objective, a detection objective, 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 a partially coherent annular light beam;
the structured light modulation module is used for carrying out structured modulation on the annular light beam to obtain +/-1-order diffracted light;
a sample is arranged between the illumination objective lens and the detection objective lens, and the sample generates non-scattered light and scattered light carrying sample information under the irradiation of +/-1 st-order diffracted 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 graphs of the sample, and the image acquisition module is used for acquiring the plurality of structured interference phase shift graphs.
In one embodiment of the invention, the annular light source is composed of a plurality of light emitting diodes, and the plurality of light emitting diodes are uniformly distributed on the 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 tube lens, and a spatial filter mask, which are sequentially disposed along 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, the illuminating light from the first thin lens is transmitted onto the optical diffraction unit through the first beam splitter, and the optical diffraction unit can load a series of binarization fringe patterns with different direction angles to perform structured modulation on the illuminating 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 tube lens, and the spatial filter mask is arranged at the confocal plane of the first tube 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 plane of the first tube lens and the first thin lens.
In one embodiment of the present 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, which are sequentially arranged along a light propagation direction,
the linear polaroid is used for adjusting the scattered light and the non-scattered light into linearly polarized light;
the second tube lens and the second thin lens form a confocal system, and the dichroic mirror is used for reflecting light rays from the second tube lens to the second thin lens;
the second beam splitter is obliquely arranged between the second thin lens and the spatial light modulator, light from the second thin lens is 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 third thin lens, and the image acquisition module is arranged at the back focal surface of the third thin lens.
In an embodiment of the present invention, the quantitative phase microscopy apparatus further includes a fluorescence excitation detection module disposed on a side of the dichroic mirror away from the second tube lens.
In another aspect of the present invention, a quantitative phase microscopy method based on partially coherent structured light illumination is provided, which includes:
s1: obtaining a plurality of structured interference phase shift maps of the sample using the apparatus for quantitative phase microscopy based on partially coherent structured light illumination according to any of the above embodiments;
s2: and calculating and obtaining phase information of the sample according to a plurality of structured interference phase shift maps.
In an 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 binary 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 pi, 0.5 pi, pi and 1.5 pi on non-scattered light of the sample by a spatial light modulator;
s14: and recording the corresponding structured interference phase shift pattern of the sample under the phase modulation by using a phase image acquisition module.
In an embodiment of the present invention, the S2 includes:
for samples having a thickness of 10 μm or less, phase information of the sample is obtained using formula (a):
where θ represents the direction angle of the fringe diffraction pattern loaded on the digital micromirror array, Iθ,0、Iθ,1、Iθ,2、Iθ,3Respectively representing interference phase shift graphs when the direction angle is theta and the modulation phases are 0, 0.5 pi, pi and 1.5 pi respectively;
for samples having a thickness of 10 μm or more, phase information of the samples is obtained using formula (b):
wherein the content of the first and second substances,
Compared with the prior art, the invention has the beneficial effects that:
the quantitative phase microscopic device and method based on the partially coherent structured light illumination combine the light-cutting effect of the partially coherent structured light illumination with the quantitative phase microscopic imaging, and have the advantages of high measurement precision and the following: firstly, the device has an optical structure with 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 an image is greatly improved through an average effect; in addition, the device utilizes an 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 capability and the transverse spatial resolution of the system, and is very suitable for three-dimensional high-resolution unmarked detection of a thick scattering sample; finally, the device can not only perform high-resolution quantitative phase microscopic imaging on a thin weak scattering sample, but also perform high-resolution light-section quantitative phase microscopic imaging on a thick strong scattering sample, and can be coupled with a fluorescence microscopy technology to form a dual-mode microscopic imaging system. Therefore, the quantitative phase microscope device and the method can carry out high-resolution label-free detection on various scattering samples, and have wide application prospect 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 structural 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 loaded on an optical diffraction unit and distributed in a vertical direction according to an embodiment of the present invention;
FIG. 3 is a schematic diagram of three demagnified versions of the ring light source produced at the back focal surface of the first tube lens when the fringe pattern of FIG. 2 is loaded on the digital micromirror array;
FIG. 4 is a schematic diagram of a reduced version of an annular light source shielded by a spatial filtering mask;
FIG. 5 is a schematic diagram of a spatial light modulator that continuously phase modulates (0, 0.5 π, π and 1.5 π) only the non-scattered light region exhibited by a class + -1 annular light source without modulating the scattered light;
FIG. 6 is a schematic structural diagram of another dual-mode system for quantitative phase microscopy and fluorescence microscopy based on partially coherent structured light illumination according to an embodiment of the present invention;
FIG. 7A is a graph showing the variation of the optical transfer function of illumination at different defocus distances;
FIG. 7B is a graph of the intensity distribution of the striped structured light in the xz plane of the sample for the selected fringe frequencies of FIG. 7A;
FIG. 8 is a comparison of four phase shift plots and a sample phase plot obtained when the fringe diffraction pattern was unloaded and loaded;
FIG. 9 is a quantitative phase imaging contrast result for 200 nm polystyrene spheres when unloaded and loaded with fringe diffraction patterns;
FIG. 10 is a comparison of quantitative phase imaging of Vero cells when unloaded and loaded with fringe diffraction patterns.
