阅读说明：本技术 基于环形led照明的高分辨定量相位显微系统 (High-resolution quantitative phase microscope system based on annular LED illumination ) 是由 马英 郜鹏 郑娟娟 马琳 刘旻 于 2021-07-22 设计创作，主要内容包括：本发明公开了一种基于环形LED照明的高分辨定量相位显微系统,包括环形照明模块以及沿环形照明模块光路方向依次设置的培养皿、探测物镜、共焦单元、空间光调制器以及图像采集模块,其中,环形照明模块包括光源安装架和均匀设置的多个LED,培养皿设置在环形照明模块的正下方且位于探测物镜的焦平面上,多个LED的光能够沿倾斜方向照射在培养皿的样品上,产生非散射光及散射光,探测物镜用于收集散射光和非散射光；空间光调制器用于对样品的非散射光进行相位调制,图像采集模块采集相移图。该系统共路径干涉的光学结构提高了系统对外界扰动的免疫性；基于LED的多角度环形超斜部分相干照明,极大地提高了图像质量和空间分辨率。(The invention discloses a high-resolution quantitative phase microscope system based on annular LED illumination, which comprises an annular illumination module, a culture dish, a detection objective lens, a confocal unit, a spatial light modulator and an image acquisition module, wherein the culture dish, the detection objective lens, the confocal unit, the spatial light modulator and the image acquisition module are sequentially arranged along the light path direction of the annular illumination module; the spatial light modulator is used for carrying out phase modulation on non-scattered light of the sample, and the image acquisition module acquires a phase shift diagram. The common-path interference optical structure of the system improves the immunity of the system to external disturbance; the multi-angle annular super-inclined part based on the LED is subjected to dry illumination, so that the image quality and the spatial resolution are greatly improved.)

1. A high-resolution quantitative phase microscope system based on annular LED illumination is characterized by comprising an annular illumination module, a culture dish (3), a detection objective lens (4), a confocal unit, a spatial light modulator (10) and an image acquisition module (12) which are sequentially arranged along the light path direction of the annular illumination module, wherein,

the annular illumination module comprises a light source mounting frame (2) and a plurality of LEDs (1) uniformly arranged on the light source mounting frame (2), the culture dish (3) is arranged right below the annular illumination module and is positioned on a focal plane of the detection objective lens (4), light of the plurality of LEDs (1) can irradiate on a sample of the culture dish (3) along an inclined direction to generate non-scattered light which is not influenced by the sample and scattered light related to the sample, and the detection objective lens (4) is used for collecting the scattered light and the non-scattered light;

the confocal unit is used for enabling the scattered light and the non-scattered light to be incident on the spatial light modulator (10), the spatial light modulator (10) is used for carrying out phase modulation on the non-scattered light of a sample, and the image acquisition module (12) acquires phase shift graphs generated according to the scattered light and the non-scattered light modulated by different phases.

2. The ring LED illumination-based high-resolution quantitative phase microscopy system according to claim 1, wherein the light source mounting frame (2) comprises one ring-shaped mounting structure or a plurality of ring-shaped mounting structures with equal height and concentricity, and the plurality of LEDs are uniformly mounted on the ring-shaped mounting structure along the circumferential direction.

3. The ring-shaped LED illumination-based high-resolution quantitative phase microscopy system according to claim 1, wherein the light source mounting frame (2) is of an arch structure or a hemispherical structure, and the plurality of LEDs are uniformly mounted on the arc surface of the arch structure or the hemispherical structure.

4. The ring-shaped LED illumination-based high-resolution quantitative phase microscopy system according to claim 1, wherein the confocal unit comprises a first reflector (5), a first tube lens (6), a second reflector (7), a linear polarizer (8) and a first thin lens (9) which are arranged in sequence along the optical path direction, wherein,

the reflecting surface of the first reflecting mirror (5) is positioned on the back focal surface of the detection objective lens (4) and the front focal surface of the first tube lens (6), and the reflecting surface of the second reflecting mirror (7) is positioned on the back focal surface of the first tube lens (6) and the front focal surface of the first thin lens (9).

5. The ring-shaped LED illumination-based high-resolution quantitative phase microscopy system according to claim 1, wherein a second thin lens (11) is further arranged between the spatial light modulator (10) and the image acquisition module (12), the spatial light modulator (10) is located at a front focal plane of the second thin lens (11), and the image acquisition module (12) is located at a rear focal plane of the second thin lens (11);

the included angles between the main shafts of the first thin lens (9) and the second thin lens (11) and the normal line of the working surface of the spatial light modulator (10) are both smaller than 5 degrees.

6. The ring LED illumination-based high-resolution quantitative phase microscopy system according to claim 5, wherein the spatial light modulator (10) is used for cyclic loading of phase modulation of 0, 0.5 pi, pi and 1.5 pi to the non-scattered light.

7. The ring LED illumination-based high-resolution quantitative phase microscopy system according to claim 6, wherein the image acquisition module (12) is configured to acquire four phase shift maps at a plurality of time pointsAndt represents time.

8. The ring LED illumination-based high-resolution quantitative phase microscopy system according to claim 7, further comprising a data processing module for acquiring four phase shift maps at a plurality of time points by using the image acquisition module (12)Andphase maps of the samples were obtained.

9. The high-resolution quantitative phase microscope system based on annular LED illumination according to claim 1, further provided with a culture dish environment control subsystem comprising a water bath heater (13), a water pump (14), a silica gel hose (15), an air pump (16), a thermostatic chamber (17), a temperature controller (18) and a control computer (19), wherein,

the control computer (19) is connected with the water bath heater (13), the water pump (14), the air pump (16) and the temperature controller (18) and is used for respectively controlling the water bath heater (13), the water pump (14), the air pump (16) and the temperature controller (18);

a water inlet of the silica gel hose (15) is connected to a water bath of the water bath heater (13), the middle section of the silica gel hose is wound on the periphery of the detection objective lens (4), the water inlet returns to the water bath of the water bath heater (13), and the water pump (14) is installed near the water inlet of the silica gel hose (15);

the temperature controller (18) is electrically connected with the thermostatic chamber (17) and is used for adjusting the temperature of the thermostatic chamber (17), and the culture dish (3) is arranged in the thermostatic chamber (17);

the constant temperature cavity (17) is communicated with a gas supply device (20) through a gas pipeline, the gas supply device (20) is used for supplying required gas into the constant temperature cavity (17), and the gas pump (16) is installed on the gas pipeline.

10. The ring LED illumination-based high-resolution quantitative phase microscopy system according to any one of claims 1 to 9, further integrated with an auxiliary fluorescence subsystem for obtaining fluorescence microscopic imaging results of the sample under illumination of a white light source.

Technical Field

The invention belongs to the technical field of optical microscopic imaging, and particularly relates to a high-resolution quantitative phase microscope system based on annular LED illumination.

