阅读说明：本技术 一种空心光束显微成像系统与显微成像方法 (Hollow beam microscopic imaging system and microscopic imaging method ) 是由 不公告发明人 于 2020-03-20 设计创作，主要内容包括：本发明提出一种空心光束显微成像系统与显微成像方法,所述系统包括：光源调节单元、空心光束调制单元和物镜成像单元,所述光源调节单元产生激光束并输出至空心光束调制单元,所述空心光束调制单元将光源调节单元产生的激光束按照预定分束比例分成非线性基础激光束和非线性调制激光束,并通过非线性基础激光束和非线性调制激光束的非线性相位调制产生空心光束,并输出至物镜成像单元,所述物镜成像单元基于所述空心光束进行成像。本发明基于非线性相位调制技术实现暗场成像,利用非线性调制激光束在非线性相位调制中产生的空心光束代替现有透射式显微成像系统中的挡光板,提高了成像光强度和成像质量。(The invention provides a hollow beam microscopic imaging system and a microscopic imaging method, wherein the system comprises: the laser imaging device comprises a light source adjusting unit, a hollow light beam modulating unit and an objective lens imaging unit, wherein the light source adjusting unit generates laser beams and outputs the laser beams to the hollow light beam modulating unit, the hollow light beam modulating unit divides the laser beams generated by the light source adjusting unit into nonlinear basic laser beams and nonlinear modulation laser beams according to a preset beam splitting ratio, the hollow light beams are generated through nonlinear phase modulation of the nonlinear basic laser beams and the nonlinear modulation laser beams and output to the objective lens imaging unit, and the objective lens imaging unit performs imaging based on the hollow light beams. The invention realizes dark field imaging based on the nonlinear phase modulation technology, and utilizes the hollow light beam generated by the nonlinear modulation laser beam in the nonlinear phase modulation to replace the light barrier in the existing transmission type microscopic imaging system, thereby improving the imaging light intensity and the imaging quality.)

1. A hollow beam microscopy imaging system, comprising: the light source adjusting unit generates a laser beam and outputs the laser beam to the hollow beam modulating unit, the hollow beam modulating unit divides the laser beam generated by the light source adjusting unit into a nonlinear basic laser beam and a nonlinear modulation laser beam according to a preset beam splitting ratio, generates a hollow beam through nonlinear phase modulation of the nonlinear basic laser beam and the nonlinear modulation laser beam and outputs the hollow beam to the dark field imaging unit, and the dark field imaging unit performs imaging based on the hollow beam.

2. The hollow beam microscopy imaging system according to claim 1, wherein the source conditioning unit comprises a laser (1), a linear polarizer (17) and a third half wave plate (16), the linear polarizer (17) being arranged between the laser (1) and the third half wave plate (16).

3. The hollow beam microscopy imaging system according to claim 1, wherein the hollow beam modulation unit comprises: the device comprises a first polarization light splitting cube (2), a focusing lens (3), a first half-wave plate (4), a second polarization light splitting cube (5), a nonlinear phase modulation unit (6), a first reflector (9), a second half-wave plate (8), a second reflector (10) and a third polarization light splitting cube (7), wherein the first polarization light splitting cube (2), the focusing lens (3), the first half-wave plate (4), the second polarization light splitting cube (5), the nonlinear phase modulation unit (6) and the third polarization light splitting cube (7) are sequentially arranged on the same straight line light path, and the first reflector (9), the second half-wave plate (8) and the second reflector (10) are sequentially arranged on the other parallel straight line light path; and a nonlinear basic laser optical path unit is formed by the first polarization beam splitting cube (2), the focusing lens (3), the first half wave plate (4) and the second polarization beam splitting cube (5) and is used for generating the nonlinear basic laser beam transmitted along the first direction, a nonlinear modulation laser optical path unit is formed by the first polarization beam splitting cube (2), the first reflecting mirror (9), the second half wave plate (8), the second reflecting mirror (10) and the third polarization beam splitting cube (7) and is used for generating the nonlinear modulation laser beam transmitted in a collinear way opposite to the first direction, and the nonlinear phase modulation unit (6) is positioned between the second polarization beam splitting cube (5) and the third polarization beam splitting cube (7).

4. The hollow beam microscopy imaging system according to claim 3, wherein the first polarizing beam splitter cube (2), the second polarizing beam splitter cube (5) and the third polarizing beam splitter cube (7) transmit horizontally polarized components in the laser beam and reflect vertically polarized components in the laser beam; the nonlinear fundamental laser beam incident into the nonlinear phase modulation unit (6) has a horizontal polarization state, and the nonlinear modulated laser beam incident into the nonlinear phase modulation unit (6) has a vertical polarization state; the first half-wave plate (4), the second half-wave plate (8) and the third half-wave plate (16) can rotationally adjust the polarization direction of the polarized light; the beam splitting ratio of the nonlinear basic laser beam and the nonlinear modulation laser beam is adjusted through a third half-wave plate (16) and the first polarization beam splitting cube (2); the power of the nonlinear fundamental laser beam incident into the nonlinear phase modulation cell (6) is adjusted by the first half-wave plate (4) and the second polarization beam splitter cube (5), and the power of the nonlinear modulated laser beam incident into the nonlinear phase modulation cell (6) is adjusted by the second half-wave plate (8) and the third polarization beam splitter cube (7).

5. Hollow beam microscopy imaging system according to claim 3 or 4, in the nonlinear modulation laser light path unit, a U-shaped nonlinear modulation laser light path is formed by the first polarization beam splitting cube (2), the first reflector (9), the second half-wave plate (8), the second reflector (10) and the third polarization beam splitting cube (7), wherein the first reflector (9) is arranged at the reflective output end of the first polarization beam splitting cube (2), and vertically reflects the nonlinear modulation laser beam reflected and output by the first polarization beam splitting cube (2) to a second reflecting mirror (10), the second reflecting mirror (10) vertically reflects the nonlinear modulation laser beam reflected by the first reflecting mirror (9) to the beam incident end of the third polarization beam splitting cube (7), the second half-wave plate (8) is arranged between the first mirror (9) and the second mirror (10).

6. The hollow beam microscopic imaging system according to any one of claims 3 to 5, wherein in the nonlinear fundamental laser light path unit, a linear nonlinear fundamental laser light path is composed of the first polarization beam splitting cube (2), the focusing lens (3), the first half-wave plate (4) and the second polarization beam splitting cube (5), the focusing lens (3) and the first half-wave plate (4) are disposed between the first polarization beam splitting cube (2) and the second polarization beam splitting cube (5), and the first half-wave plate (4) is located right behind the focusing lens (3), and the focusing lens (3) is used for focusing a waist spot of the nonlinear fundamental laser beam in the nonlinear phase modulation unit (6).

