Confocal microscopic device based on adjustable radial birefringence pupil filter

Abstract

The invention discloses a confocal microscopic device based on an adjustable radial birefringence pupil filter, and belongs to the field of optical super-resolution imaging. The device adopts a liquid crystal device as an adjustable radial birefringence pupil filter, electric field distribution of the liquid crystal device is adjusted in an electric control mode, and accurate adjustment of a pupil function is achieved. A user can flexibly select a proper pupil function according to different imaging requirements, so that the diffraction limit of an optical system is broken through, and transverse super-resolution, axial super-resolution and three-dimensional super-resolution are respectively realized. Compared with a traditional confocal microscopic device, the confocal microscopic device has higher resolution and flexibility and can meet various imaging requirements. The technology is particularly suitable for the fields of biomedicine, material science and the like, and has a wide application prospect.

Description

Technical field:

[0001] The invention relates to the field of super-resolution imaging, and in particular to a confocal microscope device based on an adjustable radial birefringence pupil filter. Background technology:

[0002] Since the confocal microscopy technology was proposed by Minsky in the 1950s, it has been widely used in biomedicine, materials science and other fields. Traditional confocal microscopy devices effectively shield scattered light outside the focus by setting a pinhole in front of the detector, enhance imaging contrast, and suppress side lobes. However, due to the constraints of optical diffraction limits, the resolution of confocal microscopes still cannot break through physical limits.

[0003] In recent years, pupil filtering technology, as an optical super-resolution technology, has become an important means to improve the resolution of microscopic imaging because it can change the diffraction characteristics of the imaging system by modulating the pupil function. Pupil filters can be divided into amplitude type, phase type and complex amplitude type according to the modulation method. Compared with amplitude type pupil filters, the use of phase type or complex amplitude type pupil filters can reduce energy loss, maintain high light intensity and improve imaging quality.

[0004] Most existing pupil filters adjust the system's resolution by rotating birefringent elements or polarizers, which usually requires additional mechanical devices, and the adjustment process is relatively slow and cannot adapt to different imaging requirements in real time. In addition, most existing pupil filters cannot achieve a relatively continuous phase distribution, so their application is subject to certain limitations.

[0005] With the development of liquid crystal spatial light modulation technology, liquid crystal devices have attracted widespread attention as pupil filter elements with good adjustment performance. Liquid crystal devices can achieve precise control of incident light by adjusting their phase delay characteristics through an external electric field. The birefringent pupil filter based on liquid crystal devices has good adjustability and continuity, and therefore has become a potential solution to improve the resolution of optical imaging. Summary of the invention:

[0006] In order to solve the problems of poor adjustability, poor flexibility and poor stability caused by mechanical movement of pupil filters in the prior art, the present invention provides a confocal microscopy device based on an adjustable radial birefringent pupil filter. The device uses a liquid crystal device as a birefringent pupil filter, and can adjust the pupil function in real time by changing the voltage of the liquid crystal device during the imaging process, thereby effectively improving the resolution of confocal microscopy imaging and improving the imaging quality.

[0007] To achieve the above-mentioned purpose, the technical scheme of the present invention is as follows: a confocal microscope device based on an adjustable radial birefringent pupil filter, comprising a light source, a collimating beam expander, an illumination pinhole, a beam splitter, a liquid crystal radial birefringent pupil filter, a microscope objective, a sample, an imaging lens, an imaging pinhole, a detector, a voltage driving device and a PC control device.

[0008] The light emitted by the light source is formed into a point light source illumination through the collimating beam expander and the illumination pinhole, and is focused on the sample surface through the microscope objective. The light returning from the sample surface returns along the original path, is separated by the beam splitter and focused on the detector through the imaging lens, the liquid crystal radial birefringent pupil filter and the imaging pinhole. The liquid crystal radial birefringent pupil filter is used to modulate the pupil function of the system to achieve super-resolution, and the imaging pinhole is used to filter out signals outside the focal plane.

[0009] Furthermore, the above-mentioned device adjusts the voltage distribution of the liquid crystal radial birefringence pupil filter through a voltage driving device to form a circularly symmetric electric field distribution. The liquid crystal molecules in the liquid crystal layer are deflected under the action of the electric field to produce a radially continuous phase distribution, thereby modulating the pupil function of the system. Different pupil functions are selected according to different imaging requirements to achieve super-resolution imaging.

[0010] Furthermore, different pupil functions are generated by adjusting the voltage of the liquid crystal radial birefringence pupil filter. Specifically, the pupil function of the liquid crystal radial birefringence pupil filter is calculated by the following formula (1):

[0011] P(r)=P*L*P=cos[δ(r) / 2]+j sin[δ(r) / 2]cos(2θ) (1)

[0012] Wherein, P(r) is the pupil function generated by the liquid crystal radial birefringence pupil filter, L is the Jones matrix of the radial birefringence element, P is the Jones matrix of the polarizer, the polarization direction of the polarizer is along the x-axis, θ is the angle between the optical axis of the birefringence element and the x-axis, and δ(r) is the phase difference generated when light passes through the birefringence element.

