Mark-free spiral phase contrast imaging module and system for biological cell tissue imaging

Abstract

The invention discloses an unmarked spiral phase contrast imaging module and system for biological cell tissue imaging. The unmarked spiral phase contrast imaging module comprises a first-order vortex half-wave plate and an optical circular baffle arranged on the light emitting face of the first-order vortex half-wave plate. Wherein the radius of the first-order vortex half-wave plate is larger than that of the optical circular baffle, and the vortex phase generated by the first-order vortex half-wave plate is coaxial with the optical circular baffle. The invention has the advantages of white light, broadband, high contrast, real-time imaging and the like, can realize efficient phase contrast imaging in broadband, is suitable for the fields of unmarked biological imaging, real-time high-throughput image processing, industrial detection and the like, and is particularly suitable for edge detection in a biological cell unmarked microscope imaging system.

Description

Technical Field

[0001] The present invention relates to the field of optical imaging, and particularly to a label-free spiral phase-contrast imaging module and system for biological cell tissue imaging. Background Art

[0002] Optical imaging is a very important direct observation research method in many research fields, such as the biological research field. During the optical microscopy imaging process, since the constituent elements of biological samples are similar, the refractive index of the observation target is similar to that of the background, and the modulation of the amplitude of light by the object when light passes through the object is very weak, resulting in small imaging contrast. Therefore, staining methods are often used to improve the contrast. Tissue staining is achieved by specific chemical reagents reacting with the target area to bind together, thereby increasing the contrast of the stained area. However, staining not only has problems such as the difficulty of specificity of the staining agent and complex staining operations, but also may damage the original structure, making the observed results untrue.

[0003] In optical microscopy imaging, the optical paths of the light passing through the object are different. Therefore, phase-contrast imaging that utilizes the phase information carried by light can be used to improve the imaging contrast. Phase-contrast imaging can convert phase information into amplitude information, thereby achieving the capture of phase information. Currently, methods for realizing phase-contrast imaging include crystal interference method, diffraction enhancement method, coaxial phase propagation method, and grating differential phase-contrast imaging method, etc. In recent years, in order to improve the isotropic detection ability of phase-contrast imaging and the sensitivity to phase detection, spiral phase modulation has been widely applied to phase-contrast imaging. Spiral phase modulation is more sensitive to the phase gradient of the sample, and the edge signal is highlighted through the vortex filtering effect, significantly enhancing the sharpness of the image edge and the visibility of details. Currently, methods for realizing spiral phase-contrast imaging include using a spiral phase plate, metasurface, nonlinear optics, and generating spiral phase modulation of light based on SLM to achieve spiral phase-contrast imaging. However, none of these methods have achieved white light spiral phase-contrast imaging. Moreover, due to the wavelength sensitivity of the optical response of the above methods, spiral phase-contrast imaging can only be realized based on monochromatic laser. However, the imaging quality of monochromatic laser is poor, and edge detection is not clear. This phenomenon reduces the imaging quality and weakens the resolution and contrast of the image. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a label-free spiral phase-contrast imaging module and system for biological cell tissue imaging to solve the above problems.

[0005] An embodiment of the present invention provides a label-free spiral phase contrast imaging module for biological cell tissue imaging, which includes a first-order vortex half-wave plate and an optical circular baffle disposed on the light-emitting surface of the first-order vortex half-wave plate; wherein, the radius of the first-order vortex half-wave plate is greater than the radius of the optical circular baffle, and the vortex phase generated by the first-order vortex wave plate is coaxial with the optical circular baffle.

[0006] Preferably, the first-order vortex half-wave plate is made of a glass substrate and a liquid crystal polymer material. The fast axes of the liquid crystal molecules of the liquid crystal polymer are oriented radially along the glass substrate and continuously change gradually along the angular direction of the liquid crystal polymer. The phase change generated in one cycle is .

[0007] Preferably, the glass substrate is an N-BK7 glass substrate.

[0008] Preferably, the vortex phase generated by the first-order vortex half-wave plate is due to the continuous and continuously gradually changing phase change generated by the first-order vortex half-wave plate along the angular direction of the glass substrate, so that the light beam passing through the center of the first-order vortex half-wave plate satisfies the condition of coherent cancellation, and the TEM00 mode Gaussian beam is converted into a "hollow hole type" hollow intensity distribution.

[0009] An embodiment of the present invention further provides a label-free spiral phase contrast imaging system for biological cell tissue imaging, which includes an illumination module, an imaging module, and the label-free spiral phase contrast imaging module for biological cell tissue imaging as described above. Among them, the label-free spiral phase contrast imaging module for biological cell tissue imaging is disposed in the imaging module. The illumination module is used to generate illumination light. After the illumination light passes through the target sample to be observed, it is received and imaged by the imaging system.