7. The hollow beam microscopic imaging system according to any one of claims 1 to 6, wherein the nonlinear phase modulation unit (6) employs a cuvette containing a nonlinear medium, the nonlinear modulation laser beam and the nonlinear fundamental laser beam jointly act on the nonlinear medium in the cuvette in a collinear reverse transmission manner, the nonlinear fundamental laser beam changes the refractive index distribution of the nonlinear medium through nonlinear action, the nonlinear modulation laser beam is subjected to phase shift on the exit end face of the nonlinear medium after passing through the nonlinear medium and forms the hollow beam comprising a central dark spot and a concentric ring in the exit far field, and the size of the hollow beam is changed by changing the power of the nonlinear fundamental laser beam incident into the nonlinear medium, and the hollow beam is output to the dark field imaging unit.

8. The hollow beam microscopy imaging system according to claim 7, wherein when the nonlinear fundamental laser beam is applied to the nonlinear medium in the cuvette, the refractive index profile of the nonlinear medium is:

n＝n₀+n₂I₁where n is the total refractive index of the non-linear medium, n₀Is a linear refractive index, I₁Is the intensity of a non-linear fundamental laser beam, n₂Is a thermally induced nonlinear refractive index, andwherein dn/dT is the temperature dependence of the refractive index of the nonlinear medium, alpha is the absorption coefficient, kappa is the heat conduction coefficient of the nonlinear medium, and omega is_pIs the radius of the nonlinear fundamental laser beam;

when the nonlinear basic laser beam acts on the nonlinear medium in the cuvette, the additional phase shift generated at the exit end face of the nonlinear medium is as follows:

wherein the exit end face of the nonlinear medium is used as the origin of coordinates, the transmission direction of the nonlinear modulation laser beam is the positive direction of the z axis, I₁(r, z) is the light intensity distribution of the nonlinear fundamental laser beam, I₁₀Is the central light intensity, k, of the nonlinear basic laser beam₀Is wave vector, ω_1p(z) is the spot radius, ω, of the nonlinear fundamental laser beam at different positions₁₀Is the beam waist radius of the nonlinear fundamental laser beam, r is the radial coordinate, and l is the effective active length of the nonlinear medium in the cuvette.

9. The hollow beam microscopy imaging system according to any one of claims 1 to 8, wherein the dark field imaging unit comprises: the device comprises a beam expander (11), an aspheric lens (12), an objective table (13), a glass slide (14) and a microscope objective (15), wherein the beam expander (11) is arranged right opposite to a hollow light beam output end of a hollow light beam modulation unit, the aspheric lens (12) is arranged right behind the beam expander (11), the microscope objective (15) is arranged right behind the aspheric lens (12), the objective table (13) is arranged between the aspheric lens (12) and the microscope objective (15), the glass slide (14) is arranged on the objective table (13), the glass slide (14) is close to or located at a focal position of the aspheric lens (12), and an emergent divergence angle of a hollow light beam is adjusted to be larger than an aperture angle of the microscope objective (15) through the beam expander (11) and the aspheric lens (12).

10. A method of microscopic imaging based on the hollow beam microscopic imaging system of any one of claims 1 to 9, comprising the steps of:

firstly, a laser beam output from a laser (1) is converted into a linearly polarized laser beam through a linear polarizer, the linearly polarized laser beam is subjected to polarization rotation adjustment through a third half-wave plate, and then penetrates through a first polarization beam splitting cube (2) to obtain a horizontally polarized nonlinear basic laser beam, the nonlinear basic laser beam sequentially passes through a focusing lens (3) and a first half-wave plate (4) and then enters a second polarization beam splitting cube (5), and the nonlinear basic laser beam penetrating through the second polarization beam splitting cube (5) is focused on a nonlinear phase modulation unit (6);

step two, the laser beam output from the laser (1) is converted into a linearly polarized laser beam by a linear polarizer, after polarization rotation adjustment is carried out on the laser beam by the third half-wave plate, a vertically polarized nonlinear modulation laser beam is obtained after the laser beam is reflected by the first polarization beam splitting cube (2), the nonlinear modulation laser beam is reflected by a first reflecting mirror (9) and a second reflecting mirror (10) and then enters a third polarization beam splitting cube (7), the nonlinear modulation laser beam passes through a second half-wave plate (8) in the period, the nonlinear modulation laser beam is reflected by the second polarization beam splitting cube (7) and then enters a nonlinear phase modulation unit (6), adjusting optical paths of the nonlinear modulation laser beam and the nonlinear fundamental laser beam so that the nonlinear modulation laser beam incident into the nonlinear phase modulation unit (6) is opposite to the propagation direction of the nonlinear fundamental laser beam and is propagated collinearly; the nonlinear modulation laser beam and the nonlinear basic laser beam are subjected to nonlinear phase modulation in the nonlinear phase modulation unit (6) to generate a hollow light beam, the hollow light beam is incident to the second polarization light-splitting cube (5), is reflected by the second polarization light-splitting cube (5), enters the beam expander (11), sequentially passes through the beam expander (11), the aspheric lens (12) and the glass slide and then enters the microscope objective lens;

thirdly, rotating a third half-wave plate (16) to adjust the beam splitting ratio of the nonlinear modulation laser beam reflected by the first polarization beam splitting cube (2) and the nonlinear basic laser beam transmitted by the first polarization beam splitting cube (2) of the laser beam output by the laser (1), and adjusting the beam splitting ratio of the nonlinear modulation laser beam and the nonlinear basic laser beam to a preset value;

fourthly, rotating the first half-wave plate (4) to dynamically adjust the power intensity of the nonlinear basic laser beam incident into the nonlinear phase modulation unit (6), and rotating the second half-wave plate (8) to adjust the power intensity of the nonlinear modulation laser beam incident into the nonlinear phase modulation unit (6) to a proper value until an obvious hollow beam is observed on the microscope objective;

fifthly, adjusting the position of the aspheric lens (12) to enable the divergence angle of the hollow light beam emitted by the aspheric lens to be larger than the aperture angle of the microscope objective (15);

and step six, placing the sample on a glass slide (14), and observing a microscopic imaging image of the sample by a microscope objective.

Technical Field

The invention relates to the field of microscopic imaging, in particular to a hollow beam microscopic imaging system and a microscopic imaging method.