Background

Optical microscopes play an important role as non-invasive imaging techniques in the field of life science research. The optical microscope has relatively simple principle and structure, low requirement on sample preparation, spatial resolution and time resolution meeting the requirements of more application occasions, can carry out long-time non-invasive detection on multi-scale living body samples, and becomes an important means for people to know and research the micro world. The fluorescence microscope realizes high-contrast imaging of a specific structure through a fluorescence signal generated by the excitation of a fluorescent substance. In recent years, through the continuous efforts of scientists, the spatial resolution of the fluorescence microscope realizes the breakthrough from diffraction limit to super-resolution, and lays a foundation for the research of the biodynamic process in living cells. However, there are still some limitations to the application of fluorescence microscopy. Firstly, when the structure in the cell is marked by using a fluorescent marker, the state of the cell is changed to a certain extent, and the detection of the biodynamic process is influenced to a certain extent; secondly, the number of channels which can be observed simultaneously by the fluorescence microscope is limited, and the number of the channels which can realize synchronous observation at present is only four to five; in addition, fluorescent markers are phototoxic and photobleaching when excited, making it difficult to continuously observe living specimens for a long period of time.

The digital holographic microscope is used as a quantitative phase microscopy technology combining optical interference and digital holography, amplitude and phase information of a sample to be detected can be recovered through single exposure, and the digital holographic microscope has good imaging speed. Although this method has high measurement accuracy, it requires an additional reference light, requires high coherence to a light source and has poor interference resistance to the environment, and thus it is difficult to continuously observe a living sample for a long time. In order to improve the anti-interference capability of the digital holographic microscope, Popescu et al propose an objective parameter common-path digital holographic microscope technology based on coaxial point diffraction. Since the object light and the reference light are subjected to identical optical elements, the system is very immune to environmental disturbances. However, the reference light in this technique is generated by filtering the object light wave, and the light intensity of the reference light is directly related to the scattering degree of the measured sample, so that it cannot be guaranteed that the interference fringes have good contrast under any sample, and the reconstruction accuracy cannot be guaranteed.

Single-beam diffraction quantitative phase microscopy has been a rapid development in recent years, and this type of technique obtains complex amplitude information of a sample by iterative computation of a series of diffraction patterns. Among them, the stacked diffraction imaging (PIE) is an imaging method that can obtain phase information of a sample without a lens. The technology illuminates different areas of a sample by transversely moving a light-transmitting small hole or the sample, sequentially records diffraction patterns of the sample, and reproduces amplitude and phase information of the sample through iterative reconstruction; fourier Ptychographic Microscopy (FPM) integrates the concepts of phase recovery and synthetic aperture, and is a significant advance over the stacked diffraction imaging technique. It is worth noting that the reconstruction of a high-resolution image using PIE and FPM requires multiple low-resolution raw images, and the imaging speed is very limited and difficult to capture fast dynamic processes. In addition, the spatial resolution of both techniques currently stays at the cellular scale, and neither technique has been applied to the detection of organelles.

In addition, the phase contrast-based quantitative phase microscopy plays an important role in recovering phase information of transparent samples. Gradient Light Interference Microscopy (GLIM) is a phase-shift quantitative phase microscopy technique based on Differential Interference microscopy (DIC). In combination with the high axial resolution of conventional DIC, GLIM can perform phase imaging on thicker samples in both transmission and reflection modes, and thus the technique is well suited for viewing thicker tissue samples. However, when imaging a sample by using this technique, the illumination beam is required to be irradiated onto the sample to be measured, and the spatial resolution of GLIM is greatly limited. Furthermore, Taewoo et al proposed a quantitative phase microscopy (SLIM) based on Zernike phase contrast, using an objective lens with a numerical aperture of 1.4 and a halogen lamp with a central wavelength of 590nm, to achieve a lateral resolution of 350nm and a temporal resolution of 16 Hz. The optical structure of common-path interference enables the SLIM to have very good immunity to environmental disturbance, meanwhile, the wide-spectrum illumination improves the image quality, and speckle noise caused by laser illumination is avoided. However, the spatial and temporal resolution of this system is not sufficient to detect the fine structure of organelles within living cells, and only the overall morphological changes of the cells can be detected.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a high-resolution quantitative phase microscope system based on annular LED illumination. The technical problem to be solved by the invention is realized by the following technical scheme:

the invention provides a high-resolution quantitative phase microscope system based on annular LED illumination, which comprises an annular illumination module, a culture dish, a detection objective, a confocal unit, a spatial light modulator and an image acquisition module, wherein the culture dish, the detection objective, the confocal unit, the spatial light modulator and the image acquisition module are sequentially arranged along the light path direction of the annular illumination module,

the annular illumination module comprises a light source mounting frame and a plurality of LEDs uniformly arranged on the light source mounting frame, the culture dish is arranged right below the annular illumination module and is positioned on a focal plane of the detection objective lens, light of the LEDs can irradiate on a sample of the culture dish along an inclined direction, non-scattered light which is not influenced by the sample and scattered light which is related to the sample are generated, and the detection objective lens is used for collecting the scattered light and the non-scattered light;

the confocal unit is used for enabling the scattered light and the non-scattered light to be incident on the spatial light modulator, the spatial light modulator is used for carrying out phase modulation on the non-scattered light of a sample, and the image acquisition module is used for acquiring a phase shift diagram generated according to the scattered light and the non-scattered light subjected to different phase modulation.

In one embodiment of the invention, the light source mounting frame comprises an annular mounting structure or a plurality of annular mounting structures with equal height and concentricity, and the plurality of LEDs are uniformly mounted on the annular mounting structure along the circumferential direction.

In an embodiment of the invention, the light source mounting frame is of an arch structure or a hemispherical structure, and the plurality of LEDs are uniformly mounted on an arc surface of the arch structure or the hemispherical structure.

In one embodiment of the present invention, the confocal unit includes a first reflecting mirror, a first tube lens, a second reflecting mirror, a linearly polarizing plate, and a first thin lens, which are sequentially arranged in an optical path direction, wherein,

the reflecting surface of the first reflecting mirror is positioned on the back focal surface of the detection objective lens and the front focal surface of the first tube lens, and the reflecting surface of the second reflecting mirror is positioned on the back focal surface of the first tube lens and the front focal surface of the first thin lens.

In an embodiment of the present invention, a second thin lens is further disposed between the spatial light modulator and the image acquisition module, and the spatial light modulator is located at a front focal plane of the second thin lens, and the image acquisition module is located at a rear focal plane of the second thin lens;

and the included angles between the main shafts of the first thin lens and the second thin lens and the normal of the working surface of the spatial light modulator are less than 5 degrees.

In one embodiment of the invention, the spatial light modulator is used for cyclically loading the non-scattered light with phase modulation of 0, 0.5 pi, pi and 1.5 pi.

In one embodiment of the invention, the image acquisition module is used for acquiring four phase shift maps at a plurality of time pointsAndt represents time.

In an embodiment of the invention, the high-resolution quantitative phase microscopy system further comprises a data processing module for acquiring four phase shift maps at a plurality of time points by using the image acquisition moduleAndphase maps of the samples were obtained.