[0010] Preferably, the illumination module includes a light source, a diaphragm, a first objective lens, and a second objective lens; the first objective lens and the second objective lens are arranged in sequence along the light propagation direction, and the diaphragm is disposed on the rear focal plane of the first objective lens; the light emitted by the light source is collected by the first objective lens, passes through the center of the diaphragm, and then is collimated and output as illumination light through the second objective lens.

[0011] Preferably, the light source is a coherent light source such as a laser or a light source with high brightness and non-coherence.

[0012] Preferably, the light source is a white light LED light source.

[0013] Preferably, the imaging module includes an achromatic objective lens, a tube lens, and a camera; the rear focal plane of the achromatic objective lens coincides with the front focal plane of the tube lens, and the camera is placed on the image plane of the imaging module.

[0014] Preferably, the label-free spiral phase-contrast imaging module for biological cell tissue imaging is inserted on the rear focal plane of the achromatic objective lens of the imaging module, and the light passes through the center of the optical vortex generated by the first-order vortex half-wave plate in the label-free spiral phase-contrast imaging module for biological cell tissue imaging.

[0015] In summary, the label-free spiral phase-contrast imaging module and system for biological cell tissue imaging proposed in the embodiments of the present invention have the advantages of white light, broadband, high contrast, and real-time imaging, etc. It can achieve efficient phase-contrast imaging within a wide wavelength band, and is applicable to fields such as label-free biological imaging, real-time high-throughput image processing, and industrial inspection, etc. It is particularly applicable to edge detection in a label-free biological cell microscope imaging system. Description of the Drawings

[0016] In order to more clearly illustrate the technical solutions of the present invention, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0017] Figure 1 It is a schematic diagram of a label-free spiral phase-contrast imaging module for biological cell tissue imaging provided by the first embodiment of the present invention.

[0018] Figure 2 It is a schematic structural diagram of a label-free spiral phase-contrast imaging system for biological cell tissue imaging provided by the second embodiment of the present invention.

[0019] Figure 3(a) - Figure 3(b) It is the experimental comparison effect of spiral phase-contrast imaging of a resolution test chart under white light LED light source illumination.

[0020] Figure 4(a) - Figure 4(b) It is the experimental comparison effect of spiral phase-contrast imaging of an amplitude object with a flower pattern under white light LED light source illumination.

[0021] Figure 5(a) - Figure 5(d) It is the experimental comparison effect of spiral phase-contrast imaging of onion cells and mouse breast cancer cells C127 cells under white light LED light source illumination. Figure 6 It is the real-time spiral phase-contrast imaging of the division process of mouse breast cancer cells (C127 cells) under white light LED light source illumination. Detailed Embodiments

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0023] Please refer to Figure 1 and Figure 2 , a first embodiment of the present invention provides a label-free spiral phase contrast imaging module 1 for biological cell tissue imaging, which includes a first-order vortex half-wave plate 2 and an optical circular baffle 3 disposed on the light-emitting surface of the first-order vortex half-wave plate 2; wherein, the radius of the first-order vortex half-wave plate 2 is greater than the radius of the optical circular baffle 3, and the vortex phase generated by the first-order vortex wave plate 2 is coaxial with the optical circular baffle 3.

[0024] In this embodiment, the first-order vortex half-wave plate 2 is made of a glass substrate and a liquid crystal polymer material. The fast axes of the liquid crystal molecules of the liquid crystal polymer are oriented uniformly along the radial direction of the glass substrate and continuously change gradually along the angular direction of the liquid crystal polymer. The phase change generated in one week is .

[0025] Among them, preferably, the glass substrate is an N-BK7 glass substrate. Of course, it can also be other glass substrates, and the present invention does not make specific limitations.

[0026] In this embodiment, the vortex phase generated by the first-order vortex half-wave plate 2 is due to the continuous and continuously gradually changing phase change generated by the first-order vortex half-wave plate 2 along the angular direction of the glass substrate, so that the light beam passing through the center of the first-order vortex half-wave plate satisfies the condition of coherent cancellation, and the TEM00 mode Gaussian light beam is converted into a "hollow hole type" hollow intensity distribution.

[0027] Please refer to Figure 2 , a second embodiment of the present invention further provides a label-free spiral phase contrast imaging system for biological cell tissue imaging, which includes an illumination module 4, an imaging module 10, and the label-free spiral phase contrast imaging module 1 as described above. Among them, the label-free spiral phase contrast imaging module 1 is disposed in the imaging module 10. The illumination module 4 is used to generate illumination light. After the illumination light passes through the target biological cells to be observed, it is received and imaged by the imaging system 10.