Background

Optical microscopy is an important tool for studying the micro world. The microscope may be classified into an optical wide-field microscope, a confocal microscope, a stereoscopic microscope, and the like according to an imaging method. In an optical wide-field microscope, bright field, dark field, polarized light, fluorescence imaging and other technologies are developed according to the structural characteristics of a specimen to be observed. Most objects can be observed by bright field microscopy, but some samples are difficult to observe by bright field microscopy because the refractive index is similar to the surrounding environment, and dark field microscopy can well solve the problem. The principle of dark field microscopy is to prevent light transmitted through the specimen from entering the objective lens directly, and to allow only light scattered by the particles to enter the objective lens. Thus, the image plane formed by the objective lens is a scene with bright particles distributed on a dark background. Dark field microscopy imaging can enhance the contrast of the image. As an effective observation and detection means for microscopic samples, dark field microscopy is widely used in various fields. The illumination mode of dark field microscopy is divided into transmission type illumination and reflection type illumination, wherein the transmission type illumination uses a circular light shielding plate to shield the middle part of an illumination light beam to form a hollow conical focusing light beam. This method is simple and easy to implement, but the light blocking plate blocks most of the illumination light, so the overall transmittance of the light flux is low, and particularly the actual amount of light irradiated onto the sample is small. In the other reflective illumination mode, a dark field reflector is arranged at the position, close to a sample, of an objective lens shell, light is incident on the sample through the dark field reflector at an angle exceeding the numerical aperture of the objective lens, and diffraction or stray light emitted by the sample is collected by the objective lens and then imaged. The reflective illumination has the advantages that the illumination light is completely irradiated on the sample, the light quantity is relatively sufficient, but the reflective illumination has a complex structure and is difficult to realize.

Disclosure of Invention

The technical problem to be solved by the present invention is to provide a novel hollow beam microscopic imaging system and method for microscopic imaging, wherein the refractive index distribution of an ethanol solution is changed by applying a nonlinear basic laser beam to a nonlinear medium such as the ethanol solution, so as to change the phase distribution of a nonlinear modulation laser beam at the exit end face of the ethanol solution, and finally the far-field light intensity of the exiting nonlinear modulation laser beam is redistributed, so as to obtain a hollow beam composed of a central dark spot and concentric circles, wherein the size of the generated hollow beam can be adjusted and controlled by adjusting the intensity of the basic light, thus the invention innovatively utilizes the generated hollow beam to replace a light barrier in transmission type illumination. In addition, the invention innovatively utilizes the aspheric lens to focus the hollow light beam on the surface of the sample, and can quickly focus different depths of the sample by adjusting the size of the hollow light beam aiming at the samples with different thicknesses, and finally the light beam scattered by the sample can be observed through the objective lens. The device can obtain the imaging conditions of sample particles at different positions by adjusting the basic optical power, thereby realizing the fast focusing dark field imaging of the sample, renovating the existing microscopic imaging system and having wide popularization and application prospects.

In order to solve the technical problems, the invention adopts the technical scheme that:

a hollow beam microscopy imaging system comprising: the light source adjusting unit generates a laser beam and outputs the laser beam to the hollow beam modulating unit, the hollow beam modulating unit divides the laser beam generated by the light source adjusting unit into a nonlinear basic laser beam and a nonlinear modulation laser beam according to a preset beam splitting ratio, generates a hollow beam through nonlinear phase modulation of the nonlinear basic laser beam and the nonlinear modulation laser beam and outputs the hollow beam to the dark field imaging unit, and the dark field imaging unit performs imaging based on the hollow beam.

Further in accordance with the present invention, the hollow beam microscopy imaging system, wherein the source adjustment unit comprises a laser 1, a linear polarizer 17 and a third half-wave plate 16, the linear polarizer 17 being arranged between the laser 1 and the third half-wave plate 16.

Further, the hollow beam microscopy imaging system according to the present invention, wherein the hollow beam modulating unit comprises: the device comprises a first polarization light splitting cube 2, a focusing lens 3, a first half-wave plate 4, a second polarization light splitting cube 5, a nonlinear phase modulation unit 6, a first reflecting mirror 9, a second half-wave plate 8, a second reflecting mirror 10 and a third polarization light splitting cube 7, wherein the first polarization light splitting cube 2, the focusing lens 3, the first half-wave plate 4, the second polarization light splitting cube 5, the nonlinear phase modulation unit 6 and the third polarization light splitting cube 7 are sequentially arranged on the same straight line light path, and the first reflecting mirror 9, the second half-wave plate 8 and the second reflecting mirror 10 are sequentially arranged on the other parallel straight line light path; and wherein the first polarization beam splitter cube 2, the focusing lens 3, the first half-wave plate 4 and the second polarization beam splitter cube 5 constitute a nonlinear basic laser optical path unit for generating the nonlinear basic laser beam transmitted along the first direction, the first polarization beam splitter cube 2, the first reflector 9, the second half-wave plate 8, the second reflector 10 and the third polarization beam splitter cube 7 constitute a nonlinear modulation laser optical path unit for generating the nonlinear modulation laser beam transmitted in the opposite direction collineation with the first direction, and the nonlinear phase modulation unit 6 is located between the second polarization beam splitter cube 5 and the third polarization beam splitter cube 7.

Further according to the hollow beam micro-imaging system of the present invention, the first polarization beam splitting cube 2, the second polarization beam splitting cube 5 and the third polarization beam splitting cube 7 transmit the horizontal polarization component in the laser beam and reflect the vertical polarization component in the laser beam; the nonlinear fundamental laser beam incident into the nonlinear phase modulation unit 6 has a horizontal polarization state, and the nonlinear modulated laser beam incident into the nonlinear phase modulation unit 6 has a vertical polarization state; the first half-wave plate 4, the second half-wave plate 8 and the third half-wave plate 16 can rotationally adjust the polarization direction of the polarized light; the beam splitting ratio of the nonlinear basic laser beam and the nonlinear modulation laser beam is adjusted through the third half-wave plate 16 and the first polarization beam splitting cube 2; the power of the nonlinear fundamental laser beam incident into the nonlinear phase modulation cell 6 is adjusted by the first half-wave plate 4 and the second polarization beam splitter cube 5, and the power of the nonlinear modulated laser beam incident into the nonlinear phase modulation cell 6 is adjusted by the second half-wave plate 8 and the third polarization beam splitter cube 7.

Further, according to the hollow beam microscopic imaging system of the present invention, in the nonlinear modulation laser optical path unit, a U-shaped nonlinear modulation laser optical path is formed by the first polarization beam splitting cube 2, the first reflecting mirror 9, the second half-wave plate 8, the second reflecting mirror 10 and the third polarization beam splitting cube 7, wherein the first reflecting mirror 9 is disposed at the reflection output end of the first polarization beam splitting cube 2, and vertically reflects the nonlinear modulation laser beam reflected and output by the first polarization beam splitting cube 2 to the second reflecting mirror 10, the second reflecting mirror 10 vertically reflects the nonlinear modulation laser beam reflected by the first reflecting mirror 9 to the beam incident end of the third polarization beam splitting cube 7, and the second half-wave plate 8 is disposed between the first reflecting mirror 9 and the second reflecting mirror 10.