Description of reference numerals:
1-a ring-shaped light source; 2-industrial lens; 3-a first thin lens; 4-a first beam splitter; 5-an optical diffraction unit; 6-a first tube lens; 7-spatial filtering mask; 8-an illumination objective; 9-sample; 10-a detection objective; 11-linear polarizer; 12-a second tube lens; 13-a dichroic mirror; 14-a second thin lens; 15-a second beam splitter; 16-a 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 of the present invention adopted to achieve the predetermined objects, a quantitative phase microscope device based on partially coherent structured light illumination according to the present invention is described in detail below with reference to the accompanying drawings and the detailed description.
The foregoing and other technical matters, features and effects of the present invention will be apparent from the following detailed description of the embodiments, which is to be read in connection with the accompanying drawings. The technical means and effects of the present invention adopted to achieve the predetermined 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 used for limiting the technical scheme of the present invention.
It is noted that, herein, relational terms such as first and second, and the like may be 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. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that an article or device 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 an … …" does not exclude the presence of additional like elements in the article or device comprising the element.
Example one
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 microscope 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 direction of a light path, wherein the annular light source 1 is used for generating a partially coherent annular light beam; the structured light modulation module is used for carrying out structured modulation on the annular light beam to obtain +/-1-order diffraction light; a sample 9 is arranged between the illumination objective lens 8 and the detection objective lens 10, and the sample 9 generates non-scattered light and scattered light carrying sample information under the irradiation of the +/-1 st order diffracted light; the spatial light modulation module is used for performing phase modulation on the non-scattered light to obtain a plurality of interference phase shift patterns of the sample 9, and the image acquisition module is used for acquiring the plurality of interference phase shift patterns.
Specifically, the ring light source 1 is composed of a plurality of identical light emitting diodes which are uniformly distributed on a ring light source skeleton. In actual operation, all the leds on the ring light source 1 are lit at the same time, and in order to clearly show the system optical path, fig. 1 only shows the light propagation when a single led is lit. 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 light source 1 is formed by 24 light emitting diodes with the same parameters, which are uniformly distributed in a ring shape and all emit white light. All the light emitting diodes are connected in parallel and are lit simultaneously 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 tube lens 6, and a spatial filter mask 7, which are sequentially arranged along the optical path direction. The number of leds on the ring light source 1 is proportional to the diameter of the ring light source, so a zoom system is required to zoom the ring light beam generated by the ring light source 1, and the industrial lens 2 and the first thin lens 3 are used to zoom and converge the ring light beam. The industrial lens 2 can collect the light beam emitted by the annular light source 1 as much as possible, and can reduce the light beam emitted by the annular light source 1. In order to maximize the utilization of 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, has a focal length of 12 mm, and has an ultra-large wide angle.
The image of the ring-shaped light source 1 reduced by the industrial lens 2 is just at the front focal plane of the first thin lens 3. After the annular light source 1 is reduced, each light emitting diode emits approximate spherical waves which are diverged at a large angle, and the approximate spherical waves are converged by the first thin lens 3. Preferably, the first thin lens 3 is a two-inch double cemented achromat having a focal length of 100 millimeters.
Further, a first beam splitter 4 is obliquely arranged 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 can load a series of binary fringe patterns with different direction angles to perform structured modulation on the illumination light, 0-order diffraction light and ± 1-order diffraction light are formed, and then the 0-order diffraction light and the ± 1-order diffraction light are reflected to the first tube 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 is obliquely irradiated onto the working surface of the optical diffraction unit 5 in the form of a plane wave, and another part of the light energy is reflected by the first beam splitter 4 and then is far away from the system. The 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 on 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.
When the microscope device works, the optical diffraction unit 5 can load a series of binarized fringe patterns with different direction angles to perform structured modulation on the illumination light, please refer to fig. 2, and fig. 2 is a binarized fringe pattern loaded on the optical diffraction unit and distributed along the 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 the specific type of the optical diffraction unit is not limited, but the digital micromirror array is merely 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 × 7.56 microns.