In one embodiment of the invention, the high-resolution quantitative phase microscope system is further provided with a culture dish environment control subsystem, which comprises a water bath heater, a water pump, a silica gel hose, an air pump, a thermostatic chamber, a temperature controller and a control computer, wherein,

the control computer is electrically connected with the water bath heater, the water pump, the air pump and the temperature controller and is used for respectively controlling the water bath heater, the water pump, the air pump and the temperature controller;

the water inlet of the silica gel hose is connected to the water bath of the water bath heater, the middle section of the silica gel hose is wound on the periphery of the detection objective lens, the water inlet returns to the water bath of the water bath heater, and the water pump is installed near the water inlet of the silica gel hose;

the temperature controller is electrically connected with the thermostatic chamber and used for adjusting the temperature of the thermostatic chamber, and the culture dish is arranged in the thermostatic chamber;

the thermostatic chamber is communicated with an air supply device through an air pipeline, the air supply device is used for supplying required air to the thermostatic chamber, and the air pump is installed on the air pipeline.

In one embodiment of the invention, the high-resolution quantitative phase microscope system is further integrated with an auxiliary fluorescence subsystem for obtaining fluorescence microscopic imaging results of the sample under the irradiation of a white light source.

Compared with the prior art, the invention has the beneficial effects that:

1. the high-resolution quantitative phase microscope system not only has high measurement precision, but also has the following advantages: firstly, the common-path interference optical structure improves the immunity of the system to external disturbance, and can be used for long-time nondestructive observation of organelles in living cells; secondly, the LED-based multi-angle annular super-inclined part is subjected to dry illumination, so that the image quality and the spatial resolution are greatly improved; in addition, the device removes a complex illumination system in the traditional quantitative phase contrast microscope, realizes large-inclination illumination by only using a simple annular illumination module consisting of LEDs, reduces the cost and the complexity of the system, improves the utilization rate of illumination light so as to improve the imaging speed of the system, simplifies the installation process and the alignment process of a sample, and is very friendly to users; finally, the device provides a temperature and gas concentration control system based on water-gas circulation for the first time, the control system is low in cost, can accurately control the cell survival conditions to be 37 ℃ and 5% of carbon dioxide, has very good flexibility and portability, and can adjust the structure according to actual conditions.

2. The high-resolution quantitative phase microscope system can carry out real-time, label-free and high-resolution in-situ detection on organelles in living cells under normal living conditions, and can be combined with various fluorescence microscopic technologies to form a multi-mode microscopic imaging system. The system has high stability and high quality of acquired images, can perform real-time phase imaging on organelles in transparent living cells, and has great application value in the fields of biomedicine and the like. The microscope system has a simple structure, has very good immunity to environmental disturbance, and has a transverse resolution of 177nm, an axial resolution of 362nm and a time resolution of 250 frames per second. The device can be used for quantitatively obtaining the phase information of the organelles in the transparent living cells under the conditions of 37 ℃ and 5% carbon dioxide, and the refractive index distribution of the organelle structure and the like can be obtained through image deconvolution processing.

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 high-resolution quantitative phase microscope system based on annular LED illumination according to an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of an LED according to an embodiment of the present invention;

fig. 3 is a schematic structural diagram of three other annular lighting modules provided by the embodiment of the present invention;

FIG. 4A is a schematic structural diagram of a living cell environment control system according to an embodiment of the present invention;

FIG. 4B is a graph showing the temperature of the cell culture fluid relative to the temperature of the water bath in the water bath heater in accordance with an embodiment of the present invention;

FIG. 5A is a phase/voltage response curve of a spatial light modulator under illumination by an annular LED illumination module in an embodiment of the present invention;

FIG. 5B is a four phase shift plot and the reconstructed phase plot of clear living cell Cos 7;

FIG. 6 is a schematic structural diagram of another high-resolution quantitative phase microscope system based on annular LED illumination according to an embodiment of the present invention;

FIG. 7A is a phase (top left) and fluorescence (bottom right) dual channel image of a 300nm diameter fluorescent polystyrene bead;

FIG. 7B is a phase image of fluorescent polystyrene beads 200nm in diameter;

FIG. 7C is a graph of the intensity variation on the white line in FIG. 7B;

FIG. 8 is a diagram illustrating the structure and dynamic process of capturing various organelles in a living cell using the high-resolution quantitative phase microscopy system according to an embodiment of the present invention;

FIG. 9 shows mitotic progression of ADSC cells captured by a high resolution quantitative phase microscopy system of an embodiment of the present invention at 37 deg.C and 5% carbon dioxide.

Description of reference numerals:

1-an LED; 2-a light source mounting frame; 3-culture dish; 4-a detection objective lens; 5-a first mirror; 6-a first tube lens; 7-a second mirror; an 8-linear polarizer; 9-a first thin lens; 10-a spatial light modulator; 11-a second thin lens; 12-an image acquisition module; 13-water bath heater; 14-a water pump; 15-a silica gel hose; 16-an air pump; 17-a thermostatic chamber; 18-a temperature controller; 19-control computer; 20-a gas supply means; 21-a third mirror; 22-a dichroic mirror; 23-an optical filter; 24-a second tube lens; 25-a second image acquisition device; 26-a third thin lens; 27-a filter set; 28-broad spectrum white light source.

Detailed Description

To further illustrate the technical means and effects of the present invention adopted to achieve the predetermined objects, a high-resolution quantitative phase microscope system based on annular LED illumination according to the present invention will be described in detail 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 high-resolution quantitative phase microscope system based on annular LED illumination according to an embodiment of the present invention. The high-resolution quantitative phase microscope system comprises an annular illumination module, a culture dish 3, a detection objective 4, a confocal unit, a spatial light modulator 10 and an image acquisition module 12, wherein the culture dish 3, the detection objective 4, the confocal unit, the spatial light modulator 10 and the image acquisition module 12 are sequentially arranged along the optical path direction of the annular illumination module.

The annular lighting module comprises a light source mounting frame 2 and a plurality of LEDs (light emitting diodes) 1 uniformly arranged on the light source mounting frame 2, a culture dish 3 is arranged under the annular lighting module and is positioned on a focal plane of a detection objective lens 4, light of the LEDs 1 can irradiate on a sample of the culture dish 3 along an inclined direction, so that extremely oblique lighting is generated, non-scattered light which is not influenced by the sample and scattered light which is related to the sample are generated, and the detection objective lens 4 is used for collecting the scattered light and the non-scattered light. As shown in fig. 1, the light source mounting frame 2 of the present embodiment includes an annular mounting structure, the LEDs 1 are uniformly mounted on the annular mounting structure along the circumferential direction, and all the LEDs 1 form an annular light source. The plurality of LEDs 1 have a wavelength in the visible light range, are required to have a small divergence angle, ensure a sufficiently high light intensity utilization ratio, and have a narrow spectral bandwidth. In the present embodiment, all the LEDs 1 have the same wavelength.