Further, according to the hollow beam microscopic imaging system of the present invention, in the nonlinear basic laser optical path unit, the first polarization beam splitter cube 2, the focusing lens 3, the first half-wave plate 4 and the second polarization beam splitter cube 5 constitute a linear nonlinear basic laser optical path, the focusing lens 3 and the first half-wave plate 4 are disposed between the first polarization beam splitter cube 2 and the second polarization beam splitter cube 5, the first half-wave plate 4 is located right behind the focusing lens 3, and the focusing lens 3 is configured to focus a waist spot of a nonlinear basic laser beam in the nonlinear phase modulation unit 6.

The hollow beam microscopic imaging system is further characterized in that the nonlinear phase modulation unit 6 adopts a cuvette filled with a nonlinear medium, the nonlinear modulation laser beam and the nonlinear basic laser beam jointly act on the nonlinear medium in the cuvette in a collinear reverse transmission mode, the nonlinear basic laser beam changes the refractive index distribution of the nonlinear medium through nonlinear action, the nonlinear modulation laser beam is subjected to phase shift on the emergent end face of the nonlinear medium after passing through the nonlinear medium, the hollow beam comprising a central dark spot and a concentric ring is formed in the emergent far field, the size of the hollow beam is changed by changing the power of the nonlinear basic laser beam incident into the nonlinear medium, and the hollow beam is output to the dark field imaging unit.

Further, according to the hollow beam microscopy imaging system of the present invention, when the nonlinear fundamental laser beam is applied to the nonlinear medium in the cuvette, the refractive index distribution of the nonlinear medium is:

n=n₀+n₂I₁where n is the total refractive index of the non-linear medium, n₀Is a linear refractive index, I₁Is the intensity of a non-linear fundamental laser beam, n₂Is a thermally induced nonlinear refractive index, and n₂=WhereinFor the temperature dependence of the refractive index of the nonlinear medium,in order to be able to take advantage of the absorption coefficient,is the coefficient of thermal conductivity of the non-linear medium,is the radius of the nonlinear fundamental laser beam;

when the nonlinear basic laser beam acts on the nonlinear medium in the cuvette, the additional phase shift generated at the exit end face of the nonlinear medium is as follows:

；

wherein the exit end face of the nonlinear medium is used as the origin of coordinates, the transmission direction of the nonlinear modulation laser beam is the positive direction of the z axis, I₁(r, z) is the light intensity distribution of the nonlinear fundamental laser beam, I₁₀Is the central light intensity, k, of the nonlinear basic laser beam₀The wave vector is the wave vector,for the spot radii of the nonlinear fundamental laser beam at different positions,is the beam waist radius of the nonlinear fundamental laser beam, r is the radial coordinate, and l is the effective active length of the nonlinear medium in the cuvette.

Further in accordance with the present invention, the hollow beam microscopy imaging system, wherein the dark field imaging unit comprises: the microscope comprises a beam expander 11, an aspheric lens 12, an objective table 13, a glass slide 14 and a microscope objective 15, wherein the beam expander 11 is arranged right opposite to a hollow light beam output end of a hollow light beam modulation unit, the aspheric lens 12 is arranged right behind the beam expander 11, the microscope objective 15 is arranged right behind the aspheric lens 12, the objective table 13 is arranged between the aspheric lens 12 and the microscope objective 15, the glass slide 14 is arranged on the objective table 13, the glass slide 14 is close to or located at a focus position of the aspheric lens 12, and an emergent divergence angle of the hollow light beam is adjusted to be larger than an aperture angle of the microscope objective 15 through the beam expander 11 and the aspheric lens 12.

The invention discloses a microscopic imaging method based on a hollow beam microscopic imaging system, which comprises the following steps:

firstly, a laser beam output from a laser 1 is converted into a linearly polarized laser beam through a linear polarizer, the linearly polarized laser beam is subjected to polarization rotation adjustment through a third half-wave plate, and then penetrates through a first polarization beam splitting cube 2 to obtain a horizontally polarized nonlinear basic laser beam, the nonlinear basic laser beam sequentially passes through a focusing lens 3 and a first half-wave plate 4 and then enters a second polarization beam splitting cube 5, and the nonlinear basic laser beam penetrating through the second polarization beam splitting cube 5 is focused on a nonlinear phase modulation unit 6;

step two, the laser beam output from the laser 1 is converted into a linearly polarized laser beam through a linear polarizer, after polarization rotation adjustment is performed through a third half-wave plate, the linearly polarized laser beam is reflected by the first polarization beam splitter cube 2 to obtain a vertically polarized nonlinear modulation laser beam, the nonlinear modulation laser beam is reflected by the first reflecting mirror 9 and the second reflecting mirror 10 and then enters the third polarization beam splitter cube 7, the nonlinear modulation laser beam passes through the second half-wave plate 8 in the period, the nonlinear modulation laser beam is reflected by the second polarization beam splitter cube 7 and then enters the nonlinear phase modulation unit 6, and the optical paths of the nonlinear modulation laser beam and the nonlinear basic laser beam are adjusted to enable the nonlinear modulation laser beam entering the nonlinear phase modulation unit 6 to be opposite to the transmission direction of the nonlinear basic laser beam and to be transmitted in a collinear mode; the nonlinear modulation laser beam and the nonlinear basic laser beam are subjected to nonlinear phase modulation in the nonlinear phase modulation unit 6 to generate a hollow light beam, the hollow light beam is incident on the second polarization light-splitting cube 5, is reflected by the second polarization light-splitting cube 5, enters the beam expander 11, and enters the microscope objective lens after passing through the beam expander 11, the aspheric lens 12 and the glass slide in sequence;

thirdly, rotating the third half-wave plate 16 to adjust the beam splitting ratio of the nonlinear modulation laser beam reflected by the first polarization beam splitting cube 2 and the nonlinear basic laser beam transmitted by the first polarization beam splitting cube 2 of the laser beam output by the laser 1, and adjusting the beam splitting ratio of the nonlinear modulation laser beam and the nonlinear basic laser beam to a preset value;

fourthly, rotating the first half-wave plate 4 to dynamically adjust the power intensity of the nonlinear basic laser beam incident into the nonlinear phase modulation unit 6, and rotating the second half-wave plate 8 to adjust the power intensity of the nonlinear basic laser beam incident into the nonlinear phase modulation unit 6 to a proper value until an obvious hollow beam is observed on the microscope objective;

fifthly, adjusting the position of the aspheric lens 12 to enable the divergence angle of the hollow light beam emitted by the aspheric lens to be larger than the aperture angle of the microscope objective 15;

and step six, placing the sample on a glass slide 14, and observing a microscopic imaging image of the sample by a microscope objective.