Specifically, after diffraction by the digital micromirror array 5, an oblique plane wave incident on its surface generates multiple orders of diffracted light (0 order diffracted light, ± 1 order diffracted light, and ± 2 order diffracted light, wherein ± 2 order diffracted light is far away from the system), and fig. 1 shows propagation of only 0 order and ± 1 order diffracted light, and the exit angle of ± 1 order diffracted light depends on the fringe frequency loaded by the digital micromirror array 5. The 0-order and + -1-order diffracted lights are converged on the back focal plane by the first tube lens 6 after being reflected by the first beam splitter 4.
It should be noted that the working surface of the dmd array 5 is at the confocal plane of the first tube lens 6 and the first thin lens 3, and at this time, three off-center demagnified ring light sources are generated at the back focal plane of the first tube lens 6 by 0 th order and ± 1 st order diffracted lights, and the connecting line of their centers is parallel to the diffraction direction of the loaded pattern of the dmd array 5, as shown in fig. 3.
The quantitative phase microscope device of the present embodiment only needs the ± 1 st order diffraction terms to illuminate the sample simultaneously, and therefore a spatial filter mask 7 is disposed at the confocal plane of the first tube lens 6 and the illumination objective lens 8 to block the 0 th order diffraction light and let only the ± 1 st order diffraction light pass through, as shown in fig. 4.
Further, the ± 1-level annular light source at the rear focal plane of the illumination objective 8 is focused by the illumination objective 8 and then simultaneously obliquely irradiates on the sample 9 placed at the confocal plane of the illumination objective 8 and the detection objective 10 in the form of a plane wave, and the sample is structurally illuminated by the stripe-shaped structured light. The fringe-structured light at this time is actually an image of a fringe diffraction pattern loaded on the digital micromirror array 5 at the front focal plane of the illumination objective lens 8 via the confocal system composed of the first tube lens 6 and the illumination objective lens 8. After the + -1 st order diffracted light is irradiated on the sample 9, non-scattered light which is not affected by the sample and scattered light carrying information on the structure and refractive index distribution of the sample are generated. Preferably, the illumination objective 8 of the present embodiment is a water immersion objective, the magnification is 25 times, and the numerical aperture NA is 1.1; the detection objective lens 10 is an oil immersion objective lens, the magnification is 60 times, and the numerical aperture NA is 1.4.
The spatial light modulation module of the present embodiment includes a linearly polarizing plate 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, which are arranged in this order along the light propagation direction. The linearly polarizing plate 11 is used to adjust scattered light and non-scattered light of the sample into linearly polarized light. Because the spatial light modulator 16 has the selectivity of the polarization direction 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 unscattered light into linearly polarized light, so that the scattered light and the unscattered light irradiated on the spatial light modulator 16 only include polarization components in the acting direction along the spatial light modulator 16, and the spatial light modulator 16 is ensured to perform accurate phase modulation on the light field.
The first tube lens 12 and the second thin lens 14 form a confocal system, and the dichroic mirror 13 is used for emitting the light from the second tube lens 12 to the second thin lens 14. The second beam splitter 15 is obliquely arranged between the second thin lens 14 and the spatial light modulator 16, light from the second thin lens 14 is transmitted to the spatial light modulator 16 through the second beam splitter 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 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 on the back focal plane of the second tube lens 12 by the confocal system composed of the detection objective lens 10 and the second tube lens 12, and imaged on the working plane of the image acquisition module 18 by the confocal system composed of the second thin lens 14 and the third thin lens 17. In this embodiment, the dichroic mirror 13 only performs a reflecting function, so that the system is more compact, and therefore the dichroic mirror 13 may be replaced by a plane mirror.
Specifically, for ± 1-order non-scattered light, the light is focused by the detection objective lens 10 and then imaged to the confocal plane of the detection objective lens 10 and the second tube lens 12 in the form of ± 1-order annular light source, and then imaged to the working plane of the spatial light modulator 16 at the back focal plane of the second thin lens 14 by the confocal system composed of the first tube lens 12 and the second thin lens 14. For the scattered light, the scattered light mainly consists of a first order spherical wave, and passes through a confocal system consisting of the detection objective lens 10 and the first tube lens 12, a confocal system consisting of the first tube lens 12 and the second thin lens 14, and a confocal system consisting of the second thin lens 14 and the third thin lens 17 in sequence, 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 is a uniform plane wave as it propagates onto the working surface of spatial light modulator 16. After passing through the second beam splitter 15, only a part of the scattered light and the non-scattered light enter the system, and after being modulated by the spatial light modulator 16, the scattered light and the non-scattered light are reflected by the second beam splitter 15 to enter the system.