[0013] Furthermore, it is assumed that the birefringent element is equivalent to a full-wave plate at the edge radial radius R and a half-wave plate at the center, and the phase delay distribution is the quadratic distribution function of an ideal lens. On this basis, by changing the parameters, the pupil function generated by the pupil filter can be calculated by the following formula (2):

[0014]

[0015] Wherein, P(r) is the pupil function generated by the liquid crystal radial birefringent pupil filter, P1, P2, θ are variable parameters, and R is the radius of the birefringent element.

[0016] On the other hand, the present invention provides a super-resolution imaging method for the above-mentioned confocal microscope device based on an adjustable radial birefringence pupil filter, specifically: imaging requirements and system parameters are input through a PC control device, and voltage parameters corresponding to the imaging requirements are searched in a database of voltages and pupil functions corresponding to each other constructed by experimental data, and then the voltage driving device is controlled to output the corresponding driving voltage, and the output driving voltage is used to control the amplitude, phase and frequency of the voltage on the electrode of the liquid crystal device, thereby achieving a phase distribution that meets the imaging requirements, thereby modulating the pupil function of the system, and then the PC control device controls the detector to collect data to achieve super-resolution imaging of the sample.

[0017] The beneficial effect of the present invention is that the phase distribution of the liquid crystal radial birefringence pupil filter is controlled by the voltage driving device to control the amplitude, frequency and phase of the liquid crystal radial birefringence pupil filter driving voltage, thereby modulating the system pupil function, and then respectively realizing lateral super-resolution, axial super-resolution and three-dimensional super-resolution. This characteristic of no mechanical movement makes the confocal microscope device more flexible and stable, and the liquid crystal radial birefringence pupil filter has a simple structure, convenient operation and lower preparation cost. Description of the drawings:

[0018] Figure 1 is a schematic structural diagram of a confocal microscope device based on an adjustable radial birefringence pupil filter provided by an embodiment of the present invention;

[0019] Figure 2 is a schematic structural diagram of an adjustable radial birefringence pupil filter provided by an embodiment of the present invention;

[0020] Figure 3 is a schematic structural diagram of a liquid crystal lens used as a radial birefringence element provided by an embodiment of the present invention;

[0021] Figure 4 θ is the variation of the axial super-resolution evaluation parameter with P1 under different θ provided by the embodiment of the present invention. (a) G A ; (b) S; (c) M A ;

[0022] Figure 5 is the variation of the lateral super-resolution evaluation parameter with P1 under different θ provided by the embodiment of the present invention. (a) G T ; (b) M T ;

[0023] Figure 6 It is a flow chart of a super-resolution imaging method of a confocal microscope device based on an adjustable radial birefringence pupil filter provided by an embodiment of the present invention;

[0024] Explanation of the reference numerals: 1. light source; 2. collimating beam expander; 3. illumination pinhole; 4. beam splitter; 5. liquid crystal radial birefringence pupil filter; 6. microscope objective; 7. sample; 8. imaging lens; 9. imaging pinhole; 10. detector; 11. voltage driving device; 12. PC control device; 13. first polarizer; 14. radial birefringence element; 15. second polarizer; 16. first transparent substrate; 17. first orientation layer; 18. liquid crystal layer; 19. second orientation layer; 20. second transparent substrate; 21. insulating layer; 22. third transparent substrate; 23. transparent circular hole electrode. Specific implementation method:

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1

[0027] like Figure 1 As shown, this embodiment provides a confocal microscope device based on an adjustable radial birefringence pupil filter, and the device includes: a light source 1, a collimating beam expander 2, an illumination pinhole 3, a beam splitter 4, a liquid crystal radial birefringence pupil filter 5, a microscope objective 6, a sample 7, an imaging lens 8, an imaging pinhole 9, a detector 10, a voltage driving device 11 and a PC control device 12.

[0028] The light emitted by the light source 1 is illuminated by a point light source through a collimating beam expander 2 and an illumination pinhole 3, and is focused on the surface of a sample 7 through a microscope objective 6. The light returning from the sample surface returns along the original path, is separated by a beam splitter 4, and is focused on a detector 10 through an imaging lens 8, a liquid crystal radial birefringent pupil filter 5, and an imaging pinhole 9. The liquid crystal radial birefringent pupil filter is used to modulate the pupil function of the system to achieve super-resolution, and the imaging pinhole is used to filter out signals outside the focal plane.