Compared with the prior art, the invention has the following advantages:

1) the invention provides a brand-new hollow beam microscopic imaging system and method, which are initiated based on a nonlinear phase modulation technology to realize fast focusing dark field imaging, change the refractive index distribution of a nonlinear medium through a nonlinear basic laser beam, further enable the nonlinear modulated laser beam to generate a hollow beam after passing through the nonlinear medium with the refractive index distribution changed, so as to replace a light baffle plate in the existing transmission type illumination, and meanwhile, the light intensity cannot be influenced, and the existing microscopic imaging system is innovated;

2) the hollow beam microscopic imaging system is realized based on the nonlinear phase modulation technology of a nonlinear medium, so that the size of the hollow beam can be changed by changing the basic light intensity, and the rapid focusing of a sample can be further realized, which is not possessed by the existing transmission type and reflection type microscopic imaging systems;

3) the hollow beam microscopic imaging system innovatively and jointly uses the hollow beam, the beam expander and the aspheric lens as the light condensing device, so that the light transmittance of the microscopic imaging system is very high, and the divergence angle of the aspheric lens is larger, thereby being beneficial to the objective lens to carry out high-quality light imaging on sample scattering;

4) the hollow light beam microscopic imaging system belongs to a great breakthrough of the existing microscopic imaging system, and has the unique advantages of simple structure, convenient operation, reasonable design, low cost and the like, thereby having wide market popularization and application prospect.

Drawings

FIG. 1 is a schematic diagram of the structure of the hollow beam microscopic imaging system according to the present invention;

the various reference numbers in the figures illustrate:

1. a laser; 2. a first polarization beam splitting cube; 3. a focusing lens; 4. a first half wave plate; 5. a second polarization beam splitting cube; 6. a nonlinear phase modulation unit; 7. a third polarization beam splitting cube; 8. a second half-wave plate; 9. a first reflector; 10. a second reflector; 11. a beam expander; 12. an aspherical lens; 13. an object stage; 14. a glass slide; 15. a microscope objective; 16. a third half-wave plate; 17. a linear polarizer.

Detailed Description

The following detailed description of the embodiments of the present invention is provided in conjunction with the accompanying drawings to enable those skilled in the art to more clearly understand the present invention, but not to limit the scope of the present invention.

The invention provides a hollow beam microscopic imaging system, which integrally comprises: the device comprises a light source adjusting unit, a hollow beam modulation unit and a dark field imaging unit.

The light source adjusting unit specifically comprises a laser 1, a linear polarizer 17 and a third half-wave plate 16, and is used for providing laser beam output, wherein the laser beam output by the laser 1 is a gaussian laser beam, is converted into linearly polarized light after passing through the linear polarizer 17, and is subjected to polarization direction rotation adjustment through the third half-wave plate 16.

The preferred laser 1 is a continuously tunable annular titanium sapphire laser with an output wavelength tuning range of 740nm-850nnm and a power of 600 mw. The laser generates a laser beam having a wavelength at an absorption wavelength of the nonlinear phase modulation unit.

The third half-wave plate 16 is disposed behind the linearly polarizing plate 17, and belongs to an optical element well known in the art, and can rotate linearly polarized light. The linearly polarized light vertically enters the half-wave plate, the transmitted light is still linearly polarized light, and if the included angle between the vibration plane and the main cross section of the crystal is theta during incidence, the vibration plane of the linearly polarized light which is transmitted out is rotated by an angle of 2 theta from the original direction. In the present optical path, the third half-wave plate 17 is rotated to change the beam splitting ratio between the horizontal polarization state and the vertical polarization state after the laser beam output by the laser passes through the first polarization beam splitting cube 2, as described below, the polarization beam splitting cube transmits a light beam in one polarization state (e.g. the horizontal polarization state) and reflects a light beam in the other polarization state (e.g. the vertical polarization state), the polarization direction of the linear polarization laser beam incident on the polarization beam splitting cube can be changed by rotating the half-wave plate, and further, the beam splitting ratio between two polarization light beams with mutually perpendicular polarization states split out by the polarization beam splitting cube of the incident linear polarization light is changed, specifically, to the present optical path, the polarization direction of the linear polarization laser beam converted by the linear polarization plate is adjusted by rotating the third half-wave plate 16, and further, the beam splitting ratio between the nonlinear base laser beam in the horizontal polarization state and the nonlinear modulation laser beam in the vertical polarization state split by the linear polarization laser beam split from the first polarization beam splitting cube 2 can be adjusted, the purpose of simultaneously changing the intensity of the nonlinear basic laser beam and the nonlinear modulation laser beam is achieved.

The hollow beam modulation unit is used for generating a hollow beam with controllable size, and comprises a nonlinear basic laser light path unit, a nonlinear modulation laser light path unit and a nonlinear phase modulation unit, and is shown in the attached drawing 1, wherein the hollow beam modulation unit specifically comprises a first polarization light-splitting cube 2, a focusing lens 3, a first half-wave plate 4, a second polarization light-splitting cube 5, a nonlinear phase modulation unit 6, a first reflector 9, a second half-wave plate 8, a second reflector 10 and a third polarization light-splitting cube 7. The nonlinear phase modulation device comprises a first polarization light splitting cube 2, a focusing lens 3, a first half-wave plate 4 and a second polarization light splitting cube 5, wherein the first polarization light splitting cube 2, the first reflector 9, a second half-wave plate 8, a second reflector 10 and a third polarization light splitting cube 7 form a nonlinear basic laser light path unit, the nonlinear phase modulation unit 6 adopts a cuvette filled with ethanol, and the ethanol is absolute ethanol and is used as an optical medium for performing nonlinear action with a laser beam. The nonlinear basic laser beam from the nonlinear basic laser optical path unit and the nonlinear modulation laser beam from the nonlinear modulation laser optical path unit are collinearly and reversely incident into the anhydrous ethanol of the cuvette, nonlinear action based on nonlinear phase modulation occurs in the nonlinear basic laser beam and the nonlinear modulation laser beam to generate a hollow light beam with controllable size, and the generated hollow light beam is reflected by the second polarization light-splitting cube 5 and is output to the dark field imaging unit.