It should be noted that the spatial light modulator 16 only performs continuous phase modulation (0, 0.5 pi, pi and 1.5 pi) on the non-scattered light region exhibited by the ± 1-order ring light source, and does not perform phase modulation on the scattered light existing in the form of a plane wave, and in the present embodiment, the spatial light modulator 16 is capable of performing phase modulation of 0, 0.5 pi, pi and 1.5 pi on the non-scattered light of the sample, as shown in fig. 5. 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 ± 1-level unscattered light is obliquely incident on the working surface of the image acquisition module 18 in the form of a plane wave after being converged by the third thin lens 17. Finally, the scattered light and the ± 1 st 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 linearly polarizing plate 11 has an average wavefront distortion caused to the transmission of light smaller than 1/4 wavelengths; 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, and the wavefront distortion caused by the reflected light to the surface thereof is less than 1/5 wavelengths, and the second thin lens 14 is a double cemented achromat lens having 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 having a focal length of 200 mm; the image acquisition module 18 is a high sensitivity sCMOS camera with individual pixel sizes of 6.5 microns by 6.5 microns.
Further, the quantitative phase microscope device further comprises a data processing module for calculating and obtaining phase information of the sample according to the plurality of interference phase shift maps, and a specific data processing process will be described in detail below.
The quantitative phase microscope device based on the partially coherent structured light illumination combines the light cutting effect of the partially coherent structured light illumination with the quantitative phase microscope imaging, and has the advantages of high measurement precision and the following advantages: firstly, the device has an optical structure with 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 an image is greatly improved through an average effect; in addition, the device utilizes an 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 capability 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
In this embodiment, another quantitative phase microscope device based on partially coherent structured light illumination is provided, as shown in fig. 6, a fluorescence excitation detection module 19 is added on the basis of the structure of the 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 passes through the second tube lens 12 and the detection objective lens 10 to excite the sample 9, and then the generated fluorescence is received by the fluorescence image acquisition unit in the fluorescence excitation detection module 19 through the detection objective lens 10 and the second tube lens 12. The other structures of the quantitative phase microscope device are completely the same as those of the first embodiment, and are not described herein again.
It should be noted that the role of the dichroic mirror 13 in the quantitative phase microscopy apparatus is different, in the structure shown in the first embodiment, the dichroic mirror 13 is to make the system more compact, and in the structure shown in the present embodiment, the dichroic mirror 13 is to couple the fluorescence excitation detection module 19 and the structure in the first embodiment into a dual-mode microscopy system. The specific optical path of the fluorescence excitation detection module is not limited in this embodiment, and the fluorescence excitation detection module is suitable for all fluorescence-related microscopic imaging systems. The quantitative phase microscope device provided by the embodiment of the invention can be combined with technologies such as fluorescence microscopic imaging and the like 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 including:
s1: a plurality of interference phase shift images of a sample are obtained by utilizing the annular light illumination-based epi-quantitative phase microscope device described in the first embodiment.
Specifically, an annular light source is turned on to generate annular illumination light; loading a series of binary 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 pi, 0.5 pi, pi and 1.5 pi on non-scattered light of the sample by a spatial light modulator; and recording the corresponding structured interference phase shift pattern of 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 the plurality of interference phase shift maps.
Specifically, during the imaging process, 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 angle to ensure that as much of the light they emit is collected by the industrial lens 2 as possible, all the leds being lit at the same time. Each led has a broad spectral bandwidth, the light emitted by different leds is incoherent, and the light emitted by each led itself is partially coherent. Considering the illumination light with wavelength λ emitted by a certain light emitting diode, after the zooming action of the industrial lens 2 and the converging action of the first thin lens 3, the illumination light is irradiated onto the working surface of the digital micromirror array 5 in the form of plane wave, and the wave vector thereof can be expressed as
Wherein the content of the first and second substances,
a unit vector of illumination directions).