Referring to fig. 2, fig. 2 is a schematic structural diagram of an LED according to an embodiment of the present invention. The LED1 of the present embodiment has a special structure, and the end thereof includes a micro lens with a small focal length, so as to converge the originally divergent light with a large angle into parallel light which is uniformly spread in a single direction. In this embodiment, a plurality of LEDs 1 of the same type are uniformly distributed on the light source mounting frame 2, unpolarized and incoherent parallel light emitted by the LEDs 1 directly irradiates the culture dish 3 with living cells attached to the bottom, and the energy is concentrated in the center of the imaging field of view.

It should be noted that, in practice, the annular illumination module is not limited to the structure shown in fig. 1, and may be formed by alternately combining LEDs with different wavelengths, or by combining annular light sources with different illumination numerical apertures, so as to meet the requirements of different applications by matching with objective lenses with different numerical apertures.

In another embodiment, the light source mounting frame 2 may include a plurality of concentric annular mounting structures with different diameters at the same height, each annular mounting structure is uniformly mounted with a plurality of LEDs, and the axis of each LED passes through the front focus of the detection objective 4. In still another embodiment, the light source mounting bracket 2 may have an arch structure or a hemispherical structure, and the plurality of LEDs 1 are uniformly mounted on an arc surface of the arch structure or the hemispherical structure.

Specifically, please refer to fig. 3, fig. 3 is a schematic structural diagram of three other annular illumination modules provided in the embodiment of the present invention, wherein (a) two-wavelength LEDs are installed in a staggered manner, so as to implement dual-wavelength annular illumination; (B) the single-wavelength LED is arranged on the arched light source mounting frame, so that multi-angle annular illumination can be realized; (C) the LED with single wavelength is arranged on the annular light source installation frames with the same height and different diameters, and multi-angle annular illumination can be realized.

Further, the confocal unit is configured to make the scattered light and the non-scattered light incident on the spatial light modulator 10, the spatial light modulator 10 is configured to perform phase modulation on the non-scattered light of the sample, and the image acquisition module 12 is configured to acquire a phase-shifted image of the sample.

The confocal unit of the present embodiment includes a first reflecting mirror 5, a first tube lens 6, a second reflecting mirror 7, a linear polarizing plate 8, and a first thin lens 9, which are arranged in this order along the optical path direction, wherein the reflecting surface of the first reflecting mirror 5 is located at the back focal surface of the detection objective lens 4 and the front focal surface of the first tube lens 6, the reflecting surface of the second reflecting mirror 7 is located at the back focal surface of the first tube lens 6 and the front focal surface of the first thin lens 9, and the first reflecting mirror 5 and the second reflecting mirror 7 are used for refraction to make the system more compact. The linearly polarizing plate 8 is used to modulate unpolarized light emitted from the LED into linearly polarized light.

Further, a second thin lens 11 is disposed between the spatial light modulator 10 and the image capture module 12, the spatial light modulator 10 is located at a front focal plane of the second thin lens 11, and the image capture module 12 is located at a rear focal plane of the second thin lens 11.

Specifically, the culture dish 3 is placed at the front focal plane of the detection objective lens 4, the detection objective lens 4 and the first tube lens 6 form a confocal telescope system, and a sample on the culture dish 3 is magnified and imaged by the detection objective lens 4 and the first tube lens 6 and then imaged on the working surface of the image acquisition module 12 by the confocal system formed by the first thin lens 9 and the second thin lens 11. After the LED1 on the annular illumination module irradiates the sample, non-scattered light which is not influenced by the sample and scattered light which is influenced by the refractive index and the thickness of the sample are generated. The non-scattered light is imaged to the back focal plane of the detection objective 4 in the form of an annular light source and is imaged by a confocal system consisting of the first tube lens 6 and the first thin lens 9 onto the spatial light modulator 10 located at the confocal plane of the first thin lens 9 and the second thin lens 11. The scattered light is collected by the detection objective 4 in the form of a spherical wave and propagated to the image acquisition module 12 at the back focal plane of the second thin lens 11, during which the scattered light is a uniform plane wave on the working surface of the spatial light modulator 10.

It is noted that both scattered and non-scattered light is reflected by spatial light modulator 10 and then interferometrically produces a phase shift pattern on image acquisition module 12. In this embodiment, the angles between the principal axes of the first thin lens 9 and the second thin lens 11 and the normal of the working surface of the spatial light modulator 10 are both required to be less than 5 degrees, so as to ensure that the spatial light modulator 10 performs accurate phase modulation on the optical field. In addition, since the spatial light modulator 10 has polarization direction selectivity for modulating the light field phase, the linear polarizer 8 with the same polarization direction is added before the spatial light modulator 10, so that the spatial light modulator 10 can perform phase modulation on all the light fields incident on the working surface of the spatial light modulator.

In this embodiment, the spatial light modulator 10 cyclically loads the non-scattered light of the sample with phase modulations of 0, 0.5 π, π and 1.5 π. Specifically, when living cells are observed for a long time, phase modulation of 0 pi, 0.5 pi, pi and 1.5 pi is cyclically loaded on non-scattered light of a sample by using the spatial light modulator 10, the non-scattered light is synchronously acquired by the image acquisition module 12, and four phase shift graphs at each time point are calculated to obtain a phase graph of the sample at the time point, so that continuous dynamic change of the transparent living cells is shown in a phase form.

In summary, the high-resolution quantitative phase microscope system has very good immunity to environmental disturbances, since scattered light and non-scattered light pass through the same optical elements; meanwhile, as a plurality of partially coherent LEDs are adopted for carrying out extremely oblique illumination, the image quality and the spatial resolution are greatly improved; in addition, the simple annular illumination module greatly simplifies the illumination module of the quantitative phase microscopic channel, and reduces the complexity of the system and the inconvenience of installing samples; after the reflector is added into the system, the volume of the device is greatly reduced, so that the device is more compact.

It is worth mentioning that the traditional phase contrast based quantitative phase microscope adopts a complex illumination system to realize multi-angle synchronous illumination. Specifically, the diverging light from a halogen lamp is filtered by a collecting lens and an annular light passing mask at the front focal plane of a condenser lens to produce a ring-shaped light source, thereby producing multi-angle oblique illumination. In order to achieve high spatial resolution, i.e. very oblique illumination, the illumination module requires a water immersion objective with a large numerical aperture to be immersed in the culture medium and to be directed towards the detection objective. Just so can realize unmarked, high-resolution quantitative phase microimaging, this has also greatly increased the complexity and the cost of system, has reduced the immunity ability of system to external disturbance (illumination module and survey the alignment of module and receive operating personnel's influence very easily) to the process of sample installation is very inconvenient, is difficult to realize the popularization of technique. In comparison, the device of the embodiment of the invention greatly simplifies the structure of the illumination module, realizes the greatly inclined illumination of the sample by only adopting the annular illumination module formed by uniformly assembling a plurality of discrete LEDs, greatly reduces the cost and the complexity of the system, removes the adverse effect on the cell state caused by the fact that the condensing lens is immersed in the cell culture solution, improves the anti-interference capability of the system to the outside, and is convenient for users to use.