The laser 1 of the light source adjusting unit is coupled with the nonlinear basic laser light path unit and the nonlinear modulation laser light path unit respectively to generate a nonlinear basic laser beam and a nonlinear modulation laser beam which are transmitted in a collinear reverse direction, and the nonlinear basic laser beam and the nonlinear modulation laser beam are incident into the nonlinear phase modulation unit, specifically, the laser 1, the linear polarizer 17, the third half-wave plate 16, the first polarization beam-splitting cube 2, the focusing lens 3, the first half-wave plate 4, the second polarization beam-splitting cube 5 and the nonlinear phase modulation unit 6 are sequentially arranged on the same linear light path, the first polarization beam-splitting cube 2, the first reflector 9, the second half-wave plate 8, the second reflector 10 and the third polarization beam-splitting cube 7 are sequentially arranged on the same U-shaped light path, wherein the optical characteristics and the light path setting positions of each optical element are as follows:

the first polarization beam splitting cube 2 is arranged right behind the third half-wave plate 16, and has the following optical characteristics: performing beam splitting based on the difference of the polarization states of the beams, specifically, the first polarization splitting cube 2 transmits the beam in the horizontal polarization direction and reflects the beam in the vertical polarization direction, the first polarization splitting cube 2 couples the horizontal polarization component in the laser beam output after passing through the third half-wave plate into the nonlinear basic laser optical path unit in a polarization splitting manner, the vertical polarization component in the laser beam is coupled into the nonlinear modulation laser optical path unit in a polarization splitting manner, and after the laser beam output specifically through the third half-wave plate enters the first polarization splitting cube 2, the laser beam penetrating through the first polarization splitting cube 2 has the horizontal polarization state which serves as the nonlinear basic laser beam; the laser beam that is vertically reflected by the first polarization beam splitter cube 2 after the output laser beam is incident on the first polarization beam splitter cube 2 has a vertical polarization state, which is a nonlinear modulated laser beam.

The focusing lens 3 is arranged right behind the transmission light splitting output end of the first polarization light splitting cube 2 and focuses the nonlinear basic laser beam from the first polarization light splitting cube 2 to a cuvette filled with ethanol solution; preferably, the focal length of the focusing lens 3 is 200 mm.

The first half-wave plate 4 is disposed right behind the focusing lens 3, and the linear polarization direction of the nonlinear basic laser beam transmitted and output by the first polarization beam splitter cube 2 can be adjusted by rotating the first half-wave plate 4, so that the polarization direction of the nonlinear basic laser beam deviates from the horizontal direction, and further, the intensity (power) of the beam in the horizontal polarization direction, which is split again by the second polarization beam splitter cube 5, of the nonlinear basic laser beam is adjusted (reduced), and therefore, the power of the nonlinear basic laser beam incident into the cross-phase modulation unit 6 can be adjusted by rotating and adjusting the first half-wave plate 4 in combination with the second polarization beam splitter cube 5.

The second polarization light splitting cube 5 is disposed right behind the first half-wave plate 4, and has optical characteristics of splitting light beams based on the difference of polarization states of the light beams, and specifically, the second polarization light splitting cube 2 transmits the light beams in the horizontal polarization state and reflects the light beams in the vertical polarization state. The function in the optical path is to transmit and output the nonlinear fundamental laser beam with the horizontal polarization component to the cross phase modulation unit 6, and to adjust the nonlinear fundamental laser beam power incident into the cross phase modulation unit 6 in cooperation with the first half-wave plate 4, while the second polarization beam splitter cube 5 can reflect and output the hollow beam with the vertical polarization state generated based on the nonlinear phase modulation to the dark field imaging unit.

The first reflecting mirror 9 is a right-angle reflecting mirror, is arranged facing the reflected light splitting output end of the first polarization light splitting cube 2, and provides 90-degree reflection for the vertically polarized light output by the first polarization light splitting cube 2.

The second reflecting mirror 10 is a right-angle reflecting mirror, the reflecting surface of the second reflecting mirror 10 is perpendicular to the reflecting surface of the first reflecting mirror 9, and the reflected light beam from the first reflecting mirror 9 enters the reflecting surface of the second reflecting mirror 10 and is reflected perpendicularly (reflected by 90 °) by the reflecting surface of the second reflecting mirror to the third polarization beam splitting cube 7.

The second half-wave plate 8 is arranged between the first reflecting mirror 9 and the second reflecting mirror 10, the action of the second half-wave plate on the first half-wave plate is similar to that of the first half-wave plate, the linear polarization direction of the nonlinear modulation laser beam reflected and output by the first polarization beam splitter cube 2 can be adjusted by rotating the second half-wave plate 8, the polarization direction of the nonlinear modulation laser beam deviates from the vertical direction, and then the intensity (power) of the nonlinear modulation laser beam in the vertical polarization direction which is split again by the third polarization beam splitter cube 7 is adjusted (reduced), so that the power of the nonlinear modulation laser beam incident into the cross phase modulation unit 6 can be adjusted by rotating and adjusting the second half-wave plate 8 and combining the third polarization beam splitter cube 7.

The light beam input end of the third polarization light splitting cube 7 is over against the reflection output end of the second reflector 10, the reflection output end of the third polarization light splitting cube 7 is over against the transmission output end of the second polarization light splitting cube 5, the optical characteristics of the third polarization light splitting cube 7 are that light beams are split based on different polarization states of the light beams, and specifically, the third polarization light splitting cube 7 transmits the light beams in the horizontal polarization state and vertically reflects the light beams in the vertical polarization state. The third polarization light splitting cube 7 has the function of reflecting and outputting the vertically polarized nonlinear modulation laser beam reflected and output by the second reflector 10 towards the transmission output end of the second polarization light splitting cube 5 in the optical path, so that the vertically polarized nonlinear modulation laser beam reflected and output by the third polarization light splitting cube 7 and the horizontally polarized nonlinear base laser beam transmitted and output by the second polarization light splitting cube 5 reach collinear reverse transmission, and are jointly incident into the nonlinear phase modulation unit 6 in a collinear reverse mode; while the third polarizing beam splitter cube 7 provides a transmission output for the horizontally polarized non-linear base laser beam from the second polarizing beam splitter cube 5, a light collector may preferably be provided at the transmission output.