The digital micromirror array 5 is loaded with a vector of frequencies in the sample space
When the wave vector in the sample space is
The plane wave is diffracted when it is irradiated to the surface of the digital micromirror array, thereby generating a series of diffracted lights. After the shielding of the spatial filtering mask 7, only the ± 1 st order diffraction term 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 through the imaging system, and the light intensity detected by the image acquisition module 18 can be expressed as:
wherein the content of the first and second substances,
is the magnitude of the plane wave impinging on the working surface of the digital micromirror array,
a spatial position vector representing the sample is determined,
indicating the initial phase of the fringe pattern loaded on the digital micromirror array,
represents the complex amplitude modulation function of the sample to be measured to the light field,
representing a frequency vector at a pupil plane;
representing a spatial position vector at the imaging plane (the working plane of the image acquisition module 18);
represents the pupil function of the quantitative phase microscopy apparatus, which continuously phase modulates (0, 0.5 pi, pi and 1.5 pi) only the non-scattered light region exhibited by a class + -1 annular light source. In the formula (1), the pairs
The integration of (a) includes integration of the illumination light wavelength (each light emitting diode has a broad spectral bandwidth) and integration of the illumination light direction vector (different light emitting diodes). Further, the left and right ends of the formula (1) are subjected to space Fourier transformObtaining:
that is:
wherein-a spatial Fourier transform of the representation variable,
represents a coordinate vector of the spectral domain, "four-stars" represents a spatial cross-correlation operation,
equation (3) contains three parts, the first part
Representing the spatial Fourier transform of an intensity signal recorded by an image acquisition module when +/-1 order diffracted light generated by the digital micromirror array modulation is used as two incoherent annular light sources to illuminate a sample; the latter two parts
And
represents the spatial Fourier transform of the mutual interference intensity signals recorded by the image acquisition module when the +/-1 st order diffracted light generated by the digital micromirror array modulation is used as two coherent light sources to illuminate the sample. Changing the phase of the fringe diffraction pattern loaded on the digital micromirror array: (
Representing the initial phase of the fringe, psi representing the current phase of the fringe, m 0, 1, 2) and collecting the corresponding raw data, three of equation (3) can be obtained by solving the system of equationsAnd (4) partial.
When reconstructing the image
Remains unchanged as a zero order, while
And
need to be moved separately in the frequency domain
And
when the digital micromirror array 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 map collected by the image collection module can be represented 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 serial number of the spatial light modulator 16 to the non-scattering light, and the corresponding phase value is n pi/2; theta denotes the azimuth angle of the fringe diffraction pattern loaded on the digital micromirror array, and typically takes 0, pi/3 and 2 pi/3). Therefore, when the digital micromirror array is loaded with a diffraction pattern in a certain direction and the spatial light modulator performs a phase modulation on the non-scattered light by a certain amount, the three components in equation (3) can be calculated as:
therefore, the corresponding high-resolution phase-shifted intensity map can be reconstructed as:
where iFFT represents an inverse spatial fourier transform. When the direction angle of the fringe diffraction pattern loaded on the digital micro-mirror array is theta, the spatial light modulator is used for carrying out phase modulation on non-scattering light for four times (n pi/2, n is 0, 1, 2 and 3), then four high-resolution phase shift intensity graphs can be reconstructed, and the high-resolution phase distribution of the sample under the current unidirectional illumination can be calculated through a phase shift algorithm. In order to facilitate the phase modulation of non-scattered light, diffraction terms of 0 order and +/-1 order generated after the annular light source is modulated by the digital micro-mirror array are not intersected pairwise, so that the zero-order annular light source can be easily filtered by a spatial filtering mask. Further, the direction angle of the fringe diffraction pattern loaded on the digital micromirror array is changed, so that the sample can be illuminated in multiple angles. Finally, the high resolution phase information of the sample is calculated as:
wherein, tan-1Denotes the arctan function, theta denotes the direction angle of the fringe diffraction pattern loaded on the digital micromirror array, Iθ,0、Iθ,1、Iθ,2、Iθ,3Respectively, represent a phase shift intensity diagram for an azimuth angle theta and modulation phases of 0, 0.5 pi, and 1.5 pi, respectively. The phase distribution of the sample calculated from equation (6) has a high lateral spatial resolution, which is determined by the numerical aperture of the detection objective, the wavelength of the illumination light and the illuminationThe angle of the light, i.e. 1/(k)g+ks+NAdetλ), wherein kg+ks≤NAillu/λ，NAdetIs the numerical aperture, NA, of the detection objectiveilluIs the numerical aperture of the illumination objective, and λ is the wavelength of the illumination light. Therefore, the quantitative phase microscopy method based on the partially coherent structured light illumination can realize high-resolution quantitative phase microscopy imaging.