Further, the observation of living cells requires that the survival conditions of the cells are 37 ℃ and 5% of carbon dioxide, which is very important for accurately observing the dynamic process of organelles in the living cells for a long time. Because the imaging system is a high-resolution microscopic device, an oil-immersed objective lens is often used as a detection objective lens, and although a mature and small cell heat preservation chamber is used for maintaining the temperature around the cell, the oil-immersed objective lens absorbs a large amount of heat in the culture solution, so that the detection objective lens needs to be heated.

Referring to fig. 4A, fig. 4A is a schematic structural diagram of a living cell survival environment control system according to an embodiment of the present invention. The living cell living environment control system comprises a water bath heater 13, a water pump 14, a silica gel hose 15, an air pump 16, a thermostatic chamber 17, a temperature controller 18 and a control computer 19. The control computer 19 is electrically connected with the water bath heater 13, the water pump 14, the air pump 16, the thermostatic chamber 17 and the temperature controller 18 so as to respectively control the water bath heater 13, the water pump 14, the air pump 16, the thermostatic chamber 17 and the temperature controller 18. The water inlet of the silica gel hose 15 is connected to the water bath in the water bath heater 13, the middle section of the silica gel hose 15 is wound around the detection objective lens 4, the water pump 14 is installed near the water inlet of the silica gel hose 15, the water inlet of the silica gel hose 15 returns to the water bath in the water bath heater 13, and the water bath heater 13 is used for heating the detection objective lens 4 by heating water. The temperature controller 18 is connected to the thermostatic chamber 17 and is used for adjusting the temperature of the thermostatic chamber 17. In this embodiment, the temperature controller 18 is under the control of the control computer 19 so that the thermostatic chamber 17 is maintained at 37 ℃ which is required for cell survival. The air pump 16 is connected to the inner cavity of the thermostatic chamber 17 through an air pipe, and the other end is connected to an air supply device 20 for supplying required air to the thermostatic chamber 17. In this embodiment, the gas supply device 20 is a commercial incubator for supplying 5% carbon dioxide gas into the thermostatic chamber 17.

Specifically, warm water heated in the water bath heater 13 is circulated to the silica gel hose 15 through the water pump 14, and the silica gel hose 15 has very good flexibility and can be easily wound around the detection objective lens 4, so that heat of the warm water in the silica gel hose 15 is transferred to the detection objective lens 4 to be heated. There is inevitably heat transfer and loss when warm water circulates through the silicone hose 15, so there is a certain difference between the temperature at the lens of the detection objective 4 (i.e. the temperature of the cell culture solution) and the temperature of the water bath in the water bath heater 13, please refer to fig. 4B, which is a corresponding curve of the temperature of the cell culture solution relative to the temperature of the water bath in the water bath heater in the embodiment of the present invention. When the water bath temperature is different, the temperature of the lens of the detection objective lens 4 is also different, and it can be observed from fig. 4B that the living cell living environment control system of the embodiment can realize accurate control of the temperature of the cell culture solution through continuous measurement for 150 minutes. On the other hand, in the present embodiment, the gas concentration is accurately controlled (e.g., 5% carbon dioxide) by circulating the adapted gas in the commercial incubator into the thermostatic chamber in which the culture dish 3 is placed by one gas pump 16. Therefore, the living conditions of the cells can be accurately controlled to be 37 ℃ and 5% of carbon dioxide, and the living cell living environment control system is low in cost, has very good flexibility and portability, and can be adjusted according to actual conditions.

Further, the high-resolution quantitative phase microscope system of the present embodiment further includes a data processing module (not shown in the drawings) for obtaining a phase map of the sample by using the phase shift maps under different phase modulations acquired by the image acquisition module 12, and the specific processing procedure is as follows.

The light emitted by each LED1 in the annular illumination module of the present embodiment is natural light with a certain wavelength range, and is composed of a large number of random wave trains, and the vibration direction, the propagation direction, and the phase difference of each wave train are random, so that the wave trains are incoherent, and the intensity is superimposed between the wave trains. But a certain train of waves are coherent, so under the illumination of the annular illumination module, firstly, coherent processing means is used for solving the light field distribution under the illumination of the certain train of waves, and then, noncoherent processing means is used for synthesizing the intensity distribution under the simultaneous illumination of the trains of waves.

Consider now a monochromatic wave light source, whose angular frequency is denoted w, and assuming that the sample to be measured is a sample having a refractive index profile ofThe scatterer of (a), wherein,is the spatial coordinates of the scatterers. The scalar light field distribution of the monochromatic light field as it passes within the scatterer isAccording to the Zernike phase contrast principle, the light field of the monochromatic illumination light after passing through the sample can be divided into scattered light which is not influenced by the sampleAnd scattering influenced by the refractive index and thickness of the sampleEmitting lightIt should be noted that, in the actual imaging process, all LEDs are turned on simultaneously, since the light fields with different frequencies are incoherent, and at the same time, the illumination of different angles is implemented by using different light sources in the actual situation, so that the light fields generated by different illumination angles are also incoherent, and therefore, under the condition of illumination by the annular illumination module without modulation by the spatial light modulator 10, the total light intensity distribution detected by the image acquisition module 12 can be represented as:

wherein, angle brackets<>Representing the average process on a time scale,which represents the wave vector of the illumination,represents the total intensity of the non-scattered light,which represents the total intensity of the scattered light,representing the mutual interference function between scattered and non-scattered light,representing the average phase modulation function of the sample over the light field.

In the present embodiment, the non-scattered light not affected by the sample is modulated (modulation phase) by the spatial light modulator 10And 1.5 pi), while the higher order scattered light with sample information is not modulated by any device,the total light intensity detected by the image acquisition module 12 at this time is represented as:

the phase distribution of the sample can be calculated by using a phase shift algorithm:

it should be noted that, since a diffraction effect inevitably exists in an actual optical system, the spatial resolution and contrast of an image are also greatly affected. Under the illumination of the annular partial coherent light of the annular illumination module, the transmission of a light field obeys a strict light diffraction theory, and the imaging process meets the partial coherent imaging theory. Since the refractive index inside a scatterer actually observed changes very slowly with position, the scalar light field inside the scatterer obeys the Helmholtz equation. Thus, the cross-spectral density (mutual interference function) between the scattered and unscattered fieldsFourier transform of) can be computed as:

here, the first and second liquid crystal display panels are,

the formula is the transfer function of the quantitative phase microscope system under the illumination of the annular partial coherent light. Where j represents the imaginary complex number, S (w) represents the intensity of the non-scattered light, k_oIs the wave number of the optical field in vacuum,is scattering potentialSpatial fourier transform of (a); the spectral coordinates of the scatterers are represented,is the scattering spectral coordinate of the scatterer,is the vector of the illumination that is,is a unit vector of the angular spectral space,the mutual interference function between the scattered field and the non-scattered field can be calculated by phase shift operation (formula (3))And then the refractive index distribution of the sample can be obtained through convolution processing, and the thickness distribution of the sample is further obtained, so that the spatial resolution and the image contrast of the system are improved.