The nonlinear phase modulation unit 6 adopts a cuvette filled with a nonlinear medium (preferably absolute ethyl alcohol (purity > 99%)), is arranged between the transmission output end of the second polarization light-splitting cube 5 and the reflection output end of the third polarization light-splitting cube 7, and jointly irradiates a nonlinear basic laser beam with a horizontal polarization state transmitted and output by the second polarization light-splitting cube 5 and a nonlinear modulation laser beam with a vertical polarization state reflected and output by the third polarization light-splitting cube 7 into the cuvette filled with ethyl alcohol, and the nonlinear basic laser beam and the nonlinear modulation laser beam are reversely and collinearly transmitted in the cuvette filled with ethyl alcohol. The nonlinear basic laser beam and the nonlinear modulation laser beam have the cross-phase nonlinear modulation effect in absolute ethyl alcohol: the cross-phase nonlinear modulation means that when two or even a plurality of light fields with the same or different frequencies act on a nonlinear medium at the same time, one strong light field causes the change of the refractive index distribution of the nonlinear medium, so that the light beams of other light fields undergo nonlinear phase shift when passing through the nonlinear medium, and the purpose of nonlinear phase modulation of the other light fields by using the strong light field is achieved. Specifically, for the invention, when a stronger nonlinear basic laser beam acts on nonlinear medium ethanol, the refractive index distribution of the ethanol is changed, so that the phase distribution of the nonlinear modulation laser beam at the position of an emergent end face of an ethanol solution is changed (phase shift), and finally the far-field emergent light intensity of the nonlinear modulation laser beam carrying the nonlinear phase shift is redistributed to obtain a hollow light beam consisting of a central dark spot and a concentric circle, wherein the central dark spot is equivalent to replace a light barrier in the prior art, thereby ensuring that dark field imaging can be realized, and the change degree of the refractive index distribution of the ethanol can be adjusted by adjusting the intensity of the nonlinear basic laser beam, so that the phase shift degree of the nonlinear modulation laser beam is adjusted and reflected in the adjustment and control of the size of the generated hollow light beam, and simultaneously, the nonlinear basic laser beam and the nonlinear modulation collinear laser beam are reversely transmitted to generate the nonlinear phase shift through the nonlinear basic laser beam The generated hollow beam is also transmitted along the direction opposite to the nonlinear basic laser beam, and the separation output of the nonlinear basic laser beam is well realized.

The following specifically shows the principle process of nonlinear phase modulation of a nonlinear fundamental laser beam and a nonlinear modulated laser beam in an ethanol solution. Theoretically, when a laser beam passes through a nonlinear medium (here, ethanol), the nonlinear medium absorbs the energy of the laser, causing local heating of the medium and temperature increase, resulting in a temperature gradient, and thus causing thermal diffusion. The refractive index is subsequently changed as a result of the density distribution of the medium being changed as a result of the propagation of the light wave in the medium.

Thermally induced nonlinear refractive index n under steady state conditions₂Is represented by the following formula:

n₂= （1）；

whereinFor a given temperature dependence of the refractive index of the medium,in order to be able to take advantage of the absorption coefficient,for a given heat transfer coefficient of the medium,is the radius of the beam.

Considering the thermally induced nonlinear process, the dependence of the refractive index of the medium on the light intensity is expressed by the following formula:

n=n₀+n₂I，I=I₁+I₂ （2）。

where n is the total refractive index, n₀Is the linear refractive index, I is the total light intensity, I₁Based on the intensity of the laser light, I₂To modulate the laser intensity.

Since the power of the modulated laser is very weak compared to the basic laser, the effect of the non-linearly modulated laser beam on the whole process can be neglected, so that: n = n₀+n₂I₁ （3）。

At this time, when the nonlinear modulation laser beam passes through the nonlinear medium ethanol, an additional phase shift is generated at the exit surface of the medium. The modulated light transmission direction is assumed to be the positive direction, i.e., the z-axis, and the right end surface of the medium is assumed to be the origin of coordinates. The additional phase shift at the exit end face of the medium due to the non-linear refractive index is represented by the following formula:

（4）。

wherein I₁(r, z) is the intensity distribution of the basic light, I₁₀Based on the central intensity of the light, k₀The wave vector is the wave vector,the spot radii at different positions of the basis light,is the beam waist radius of the base light, r is the radial coordinate, and l is the effective length of the sample cell.

Thus, the complex amplitude E of the optical field of the modulated light transmitted through the nonlinear medium₂(r, z) can be represented as:

（5）

where E20 is the complex amplitude of the optical electric field of the incident modulated light and R (z) is the wavefront radius of curvature of the corresponding location.

Since it is mainly the focus of the fundamental light that plays a role in the phase-shift modulation of the modulated light by the fundamental light, equation 4 can be approximated asWherein。

Based on the above principle, it is known that the phenomenon of generating a hollow beam is a non-linear phase modulation due to thermally induced non-linear effects. When the fundamental light laser passes through the nonlinear medium, the refractive index of the medium changes, and when the modulated light passes through the nonlinear medium, a negative lateral additional phase shift is generated on the exit surface of the medium. Because of the additional phase shift, the modulated light will transform the original incident gaussian beam distribution into a hollow beam in the far field. Meanwhile, the refractive index and the phase shift distribution formula are both related to the intensity of the basic beam, so that the size of the hollow beam generated by the nonlinear modulation laser beam can be adjusted by adjusting the intensity of the nonlinear basic beam.

The dark field imaging unit comprises a beam expander 11, an aspheric lens 12, an objective table 13, a glass slide 14 and a microscope objective 15, wherein the beam expander 11 is arranged right opposite to the reflection output end of the second polarization light-splitting cube 5, a hollow light beam with a vertical polarization state, which is generated by nonlinear phase modulation of a nonlinear basic laser beam and a nonlinear modulation laser beam in a cuvette through the nonlinear phase modulation, is reflected by the second polarization light-splitting cube 5 and then output to the beam expander 11, and the aspheric lens 12 is arranged right behind the beam expander 11. The exit divergence angle of the hollow light beam is adjusted through the beam expander 11 and the aspheric lens 12, so that the exit divergence angle of the hollow light beam is larger than the aperture angle of the selected microscope objective 15, and dark field imaging is ensured. The specific method for adjusting the outgoing divergence angle of the hollow light beam comprises the following steps: the beam expanding proportion of the beam expander 11 and the focal length of the aspheric lens 12 are selected according to the aperture angle of the microscope objective 15, the generated hollow light beam sequentially passes through the beam expander 11 and the aspheric lens 12, the position of the aspheric lens 12 is adjusted to enable the emergent divergence angle of the hollow light beam output by the aspheric lens 12 to be larger than the aperture angle of the microscope objective 15, and after the hollow light beam preferentially passes through the beam expander, the radius of the hollow light beam is about 2-3 cm. The glass slide 14 is arranged behind the light beam output end of the aspheric lens 12, the glass slide 14 is arranged on the object stage 13, a sample to be imaged is arranged on the glass slide 14, and the sample to be imaged is positioned near the focus of the light beam output by the aspheric lens 12 on the glass slide 14, so that the hollow light beam focused on the surface of the glass slide by the aspheric lens 12 is diverged and output after passing through the sample. The light beam input end of the microscope objective 15 is arranged right opposite to a sample on the glass slide, and the input aperture angle of the microscope objective 15 relative to the glass slide is smaller than the emergent divergence angle of the hollow light beam passing through the glass slide, so that the central dark field of the hollow light beam plays a role of being equivalent to a light baffle plate in the existing transmission type illumination, but the weakening effect of the light baffle plate on the illumination light intensity is avoided, meanwhile, the larger emergent divergence angle of the hollow light beam ensures that the effective light beam entering the microscope objective is a sample particle scattering light beam, and further the dark field imaging quality is improved. In addition, the aspheric lens is utilized to focus the hollow light beam on the surface of the sample, and for samples with different thicknesses, the size of the hollow light beam can be adjusted by adjusting the intensity of basic light, so that the samples are quickly focused at different depths, and the quick focusing dark field imaging of the sample is realized.