It should be noted that when a thick sample (usually, a sample of 10 μm or more) is imaged by using the above theory, the quality of an in-focus image is seriously affected by a defocus signal out of the focal plane. Therefore, for thick and strongly scattering samples, new reconstruction models need to be further developed to realize three-dimensional high-resolution quantitative phase imaging. For simplicity, consider an extreme case where the light emitted from each led on the ring light source is incoherent, and the intensity distribution generated in the sample space after the light emitted from the ring light source is modulated by the digital micromirror array can be expressed as:
wherein m represents the phase number of the stripe-structured light,
is shown in sample space
Three-dimensional intensity distribution of structured light, I0Indicating the amplitude of the structured light in stripes,
representing the frequency vector in sample space of the fringe pattern loaded on the digital micromirror array,
representing the optical transfer function of the illumination at a distance z from the focal plane of the sample
At frequency
Value of (d), phimA phase value representing the light of the fringe structure corresponding to
As can be seen from equation (7), the frequency of the structured light remains spatially constant, but the contrast of the structured light at different axial positions is different, depending on the illumination optical transfer function and the fringe frequency. From the Stokseth approximation, the illumination optical transfer functions at different axial locations can be plotted approximately, as shown in FIG. 7A. It can be seen that the amplitude of the illumination optical transfer function decreases progressively with increasing fringe frequency in the same axial plane, meaning that the contrast of the fringes decreases progressively with increasing fringe frequency in the axial plane. On the other hand, when the frequency of the fringes is kept constant, the amplitude of the illumination optical transfer function gradually decreases as the axial position is deviated (the absolute value of z gradually becomes larger), which means that the contrast of the fringe-structured light becomes lower the greater the deviation from the focal plane with the frequency of the fringes kept constant. For the fringe frequency selected in FIG. 7A
FIG. 7B shows the intensity distribution of the structured light in the xz plane of the sample. It is clear from the figure that the contrast of the structured light is relatively good in a very small area near the focal plane, while the contrast of the structured light is almost zero in an area far from the focal plane. Therefore, structured illumination of the sample in a narrow region near the focal plane and uniform illumination of the sample in regions far from the focal plane are important bases for light-cut quantitative phase imaging with partially coherent structured light illumination.
Under the structured light illumination shown in equation (7), the intensity signal recorded by the image acquisition module can be expressed as:
wherein the content of the first and second substances,
represents the three-dimensional structural 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 ismRespectively take
During the process, the image acquisition module at the imaging surface acquires a series of intensity signals, and the signals are synthesized to obtain a result with the light section performance:
that is:
to verify the optical sectioning of the imaging system under incoherent structured light illumination, this embodiment considers a special sample whose structural information can be expressed as
Substituting this sample information into equation (10) yields:
further, only the sample is consideredBoth in the focal plane and away from the focal plane. When the two-dimensional sample is in the focal plane, zsThe value of (2) is 0, and the image acquisition module at the imaging plane acquires clear images. When the two-dimensional sample is far away from the focusing surface, for convenience, let
C is constant, that is, the sample is a uniform two-dimensional sample, and in this case, equation (9) can be calculated as:
wherein, OTFdetIs PSFdetSpatial fourier transform of (a). As can be seen from equation (12), when the structural information in the defocus plane of the sample propagates through the system to the imaging plane, the signal is attenuated by the third power of the defocus distance. Compared with confocal microscopy (quadratic attenuation), the quantitative phase microscopy system device in the embodiment of the invention has better light section performance.
Equations (7) through (12) quantitatively demonstrate the capability of the quantitative phase microscopy system apparatus of the present invention to perform three-dimensional tomography under illumination of a ring light source and structured modulation of a digital micromirror array. Thus, the photo-cut quantitative phase image of a sample can be calculated as:
wherein the content of the first and second substances,
wherein the content of the first and second substances,
the phase shift intensity graph acquired by the image acquisition module is shown, m takes 0, 1 and 2 and represents fringe diffractionThe phase sequence number of the shot pattern corresponds to the phase value
n is 0, 1, 2 and 3, which represents the phase modulation serial number of the spatial light modulator to the non-scattering light, and the corresponding phase value is n pi/2; theta denotes the azimuth angle of the fringe diffraction pattern loaded on the digital micromirror array and typically takes 0, pi/3 and 2 pi/3.
The annular light source of this embodiment consists of a plurality of white light emitting diodes, and although the annular light source is partially coherent, there is no excessive loss in light cutting capability. In summary, the quantitative phase microscopy method based on partially coherent structured light illumination according to the embodiments of the present invention can obtain the high resolution phase image of the thin sample by using the equations (4) to (6) and obtain the high resolution light-cut quantitative phase image of the thick sample by using the equations (13) and (14). For high resolution quantitative phase imaging as described in equations (4) to (6), the fringe pattern frequency loaded by the digital micromirror array should be as high as possible, as the illumination objective numerical aperture allows, in order to improve the lateral spatial resolution as much as possible. For the photo-shear quantitative phase imaging described in the formulas (13) and (14), the frequency of the fringe diffraction pattern needs to be adjusted by the digital micromirror array to improve the photo-shear capability of the system as much as possible, and generally, the frequency of the fringe pattern loaded by the digital micromirror array on the sample surface should be half of the cut-off frequency of the detection objective lens, namely 0.5NAdet/λ。
To further reflect the feasibility of the quantitative phase microscopy imaging apparatus based on partially coherent illumination of the present embodiment, the feasibility of the quantitative phase microscopy imaging apparatus shown in the first embodiment was experimentally verified, and the selected device models and parameters were as follows.