[0028] In this embodiment, the illumination module 4 includes a light source 5, a diaphragm 7, a first objective lens 6, and a second objective lens 8; the first objective lens 6, the diaphragm 7, and the second objective lens 8 are sequentially arranged along the light propagation direction. The diaphragm 7 is disposed on the rear focal plane of the first objective lens 6; the light emitted by the light source 5 is collected by the first objective lens 6, passes through the center of the diaphragm 7, and then is collimated and output as illumination light through the second objective lens 8.

[0029] In this embodiment, the light source 5 can be an LED, a halogen lamp, a laser, etc., especially a broadband incoherent light source, such as a white light LED light source. Compared with laser illumination, the white light LED light source is more uniform and has a lower cost.

[0030] In this embodiment, the spiral phase contrast imaging system further includes a stage 9 for placing a target sample to be observed, such as a target biological cell.

[0031] In this embodiment, the imaging module 10 includes an achromatic objective lens 11, a tube lens 12, and a camera 13; the rear focal plane of the achromatic objective lens 11 coincides with the front focal plane of the tube lens 12, the camera 13 is placed on the image plane of the imaging module 10, and the label-free spiral phase contrast imaging module 1 is inserted on the rear focal plane of the achromatic objective lens 11 of the imaging module 10, and the light passes through the center of the optical vortex generated by the first-order vortex half-wave plate 2 in the label-free spiral phase contrast imaging module 1 for biological cell tissue imaging.

[0032] In this embodiment, the collimated light emitted by the illumination module 4 hits the target biological cell on the stage 9, and the transmitted light is received by the achromatic objective lens 11 in the imaging system 10. The spatial frequency of the target biological cell is formed on the rear focal plane (i.e., the Fourier plane) of the achromatic objective lens 11. By inserting the label-free spiral phase contrast module 1, the low-frequency components are filtered out, and finally, the tube lens 12 performs an inverse Fourier transform, and the image is formed on the camera 13 to complete differential imaging.

[0033] In this embodiment, the function of the achromatic objective lens 11 is to avoid chromatic aberration caused by imaging and improve the imaging quality.

[0034] In this embodiment, the type of the camera 13 can be a CCD, a CMOS, etc., and the specific model thereof is not limited in the present invention.

[0035] The specific working principle of this embodiment is described in detail below: As Figure 1 and Figure 2 shown, in a 4f system, due to the special structure of the objective lens, when the light passing through the object passes through the objective lens, the spatial frequency of the object is formed on the rear focal plane (Fourier plane) of the objective lens, with low-frequency components in the middle of the plane and high-frequency components around. Insert a first-order vortex half-wave plate 2 on the Fourier plane. The transfer function of the first-order vortex half-wave plate 2 is , then the output spatial frequency distribution is expressed as: , according to the convolution theorem, the output light can be expressed as: , the transfer function of the first-order vortex half-wave plate 2 in the 4f system can be expressed as: , then the point spread function (PSF) is obtained through Fourier transform:

[0036] Integrating the above formula with respect to gives the point spread function (PSF) of the vortex half-wave plate:

[0037] It can be seen that the vortex phase generated by the first-order vortex half-wave plate 2 causes some low-frequency components on the Fourier plane to cancel each other out coherently. On this basis, an optical circular baffle 3 is added on the Fourier plane, and the two together form the spiral phase-contrast module 1. The PSF of this spiral phase-contrast module 1 is equivalent to the PSF of a first-order vortex half-wave plate with a radius of R h 1( x, y ) minus the PSF of the central region of the vortex half-wave plate with a radius of R0 h 2( x, y ) (R0 is the radius of the optical circular baffle 3), which is expressed as:

[0038] Using the optical circular baffle 3 to filter out more low-frequency components, h The 2( x, y ) part is the frequency components subtracted more by this embodiment than the traditional spiral phase-contrast imaging, further reducing the ambient light and improving the contrast to obtain the edge contour information of the object. Due to the low sensitivity of the first-order vortex wave plate 2 to the wavelength, the chromatic aberration of the object edge obtained is small, realizing high-contrast spiral phase-contrast imaging under white light illumination.

[0039] To better understand the present invention, the following shows the application of the embodiments of the present invention in spiral phase-contrast imaging through some practical examples. It should be noted that these practical applications only represent a part of the applications of the present invention and cannot be regarded as a limitation of the present invention.

[0040] In this embodiment, first, the square pattern and the amplitude object above the resolution plate are subjected to spiral phase-contrast imaging under white light illumination using the label-free spiral phase-contrast imaging system of the above embodiment. Figures 3(a) and 4(a) are the original images of the square pattern and the amplitude object, and Figures 3(b) and 4(b) are the spiral phase-contrast imaging patterns of the square pattern and the amplitude object. It can be seen that the edges of the phase-contrast imaging are clearly and completely detected.