From the formula (5), the transfer function of the high-resolution quantitative phase microscope system and the numerical aperture NA of the detection objective lens in this embodiment can be known_detThe angle of illumination of the sample (i.e. the numerical aperture of illumination NA)_s) And the wavelength lambda of the illumination light. Specifically, the larger the numerical aperture of the detection objective lens, the larger the inclination angle of the illumination light, and the shorter the wavelength of the illumination light, the higher the spatial resolution. The lateral spatial resolution can be calculated as 0.82 λ/(NA) according to the Rayleigh criterion_s+NA_det) The axial resolution can be calculated as 0.82 λ/(n)_m·cos(asin(NA_s/n_m) In) wherein n) is_mThe refractive index of the culture medium is shown. It should be noted that the annular illumination module in the device of the present embodiment is formed by combining a plurality of individual LEDs of the same type, so that the image quality can be effectively improved by the average effect of the partially coherent illumination and the multi-angle illumination of a single LED. In addition, in the conventional quantitative phase microscopy based on phase contrast, besides a series of phase modulation is performed on the non-scattered light, certain amplitude attenuation (transmittance of 0.1-0.3) needs to be performed on the non-scattered light, and the complexity of the system is greatly increased. As can be seen from the formula (3), the subtraction and the division of the denominator and the numerator completely remove the amplitude modulation of the non-scattered light, so that the amplitude modulation module of the non-scattered light is removed in the device for the first time, and the imaging system is greatly simplified.

Further, when a living cell sample is observed for a long time, phase modulation of 0 pi, 0.5 pi, pi and 1.5 pi is cyclically loaded to non-scattered light of the sample by using the spatial light modulator 10, the non-scattered light is synchronously acquired by the image acquisition module 12, and then the phase diagram of the sample at each time point is obtained by calculating four phase shift diagrams at each time point by using the formula (3), so that continuous dynamic change of the transparent living cells can be shown in the form of phase. Specifically, for the conventional reconstruction method, it is assumed that four phase shift maps at the first time point t1 are represented asAndthe phase map generated from these four maps according to equation (3) is represented asThe four phase shift diagrams at the second time point t2 are shown asAndthe phase map generated from these four maps according to equation (3) is represented asThe four phase shift graphs at the third time point t3 are shown asAndthe phase map generated from these four maps according to equation (3) is represented asThereby generating a time-series phase mapAnd the like.

In the system of the embodiment, the time resolution is improved to 4 times of that of the conventional reconstruction method by repeatedly using the original phase shift map of each time point, and the time resolution of the system is only related to the switching time of the liquid crystal of the spatial light modulator and the exposure time of the image acquisition module. In particular, a first phase diagramUsing the pair of formula (3)Andprocessing the first phase image to obtain a first phase imageThe same; second phase diagramUsing the pair of formula (3)Andprocessing to obtain; third phase diagramUsing the pair of formula (3)Andprocessing to obtain; the fourth phase diagramUsing the pair of formula (3)Andprocessing to obtain; fifth phase diagramUsing the pair of formula (3)Andprocessing the second phase map to obtain a second phase mapThe same is true. Compared with the conventional reconstruction method, the reconstruction method adopted by the embodiment obtains three additional phase maps between two time points t1 and t2, so that the time resolution is effectively improved to 4 times, and only the time of switching the liquid crystal by the spatial light modulator and the image acquisition are carried outThe exposure time of the module.

In this embodiment, the selection of the LED1 needs to consider that the LED has a small divergence angle, a large power, a short central wavelength, and a narrow spectral range, so that the high-resolution quantitative phase microscope system has a high temporal and spatial resolution and ensures that the spatial light modulator can perform accurate phase modulation on the optical field. Preferably, the LED1 has a diameter of 5mm, a wavelength in the range of 470nm + -10 nm, and a single LED power of 0.36W.

The design of the annular illumination module (annular light source) considers three aspects, on one hand, the number of LEDs is increased as much as possible so as to improve the illumination intensity of a sample to improve the imaging speed, and meanwhile, the image quality is improved through an average effect; on one hand, the illumination angle is increased as much as possible so as to improve the spatial resolution, namely the diameter of the light source mounting frame 2 is increased and the distance between the annular light source and the sample is reduced; on the other hand, considering that the culture dish 3 has a certain depth, it is ensured that the sample has a sufficient imaging field of view, that is, when the sample moves laterally in a certain range, the illumination light of each angle of the annular light source composed of a plurality of LEDs can not be shielded by the side part of the culture dish. In this regard, in the present embodiment, the light source mounting frame 2 preferably has a diameter of 100mm and a distance of 50mm from the bottom of the culture dish 3, so as to realize annular large-angle illumination of 45 degrees on the sample (the numerical aperture of the illumination is sin45 ° -0.71, when the lateral spatial resolution of the system is 0.82 λ/(NA)_s+NA_det) 177nm, axial spatial resolution of 0.82 λ/(n)_m·cos(asin(NA_s/n_m) 362nm) while mounting as many LEDs as possible. In this embodiment, as shown in fig. 1, the light source mounting frame 2 is implemented by 3D printing, and 38 LEDs 1 are mounted, so that 45-degree annular illumination can be implemented on a sample. Under the condition, the imaging field of the high-resolution quantitative phase microscope system is at least a circular area with the diameter of 20 mm.

The detection objective lens 4 is selected in consideration of its magnification and numerical aperture, and the detection objective lens 4 of the present embodiment is an oil immersion objective lens, the magnification is 100X, and the numerical aperture NA is 1.44.

The first tube lens 6 is matched with the detection objective lens 4 to form an infinite optical system with good correction, apochromatic correction is realized on the whole field of view, and meanwhile, a specific magnification is realized by matching the detection objective lens 4, so that the first tube lens has a better integral aberration correction function compared with a standard achromatic lens. And therefore, an infinity corrected sleeve lens is chosen. Preferably, the first tube lens 6 is a tube lens with a focal length of 200mm, so as to realize a magnification of 100X in cooperation with the selected detection objective 4.

The image capturing module 12 needs to consider its imaging speed and quantum efficiency when selecting, and is preferably an sCOMS image capturing module, the number of pixels is 2048 × 2048, the size of a single pixel is 6.5 μm × 6.5 μm, the quantum efficiency reaches 82%, and the imaging speed reaches 1000 frames per second. The linear polarizer 8 is a linear polarizer, through which the optical field undergoes less than a quarter wavelength deformation in front of the wave.