The annular light source 1 consists of 24 light-emitting diodes with the same parameters which are uniformly distributed in an annular mode and all emit white light. All the light emitting diodes are connected in parallel and lighted simultaneously when the system is in operation. The small-focus industrial lens 2 has a focal length of 12 mm and 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 ratios of the first beam splitter 4 and the second beam splitter 15 are both 45: 55; the resolution of the digital micromirror array 5 is 1920 × 1080, and the size of each pixel is 7.56 × 7.56 micrometers; the first barrel lens 6 and the second barrel lens 12 are barrel lenses having a focal length of 200 mm; the illumination objective lens 8 is a water immersion objective lens, the magnification is 25 times, and the numerical aperture NA is 1.1; the detection objective lens 10 is an oil immersion objective lens, the magnification is 60 times, and the numerical aperture NA is 1.4; the linearly polarizing plate 11 has an average wavefront distortion to the transmitted light of less than 1/4 wavelengths; the second thin lens 14 is a double cemented achromat having a focal length of 150 mm; the dichroic mirror 13 has a thickness of 3 mm, and the wavefront distortion caused by the light reflected from its surface is smaller than 1/5 wavelengths; the phase modulation resolution of the spatial light modulator 16 is 8 bits; the third thin lens 17 is a double cemented achromat having a focal length of 200 mm; the image acquisition device 18 is a high-sensitivity sCMOS camera with individual pixel sizes of 6.5 microns by 6.5 microns.
Under this structure, the embodiment of the present invention first verifies the high resolution quantitative phase imaging described in equations (4) to (6) through simulation experiments. In the simulation experiment, the spectrum of each light emitting point on the annular light source 1 adopts the real 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 orientation 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 gathering surface is setgIs 2.16um-1Illumination wave vector k of annular light sourcesIs 0.72um-1。
Using the standard structure (siemens) as shown in fig. 8(a) as a sample, the illumination source was a conventional annular light source when the digital micromirror array was not loaded with a fringe diffraction pattern, as shown in fig. 8 (B). At this time, the detector acquires four phase shift maps (0 π, 0.5 π, π and 1.5 π) as shown in FIGS. 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 fringe diffraction patterns having direction angles θ of 0 °, 60 °, and 120 °, respectively, the resultant image of the 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 π, 0.5 π, π and 1.5 π) obtained by reconstruction are shown in FIGS. 8(I) - (L), and a high-resolution sample phase map calculated using equation (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 fan-shaped fringe structure cannot be resolved under normal ring illumination, but can be clearly identified under the partially coherent structured light illumination of the embodiment of the present invention. Therefore, the high-resolution quantitative phase imaging based on the partially coherent structured light illumination proposed by the present embodiment is feasible in both theoretical and analog verification.
Further, the high resolution quantitative phase imaging described by equations (4) to (6) was experimentally verified. In the experiment, 200 nm polystyrene spheres and living cells are subjected to unmarked quantitative phase imaging under two conditions of structured light and structured light adopted in the embodiment of the invention. The result shown in fig. 9(a) is a high-resolution quantitative phase map reconstructed using equations (4) to (6) when the digital micromirror array is loaded with a series of fringe patterns having different orientation angles, while the result shown in fig. 9(B) is a quantitative phase map calculated when the digital micromirror array is not loaded with fringe patterns from 200 nm polystyrene pellets. FIG. 9(C) depicts the outline of a single polystyrene bead in FIGS. 9(A) and (B), where the solid line represents the case without structured light and 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 nm, while the lateral spatial resolution of the system without structured light modulation is 326 nm.
Further, in this example, quantitative phase imaging is performed on living vero cells, and the result shown in fig. 9(D) is a high-resolution quantitative phase map reconstructed by using equations (4) to (6) when the digital micromirror array is loaded with a series of fringe patterns having different orientation angles, whereas the result shown in fig. 9(E) is a quantitative phase map calculated when the digital micromirror array is not loaded with a fringe pattern, and it can be seen that the resolution of the result shown in fig. 9(D) is higher. Therefore, the results shown in fig. 9 experimentally verify the high resolution quantitative phase imaging described in equations (4) to (6).