The invention further provides a hollow beam microscopic imaging method based on the hollow beam microscopic imaging system, which comprises the following steps:

the method comprises the following steps that firstly, a linear polarization Gaussian laser beam output from a light source adjusting unit passes through a first polarization light-splitting cube 2 to obtain a horizontally polarized nonlinear basic laser beam, the nonlinear basic laser beam passes through a focusing lens 3 and a first half-wave plate 4 in sequence and then enters a second polarization light-splitting cube 5, and the nonlinear basic laser beam passing through the second polarization light-splitting cube 5 is focused in a cuvette filled with absolute ethyl alcohol;

step two, a laser beam output from the light source adjusting unit is reflected by the first polarization beam splitting cube 2 to obtain a vertically polarized nonlinear modulation laser beam, the nonlinear modulation laser beam is reflected by the first reflecting mirror 9 and the second reflecting mirror 10 and then enters the third polarization beam splitting cube 7, the nonlinear modulation laser beam passes through the second half-wave plate 8 in the period, the nonlinear modulation laser beam is vertically reflected by the second polarization beam splitting cube 7 and then enters a cuvette filled with absolute ethyl alcohol, and the nonlinear modulation laser beam entering the cuvette is opposite to the transmission direction of the nonlinear modulation laser beam and the nonlinear basic laser beam and is transmitted in a collinear way by adjusting the light paths of the nonlinear modulation laser beam and the nonlinear basic laser beam; the nonlinear basic laser beam acts on the absolute ethyl alcohol in the cuvette and changes the refractive index distribution of the absolute ethyl alcohol based on a nonlinear effect, then after the nonlinear modulation laser beam passes through the absolute ethyl alcohol with the changed refractive index distribution, the phase distribution of the nonlinear modulation laser beam at the position of an ethanol emergent end face is correspondingly changed, finally, the far-field light intensity of the nonlinear modulation laser beam emergent from the cuvette is redistributed, a hollow light beam consisting of a central dark spot and a concentric circle is obtained, the generated hollow light beam has a vertical polarization state and is incident into a second polarization light-splitting cube 5 along the transmission direction of the nonlinear modulation laser beam, the hollow light beam is reflected by the second polarization light-splitting cube 5 and then enters a beam expander 11, and the hollow light beam sequentially passes through the beam expander 11 and an aspheric lens 12 and then enters a microscope objective;

thirdly, rotating the third half-wave plate 16 to adjust the beam splitting ratio of the nonlinear modulation laser beam reflected by the first polarization beam splitting cube 2 and the nonlinear basic laser beam transmitted by the first polarization beam splitting cube 3 of the laser beam output by the laser 1, and adjusting the beam splitting ratio of the nonlinear modulation laser beam and the nonlinear basic laser beam to a preset value;

and step four, dynamically adjusting the power intensity of the nonlinear basic laser beam incident into the absolute ethyl alcohol in the cuvette by rotating the first half-wave plate 4, and adjusting the power intensity of the nonlinear modulation laser beam incident into the absolute ethyl alcohol in the cuvette to a proper value (fixed) by rotating the second half-wave plate 8 until an obvious hollow beam is observed on the microscope objective.

And step five, further adjusting the position of the aspheric lens 12 to enable the divergence angle of the emergent light of the aspheric lens to be larger than the aperture angle of the microscope objective 15, and when only a dark background with a bright point is seen in the microscope objective, determining that the divergence angle is larger than the aperture angle.

Step five, then the sample is placed on a glass slide 14 near the focal point of the aspherical lens, and the sample is observed with a microscope objective 15.

Step six, after the positions of the aspheric lens 12, the objective table 13 and the microscope objective 15 are determined, the size of the hollow light beam is adjusted by adjusting the angle of each half-wave plate, and finally the rapid focusing of the sample is realized (as can be known from the formula 3, the nonlinear refractive index is increased along with the increase of the basic light intensity, and as can be known from the formula 4, when the basic light power is increased, the nonlinear additional phase shift is also increased, and the size of the middle dark spot corresponding to the hollow light beam is also correspondingly increased, therefore, the size of the hollow light beam generated by the nonlinear modulation laser beam through the nonlinear action can be adjusted by adjusting the power of the nonlinear basic laser beam by rotating the half-wave plate, and the change of the size of the hollow light beam can change the light flux focused on the sample, thereby realizing the imaging of the sample at different positions in the sample pool, and realizing the imaging of the sample at different positions without moving the sample pool, and the fast focusing under dark field imaging is realized.

To sum up, the invention provides a brand-new hollow light beam microscopic imaging system and an imaging method, which are initiated to realize fast focusing dark field imaging based on a nonlinear phase modulation technology, change the refractive index of a nonlinear medium through a nonlinear basic laser beam, further enable the nonlinear modulated laser beam to generate a hollow light beam after passing through the nonlinear medium with the changed refractive index, so as to replace a light baffle plate in the existing transmission type illumination, simultaneously not to influence the light intensity of the dark field, simultaneously enable the size of the hollow light beam to be changed by changing the basic light intensity based on the cross phase debugging of the nonlinear medium, further realize fast focusing on a sample, simultaneously innovatively and combinatively use the hollow light beam, a beam expander and an aspheric lens as a condensing device, enable the light transmittance of the microscopic imaging system to be very high, and the divergence angle of the aspheric lens to be larger, thus being beneficial for an objective lens to carry out high-quality light imaging on the sample scattering, therefore, the hollow light beam microscopic imaging system belongs to a great breakthrough of the existing microscopic imaging system and has wide market popularization and application prospect.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, changes and equivalent structural changes made to the above embodiments according to the technical spirit of the present invention still fall within the protection scope of the technical solution of the present invention.