The first thin lens 9 and the second thin lens 11 are selected in consideration of three aspects, so that on one hand, the imaging of the system of the embodiment meets the sampling law; on one hand, the spatial light modulator 10 is required to completely perform phase modulation on the system pupil aperture (that is, the clear aperture of the detection objective 4 is within the working range of the spatial light modulator 10 after being imaged by the first tube lens 6 and the first thin lens 9); on the other hand, the main axes of the first thin lens 9 and the second thin lens 11 are ensured to form an angle smaller than 5 degrees with the normal of the working surface of the spatial light modulator 10. Preferably, the first thin lens 9 and the second thin lens 11 of the present embodiment use two-inch achromatic lenses to reduce aberrations during high-resolution imaging, and the first thin lens 9 is a double-cemented achromatic lens having a focal length of 250 mm; lens 11 is a double cemented achromatic lens with a focal length of 300 mm.

The liquid crystal switching time of the spatial light modulator 10 is 2 milliseconds with 8-bit phase modulation resolution. Specifically, the working range of the spatial light modulator 10 is 17.7mm × 10.6mm, the pupil diameter of the detection objective 4 is 5.76mm, and after imaging through the first tube lens 6 and the first thin lens 9, the diameter of the pupil aperture of the detection objective 4 on the working surface of the spatial light modulator 10 is 7.2mm (much less than 10.6 mm); secondly, the transverse resolution of the system is 177nm, the size of the working surface imaged to the image acquisition module 12 is 21.24 μm, which is 3.3 times of the size of a single pixel of the image acquisition module, and the sampling law is satisfied.

As described above, in the high-resolution quantitative phase microscope system of the present embodiment, the modulation of the phase of the sample scattered light and the phase of the non-scattered light by the spatial light modulator 10 is the key to realize the accurate measurement of the phase of the sample. Since the liquid crystal response to the voltage applied to each pixel is non-linear, phase/voltage response curve calibration is required. Theoretically, the narrower the spectral bandwidth of the light source, the higher the modulation accuracy of the spatial light modulator on the light field phase. Compared with a halogen lamp used by a traditional quantitative phase contrast microscope, the spectral bandwidth of the LED used by the high-resolution quantitative phase microscope system of the embodiment is very narrow and is only 20nm, so that the spatial light modulator can perform accurate phase modulation on a sample light field. By using the gamma calibration method, the response curve of the spatial light modulation phase/voltage is obtained under the illumination of the annular illumination module, as shown in fig. 5A. In this example, transparent Cos7 live cells were imaged, the four images on the left side of FIG. 5B are phase shift images acquired by the image acquisition module when phase delays of 0, 0.5 π, and 1.5 π were added to the non-scattered light, respectively, and the image on the right side of FIG. 5B is a phase image reconstructed using a phase shift algorithm. The results show that the phase images well demonstrate mitochondrial images that cannot be displayed by conventional bright field microscopy.

The high-resolution quantitative phase microscope system of the embodiment not only has high measurement precision, but also has the following advantages: firstly, the common-path interference optical structure improves the immunity of the system to external disturbance, and can be used for long-time nondestructive observation of organelles in living cells; secondly, the LED-based multi-angle annular super-inclined part is subjected to dry illumination, so that the image quality and the spatial resolution are greatly improved; in addition, the device removes a complex illumination system in the traditional quantitative phase contrast microscope, realizes large-inclination illumination by only using a simple annular illumination module consisting of LEDs, reduces the cost and the complexity of the system, improves the utilization rate of illumination light so as to improve the imaging speed of the system, simplifies the installation process and the alignment process of a sample, and is very friendly to users; finally, the device provides a temperature and gas concentration control system based on water-gas circulation for the first time, the control system is low in cost, can accurately control the cell survival conditions to be 37 ℃ and 5% of carbon dioxide, has very good flexibility and portability, and can adjust the structure according to actual conditions.

Example two

On the basis of the above embodiment, the present embodiment provides another high-resolution quantitative phase microscope system based on annular LED illumination. The high-resolution quantitative phase microscope system comprises a quantitative phase microscope subsystem and an auxiliary fluorescence subsystem.

The quantitative phase microscope subsystem comprises an annular illumination module, a culture dish 3, a detection objective 4, a confocal unit, a spatial light modulator 10 and an image acquisition module 12, wherein the culture dish 3, the detection objective 4, the confocal unit, the spatial light modulator 10 and the image acquisition module 12 are sequentially arranged along the light path direction of the annular illumination module.

The annular lighting module comprises a light source mounting frame 2 and a plurality of LEDs 1 uniformly arranged on the light source mounting frame 2, a culture dish 3 is arranged right below the annular lighting module and is positioned on a focal plane of a detection objective lens 4, light of the LEDs 1 can irradiate on a sample of the culture dish 3 along an inclined direction, so that extremely oblique lighting is generated, non-scattered light which is not influenced by the sample and scattered light which is related to the sample are generated, and the detection objective lens 4 is used for collecting the scattered light and the non-scattered light.

The confocal unit is configured to make the scattered light and the non-scattered light incident on the spatial light modulator 10, the spatial light modulator 10 is configured to perform phase modulation on the non-scattered light of the sample, and the image acquisition module 12 is configured to acquire a phase-shifted image of the sample.

The confocal unit of the present embodiment includes a dichroic mirror, a first tube lens 6, a second reflecting mirror 7, a linear polarizing plate 8, and a first thin lens 9, which are sequentially arranged along the optical path direction, wherein the reflecting surface of the first reflecting mirror 5 is located at the back focal surface of the detection objective lens 4 and the front focal surface of the first tube lens 6, the reflecting surface of the second reflecting mirror 7 is located at the back focal surface of the first tube lens 6 and the front focal surface of the first thin lens 9, and the first reflecting mirror 5 and the second reflecting mirror 7 are used for refraction to make the system more compact. The linearly polarizing plate 8 is used to modulate unpolarized light emitted from the LED into linearly polarized light. For a specific structure and a working process of the quantitative phase microscope subsystem, please refer to embodiment one, which is not described herein again.

It should be noted that, in this embodiment, the first reflecting mirror 5 in the first embodiment is replaced by a dichroic mirror, which is used to isolate the high-resolution quantitative phase system in the first embodiment from the auxiliary fluorescence subsystem in this embodiment, and the radius of curvature of its surface is required to be about one kilometer, that is, the wavefront deformation caused by the reflected light on its surface is less than one fifth wavelength, so as to avoid causing aberration to the reflected phase channel.

The auxiliary fluorescence subsystem is used for obtaining a fluorescence microscopic imaging result of the sample under the irradiation of the white light source. The auxiliary fluorescence subsystem consists of fluorescence excitation and fluorescence collection, and the specific structure of fluorescence microscopic imaging is not limited here, and only a common wide-field fluorescence microscope is taken as an example.

In the present embodiment, as shown in fig. 5, the auxiliary fluorescence subsystem includes a third reflector 21, a dichroic mirror 22, a filter 23, a second tube lens 24, a second image capture device 25, a third thin lens 26, a filter set 27, and a broad spectrum white light source 28.