[0041] Figure 5(a) - Figure 5(d)They are respectively to perform spiral phase-contrast imaging on the outer epidermal cells of onions and mouse breast cancer cells (C127 cells) under white light illumination using the spiral phase-contrast imaging system described above. It can be seen that the parts that cannot be detected due to cell transparency in the original image can also be detected under the spiral phase-contrast imaging described above. Not only can the cell boundaries be detected, but also the details inside the cells (such as cell nuclei, etc.) can be clearly detected.

[0042] The observation of cancer cells involves pathological judgment, determination of the location of cancer cells, etc., and plays an important role in biological imaging. Figure 6 It is to perform real-time spiral phase-contrast imaging on the division process of mouse breast cancer cells (C127 cells) using the optical spatial differential imaging system described above. The division process can be clearly captured, indicating that the present invention can achieve real-time spiral phase-contrast imaging. The application results of the present invention will have a significant impact on biological imaging.

[0043] In summary, the label-free spiral phase-contrast imaging module and system proposed in the embodiments of the present invention have the advantages of white light, broadband, high contrast, and real-time imaging, etc. It can achieve efficient phase-contrast imaging within a wide wavelength band, and is applicable to fields such as label-free biological imaging, real-time high-throughput image processing, and industrial inspection, and is particularly suitable for edge detection in a label-free biological cell microscope imaging system.

[0044] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A label-free spiral phase contrast imaging module for biological cell tissue imaging, characterized in that: It comprises a first-order vortex half-wave plate and an optical circular baffle arranged on the light-emitting surface of the first-order vortex half-wave plate; wherein the radius of the first-order vortex half-wave plate is larger than the radius of the optical circular baffle, and the vortex phase generated by the first-order vortex wave plate is coaxial with the optical circular baffle.

2. The label-free spiral phase contrast imaging module for biological cell tissue imaging according to claim 1, characterized in that: The first-order vortex half-wave plate is made of glass substrate and liquid crystal polymer material. The fast axis orientation of the liquid crystal molecules of the liquid crystal polymer is consistent along the radial direction of the glass substrate and continuously changes along the angular direction of the liquid crystal polymer. The phase change produced in one cycle is 2π.

3. The label-free spiral phase contrast imaging module for biological cell tissue imaging according to claim 2, characterized in that: The vortex phase generated by the first-order vortex half-wave plate is due to the first-order vortex half-wave plate producing a continuous and gradual phase change along the angular direction of the glass substrate, so that the light beam passing through the center of the first-order vortex half-wave plate meets the coherent destructive condition, and the TEM00 mode Gaussian beam is converted into a hollow intensity distribution of a "hollow hole type".

4. A label-free spiral phase contrast imaging system for biological cell tissue imaging, characterized in that: It comprises an illumination module, an imaging module and a label-free spiral phase contrast imaging module for biological cell tissue imaging as claimed in any one of claims 1 to 3, wherein the label-free spiral phase contrast imaging module for biological cell tissue imaging is arranged in the imaging module, and the illumination module is used to generate illumination light, and the illumination light is received and imaged by the imaging system after passing through the target sample to be observed.

5. The label-free spiral phase contrast imaging system for biological cell tissue imaging according to claim 4, characterized in that: The lighting module includes a light source, an aperture, a first objective lens and a second objective lens; the first objective lens and the second objective lens are placed in sequence along the light propagation direction, and the aperture is arranged on the rear focal plane of the first objective lens; the light emitted by the light source is collected by the first objective lens, passes through the center of the aperture, and is then collimated by the second objective lens and output as illumination light.

6. The label-free spiral phase contrast imaging system for biological cell tissue imaging according to claim 5, characterized in that: The light source is a coherent light source such as laser or a light source with high brightness and incoherence.

7. The label-free spiral phase contrast imaging system for biological cell tissue imaging according to claim 6, characterized in that: The light source is a white light LED light source.

8. The label-free spiral phase contrast imaging system for biological cell tissue imaging according to claim 4, characterized in that: The imaging module comprises an achromatic objective lens, a tube lens and a camera; the rear focal plane of the achromatic objective lens coincides with the front focal plane of the tube lens, and the camera is placed on the image plane of the imaging module.

9. The label-free spiral phase contrast imaging system for biological cell tissue imaging according to claim 8, characterized in that: The label-free spiral phase contrast imaging module for biological cell tissue imaging is inserted into the rear focal plane of the achromatic objective lens of the imaging module, and the light passes through the center of the optical vortex generated by the first-order vortex half-wave plate in the label-free spiral phase contrast imaging module for biological cell tissue imaging.

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