In addition, this embodiment experimentally verifies the high-resolution photo-shear quantitative phase imaging described in equations (13) and (14). In the experimental process, a sample (in-vivo breast cancer cells cultured for 24 hours) was scanned in three dimensions, the number of scanning layers was 80, and the axial distance between two adjacent layers was 200 nm. The structure shown in fig. 10(a) is a high-resolution photo-cut quantitative phase map reconstructed by equations (13) and (14) when the digital micromirror array is loaded with a series of fringe patterns having different azimuth angles, while the result shown in fig. 10(B) is a quantitative phase map calculated when the digital micromirror array is not loaded with a fringe pattern. As can be seen from the results shown in fig. 10, for relatively thick breast cancer cells, the conventional quantitative phase imaging is affected by structures outside the focal plane, and three-dimensional tomography cannot be realized; by using the light-cut quantitative phase imaging based on the partially coherent structured light illumination provided by the embodiment of the invention, namely using the formulas (13) and (14), the lipid droplets at different layers in the breast cancer cell can be subjected to three-dimensional tomography, and various organelles in a living cell, such as mitochondria, cell nucleus, pseudopodia and the like, can be clearly identified.
The simulation and experiment 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-section quantitative phase imaging on a thick sample by combining the light-section effect of partial coherent structured light illumination and quantitative phase-contrast microscopic imaging. The annular light source used by the device improves the image quality greatly, and avoids coherent noise caused by coherent light sources such as laser.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (10)
1. A quantitative phase microscope device based on partially coherent structured light illumination is characterized by comprising an annular light source (1), a structured 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 direction of a light path, wherein,
the annular light source (1) is used for generating a partially coherent annular light beam;
the structured light modulation module is used for carrying out structured modulation on the annular light beam to obtain +/-1-order diffracted light;
a sample (9) is arranged between the illumination objective lens (8) and the detection objective lens (10), and the sample (9) generates non-scattered light and scattered light carrying sample information under the irradiation of +/-1 st-order diffracted 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 graphs of the sample (9), and the image acquisition module is used for acquiring the plurality of structured interference phase shift graphs.
2. Quantitative phase microscopy device based on partially coherent structured light illumination according to claim 1 characterized in that the ring light source (1) consists of a plurality of light emitting diodes which are evenly distributed on the ring light source skeleton.
3. The quantitative phase microscopy device based on partially coherent structured light illumination 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) arranged in sequence along the optical path direction, wherein,
the industrial lens (2) and the first thin lens (3) are used for zooming and converging the 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 onto the optical diffraction unit (5) through the first beam splitter (4), and the optical diffraction unit (5) can be loaded with a series of binaryzation stripe patterns with different direction angles to structurally modulate 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 diffracted light and the +/-1 st-order diffracted light to a first tube lens (6), and the spatial filter 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 diffracted light.
4. Quantitative phase microscopy device based on partially 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. Quantitative phase microscopy device based on partially coherent structured light illumination according to claim 1 characterized in that the optical diffraction unit (5) is any of a digital micro-mirror 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 1, 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) arranged in sequence along the 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 splitter (15) is obliquely arranged between the second thin lens (14) and the spatial light modulator (16), light from the second thin lens (14) is transmitted to the spatial light modulator (16) through the second beam splitter (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 splitter (15);
the second thin lens (14) and a third thin lens (17) form a confocal system, the spatial light modulator (16) is positioned at a confocal plane of the second thin lens (14) and the third thin lens (17), and the image acquisition module (18) is arranged at a back focal plane of the third thin lens (17).
7. The device according to claim 1, further comprising a fluorescence excitation detection module disposed on a side of the dichroic mirror (13) away from the second tube lens (12).
8. A quantitative phase microscopy method based on partially coherent structured light illumination is characterized by comprising the following steps:
s1: obtaining a plurality of structured interference phase shift maps of a sample using the device for quantitative phase microscopy based on partially coherent structured light illumination according to any one of claims 1 to 6;
s2: and calculating and obtaining phase information of the sample according to a plurality of structured interference phase shift maps.
9. The method for quantitative phase microscopy based on partially coherent structured light illumination according to claim 8, wherein the step S1 comprises:
s11: turning on the annular light source to generate annular illumination light;
s12: loading a series of binary 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 pi, 0.5 pi, pi and 1.5 pi on non-scattered light of the sample by a spatial light modulator;
s14: and recording the corresponding structured interference phase shift pattern of the sample under the phase modulation by using a phase image acquisition module.
10. The method for quantitative phase microscopy based on partially coherent structured light illumination according to claim 8, wherein the step S2 comprises:
for samples having a thickness of 10 μm or less, phase information of the sample is obtained using formula (a):
where θ represents the direction angle of the fringe diffraction pattern loaded on the digital micromirror array, Iθ,0、Iθ,1、Iθ,2、Iθ,3Respectively representing interference phase shift graphs when the direction angle is theta and the modulation phases are 0, 0.5 pi, pi and 1.5 pi respectively;
for samples having a thickness of 10 μm or more, phase information of the samples is obtained using formula (b):
wherein the content of the first and second substances,
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