[0029] In this example, the voltage distribution of the liquid crystal radial birefringence pupil filter is adjusted by the voltage driving device 11 to form a circularly symmetric electric field distribution. The liquid crystal molecules in the liquid crystal layer are deflected under the action of the electric field to produce a radially continuous phase distribution, thereby modulating the pupil function of the system. Different pupil functions are selected according to different imaging requirements to achieve super-resolution imaging.

[0030] The liquid crystal radial birefringence pupil filter is composed of a first polarizer 13, a radial birefringence element 14, and a second polarizer 15. The radial birefringence element adopts a circular hole liquid crystal lens with an aperture of 2 mm. The liquid crystal lens is composed of a first transparent substrate 16, a first orientation layer 17, a liquid crystal layer 18, a second orientation layer 19, a second transparent substrate 20, an insulating layer 21, and a third transparent substrate 22. The first transparent substrate is coated with a transparent conductive film on one side, the second transparent substrate is coated with a circular hole-shaped transparent conductive film 23 on one side, and the third transparent substrate is coated with a transparent conductive film on one side.

[0031] In this embodiment, when the liquid crystal radial birefringence pupil filter is driven, the polarization directions of the first polarizer and the second polarizer are parallel, the angle between the optical axis of the liquid crystal lens and the polarizer is θ, the first transparent electrode is grounded, and there is a certain voltage difference between the second transparent electrode and the third transparent electrode.

[0032] like Figure 2 As shown, the pupil function of the liquid crystal radial birefringence pupil filter is calculated by the following formula:

[0033] P(r)=P*L*P=cos[δ(r) / 2]+j sin[δ(r) / 2]cos(2θ)

[0034] Wherein, P(r) is the pupil function generated by the liquid crystal radial birefringence pupil filter, L is the Jones matrix of the radial birefringence element, P is the Jones matrix of the polarizer, the polarization direction of the polarizer is along the x-axis, θ is the angle between the optical axis of the birefringence element and the x-axis, and δ(r) is the phase difference generated when light passes through the birefringence element.

[0035] Assume that the birefringent element is equivalent to a full-wave plate at the edge radial radius R and a half-wave plate at the center, and the phase delay distribution is the quadratic distribution function of an ideal lens. On this basis, the parameters are changed, and the pupil function generated by the pupil filter can be calculated by the following formula:

[0036]

[0037] Wherein, P(r) is the pupil function generated by the liquid crystal radial birefringent pupil filter, P1, P2, θ are variable parameters, and R is the radius of the birefringent element.

[0038] Example 2

[0039] In this embodiment, the values ​​of parameters P1, P2 and θ are adjusted according to the evaluation parameters of spot size ratio G, Strehl ratio S and maximum sidelobe intensity ratio M, so as to realize axial super-resolution, lateral super-resolution and three-dimensional super-resolution of the system respectively. The difference of the additional phase difference between the edge and the center of the liquid crystal birefringent element is fixed as π, that is, P2 is 1. Considering the focal shift introduced by the pupil filter element under certain θ, the imaging quality of axial super-resolution is first studied with the change of parameters. P1 changes from 0.5 to 1.5 with a step size of 0.01, and θ changes from 0π to 0.5π with a step size of 0.05π. The change of each parameter is as follows: Figure 4 As shown. Taking S greater than 0.5 as the design index, find G A The minimum case. When P1 = 1.13 to 1.37, θ = 0.3π and 0.35π, the axial super-resolution factor can be less than 0.9, and the G of 0.3π A Minimum G is 0.804, 0.35π A The minimum is 0.898. For the part with larger axial resolution when θ=0.3π, the sidelobe intensity is higher, and the maximum M=0.23 is greater than 0.1, while M is less than 0.1 at 0.35π, which has a smaller impact. Although good axial super-resolution can be achieved at 0.3π, the corresponding S is around 0.24, which is relatively low, while 0.35π can maintain a G of 0.898 at a relatively high S of 0.461. A Therefore, choosing θ = 0.35π and P1 = 1.38 can achieve axial super-resolution with better imaging quality in the system. A is 0.938, S is 0.512, M A It is 0.073.

[0040] Next, we study the changes of lateral super-resolution imaging quality with θ and P1, considering the influence of axial focal plane movement. Figure 5 As shown in the figure, when θ = 0.25π, the super-resolution factor between P1 = 0.5 and 0.62 is low. Then, when θ = 0.25π and P1 = 0.54, the lateral super-resolution with good imaging quality can be achieved in the system. At this time, G T is 0.85, S is 0.506, M T It is 0.062.

[0041] For the 3D super-resolution capability that considers both lateral and axial super-resolution, we also select the parameters with better axial super-resolution capability, θ = 0.35π, P1 = 1.38. At this time, G T is 0.938, G A is 0.922, and S is 0.51, which can achieve three-dimensional super-resolution with good imaging quality in the system.