The broad spectrum white light source 28 combines with the filter set 27 to provide excitation light with different wavelengths for the auxiliary fluorescence subsystem, the dichroic mirror 22 is used for coupling an excitation light path and a signal collection light path of a fluorescence channel, the second tube lens 24 is used for reducing system aberration, the second image acquisition device 25 is used for acquiring a fluorescence channel image of a sample, and the size and the number of pixels of the second image acquisition device should meet the requirements of the sampling rate and the field of view of an imaging system.

Specifically, the white light generated by the broad spectrum white light source 28 is filtered by the filter set 27 to generate the required illumination light with different wavelengths, and the illumination light is transmitted through the third thin lens 26, the dichroic mirror 22, the third reflecting mirror 21 and the microscope objective 4 and then irradiated onto the sample of the culture dish 3. The generated fluorescence signal passes through the detection objective 4, the first reflector 5, the third reflector 21, the dichroic mirror 22, the optical filter 23 and the second tube lens 24, and is collected by the second image collecting device 25.

In the present embodiment, the second image capturing device 25 is an Andor camera, the number of pixels is 2048 × 2048, the size of a single pixel is 6.5 μm × 6.5 μm, the quantum efficiency reaches 82%, and the imaging speed reaches 1000 frames per second.

Further, in order to verify the spatial resolution of the high-resolution quantitative phase microscopy system and the accuracy of phase retrieval of the sample, the high-resolution quantitative phase microscopy system (including the auxiliary fluorescence subsystem) of the present embodiment was used to simultaneously image the fluorescent polystyrene beads (Huge Biotechnology, RF300C) with a diameter of 300nm, as shown in fig. 7A, and fig. 7A is a phase (upper left) and fluorescence (lower right) two-channel imaging map of the fluorescent polystyrene beads with a diameter of 300 nm. Considering that the refractive index n of polystyrene is about 1.55, the refractive index n of the culture solution_mIs 1.33, so the theoretical maximum phase change caused by 300nm polystyrene spheres is 2 π (n-n)_m) D/λ 2 pi (1.55-1.33) · 300/470 0.88rad, and the maximum phase change actually measured is 0.9rad, so the high-resolution quantitative phase microscope system of this embodiment has very high phase recovery accuracy. On the other hand, the theoretical transverse spatial resolution of the high-resolution quantitative phase microscope system is 0.82 lambda/(NA)_s+NA_det) 177nm, axial spatial resolution of 0.82 λ/(n)_m·cos(asin(NA_s/n_m) 362 nm). Actual measurements were made here with polystyrene spheres of 200nm diameter (Huge Biotechnology, RF200C) and the results are shown in FIGS. 7B and 7C, where FIG. 7B is a phase image of fluorescent polystyrene spheres of 200nm diameter; fig. 7C is an intensity variation curve on the white line in fig. 7B. This example performed random measurements on 20 spheres whose contours were drawn using a gaussian function, and the results showed that the actual measured lateral spatial resolution was 245nm and the axial resolution was 500 nm. On the one hand, optical aberrations inevitably exist during the imaging of the system, and on the other hand, the actual imaging process of the sample is a convolution between the optical aberrations and the system point spread function, so the spatial resolution measured with the 200nm sphere is somewhat larger than theoretical.

Further, referring to fig. 8, fig. 8 is a diagram illustrating the structure and dynamic process of capturing various organelles in a living cell by using the high-resolution quantitative phase microscope system according to an embodiment of the present invention, wherein (a) is the interaction between various organelles in an adipose-derived stem cell; (B) is a Cos7 cell filopodia structure; (C) distribution of mitochondria and lipid droplets in macrophages; (D) the adipose-derived stem cells can generate a large amount of vacuoles around cell nuclei through endocytosis when the adipose-derived stem cells die at the temperature of 20 ℃; (E) to capture the high-speed vibration process of endoplasmic reticulum network during Cos7 apoptosis at 250Hz imaging speed. With the high-resolution quantitative phase microscopy system of this embodiment, the fine structures of various organelles in living cells, such as endoplasmic reticulum network structure, mitochondria (inner cristae), nucleus, cytoskeleton, pseudopodia, vacuole, lysosome (lipid drop, endosome), can be captured without fluorescent labeling, as shown in (a), (B), (C), and (D) of fig. 8. Meanwhile, the dynamic processes of various organelles, such as the high-speed vibration process of the endoplasmic reticulum network (the imaging speed of 250 Hz), can be successfully captured by using the high-resolution quantitative phase microscope system of the embodiment, as shown in fig. 8 (E); during apoptosis of the adipose-derived stem cells, a large number of vacuoles are generated through endocytosis, and the vacuoles have interaction with lysosomes, mitochondria and the like; when the endoplasmic reticulum transports substances, behaviors such as 'taking a vehicle for convenience' and 'giving way' exist; mitochondria undergo fusion, division, etc. under the action of cytoskeleton and endoplasmic reticulum.

As shown above, the system of this example allows the continuous observation of living cells under normal conditions (37 ℃ C. + 5% carbon dioxide) for a long period of time without labeling and with high resolution. Meanwhile, cell division is known as the basis of life survival and is a necessary condition for the alternation of organism multiplication and life and death. Therefore, the dynamic process of mitosis of mouse stem cells (ADSCs) is captured by the system without any fluorescent label, as shown in FIG. 9. In the whole observation process, the living cell environment control system of the embodiment accurately maintains the normal living conditions of the cells, namely 37 ℃ and 5% CO₂. From the results shown in FIG. 9, it can be found that cell division starts from chromosome division. Specifically, chromosomes produced by chromatin condensation separate into identical sister chromatids under the action of spindle microtubules and eventually divide evenly into two daughter cells. Around the 12 th minute, a contraction loop appears in the middle of the dividing cell to addAnd (5) a rapid splitting process. Around 26 minutes, nuclear membranes appear and coat the chromosomes to form new nuclei (surrounded by white lines), which means that mitosis is complete. Interestingly, it was found that during cytokinesis, as the cleavage furrow appears, many prominent bubbles (positions indicated by arrows at time 9.35, etc.) appear around the cell, which disappear at the end of mitosis. The presence of protruding bubbles helps the adhesion of new cells to the bottom of the culture dish, and the mechanism of the presence of protruding bubbles needs to be excavated with the help of biologists.

In conclusion, the high-resolution quantitative phase microscope system can carry out real-time, label-free and high-resolution in-situ detection on organelles in living cells under normal living conditions, and can be combined with various fluorescence microscopy technologies to form a multi-mode microscopic imaging system. The system has high stability and high quality of acquired images, can perform real-time phase imaging on organelles in transparent living cells, and has great application value in the fields of biomedicine and the like. The microscope system has a simple structure, has very good immunity to environmental disturbance, and has a transverse resolution of 177nm, an axial resolution of 362nm and a time resolution of 250 frames per second. The device can be used for quantitatively obtaining the phase information of the organelles in the transparent living cells under the conditions of 37 ℃ and 5% carbon dioxide, and the refractive index distribution of the organelle structure and the like can be obtained through image deconvolution processing.

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.