[0042] Example 3

[0043] like Figure 6 As shown, this embodiment provides a super-resolution imaging method for the above-mentioned confocal microscope device based on an adjustable radial birefringence pupil filter, comprising the following steps: inputting imaging requirements and system parameters through a PC control device 12, searching for voltage parameters corresponding to the imaging requirements in a database of voltages and pupil functions corresponding to each other constructed by experimental data, and then controlling the voltage driving device to output a corresponding driving voltage, and using the output driving voltage to control the amplitude, phase and frequency of the voltage on the electrode of the liquid crystal device, thereby achieving a phase distribution that meets the imaging requirements, thereby modulating the pupil function of the system, and then the PC control device controls the detector to collect data to achieve super-resolution imaging of the sample.

[0044] The description and application of the present invention here are illustrative and are not intended to limit the scope of the present invention to the above-mentioned embodiments. Variations and changes of the embodiments disclosed here are possible, and replacements and various equivalent components of the embodiments are well known to those of ordinary skill in the art. It should be clear to those skilled in the art that the present invention can be implemented in other forms, structures, arrangements, proportions, and with other components, materials and parts without departing from the spirit or essential features of the present invention. Other variations and changes can be made to the embodiments disclosed here without departing from the scope and spirit of the present invention. The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments, and similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables professionals and technicians in this field to implement or use the present invention. Various modifications to these embodiments will be obvious to professionals and technicians in this field, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A confocal microscope device based on an adjustable radial birefringence pupil filter, comprising a light source, a collimating beam expander, an illumination pinhole, a beam splitter, a liquid crystal radial birefringence pupil filter, a microscope objective, a sample, an imaging lens, an imaging pinhole, a detector, a voltage driving device and a PC control device, characterized in that: The voltage distribution of the liquid crystal radial birefringence pupil filter is adjusted by a voltage driving device. The liquid crystal molecules are deflected under the action of the electric field to produce a continuous phase distribution, thereby modulating the pupil function of the system. Different pupil functions are selected according to different imaging requirements to achieve super-resolution imaging.

2. A confocal microscope device based on an adjustable radial birefringence pupil filter according to claim 1, characterized in that: The adjustable radial birefringence pupil filter is composed of a first polarizing plate, a radial birefringence element and a second polarizing plate.

3. A confocal microscope device based on an adjustable radial birefringence pupil filter according to claim 2, characterized in that: The pupil function generated by the liquid crystal radial birefringence pupil filter can be calculated by the following formula: P(r)=P*L*P=cos[δ(r) / 2]+j sin[δ(r) / 2]cos(2θ) Wherein, P(r) is the pupil function generated by the liquid crystal radial birefringence pupil filter, L is the Jones matrix of the radial birefringence element, P is the Jones matrix of the polarizer, the polarization direction of the polarizer is along the x-axis, θ is the angle between the optical axis of the birefringence element and the x-axis, and δ(r) is the phase difference generated when light passes through the birefringence element. Assume that the birefringent element is equivalent to a full-wave plate at the edge radial radius R and a half-wave plate at the center, and the phase delay distribution is the quadratic distribution function of the ideal lens. On this basis, the parameters are changed, and the pupil function generated by the pupil filter can be calculated by the following formula: Wherein, P(r) is the pupil function generated by the liquid crystal radial birefringent pupil filter, P1, P2, θ are variable parameters, and R is the radius of the birefringent element.

4. A confocal microscope device based on an adjustable radial birefringence pupil filter according to claim 2, characterized in that: The radial birefringence element adopts a liquid crystal lens, which is composed of a first transparent substrate, a first orientation layer, a liquid crystal layer, a second orientation layer, a second transparent substrate, an insulating layer and a third transparent substrate. The first transparent substrate is coated with a transparent conductive film on one side, the second transparent substrate is coated with a circular hole-shaped transparent conductive film on one side, and the third transparent substrate is coated with a transparent conductive film on one side.

5. A confocal microscope device based on an adjustable radial birefringence pupil filter according to claim 4, characterized in that: The electrode materials in the liquid crystal lens can be selected from indium tin oxide, graphene, metal nanowires, carbon nanotubes, conductive polymers, silver, copper and aluminum films, etc. The material of the orientation layer is the friction orientation agent PI, and the material of the liquid crystal layer is positive nematic phase liquid crystal.

6. A super-resolution imaging method of the confocal microscope device based on an adjustable radial birefringence pupil filter, characterized in that: The method comprises: inputting imaging requirements and system parameters through a PC control device, searching for voltage parameters corresponding to the imaging requirements in a database corresponding to voltages and pupil functions constructed by experimental data, then controlling a voltage driving device to output a corresponding driving voltage, using the output driving voltage to control the amplitude, phase and frequency of the voltage on the electrode of the liquid crystal device, and then achieving a phase distribution that meets the imaging requirements, thereby modulating the pupil function of the system to achieve super-resolution imaging of the sample, and then the PC control device controls the detector to collect data.

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