CN113433681B - Structured light illumination microscopic imaging system and method - Google Patents

Abstract

The application relates to a structured light illumination microscopic imaging system and a structured light illumination microscopic imaging method, and relates to the technical field of structured light illumination microscopic imaging. In the structured light illumination microscopic imaging system, an illumination system is used for emitting light wave signals to a diffraction optical system. The diffraction optical system is used for modulating the received light wave signals, generating two-dimensional sinusoidal periodic dot matrix light spots, and scanning the target sample by using the two-dimensional sinusoidal periodic dot matrix light spots. And the displacement platform is used for moving the target sample so that the two-dimensional sinusoidal periodic lattice light spot performs phase-shift scanning on the target sample. The microscopic imaging system is used for acquiring a scanning image of the target sample after the displacement table moves the target sample every time, and performing super-resolution reconstruction based on the acquired multiple scanning images. The structured light illumination microscopic imaging system has a relatively simple illumination light path, and only a diffraction optical element needs to be added in the illumination light path, so that the structural complexity of the structured light illumination microscopic imaging system is simplified.

Description

Structured light illumination microscopic imaging system and method

Technical Field

The present application relates to the technical field of structured light illumination microscopic imaging, and in particular to a structured light illumination microscopic imaging system and method.

Background Art

Structured illumination microscopy (SIM) is a super-resolution technology that uses periodic structured light patterns to illuminate samples to improve imaging resolution.

Current SIM systems usually need to generate periodic structured light to illuminate samples through interference or projection methods, and their working process includes many links such as periodic modulation of the light field, spatial filtering, polarization control, and coupling with the microscope optical path. Each link needs to be completed based on specific equipment, such as spatial light modulator (SLM), digital micromirror device (DMD), etc.

Due to the complex structure of the SIM system, it is bulky. The current commercial Nikon microscope SIM system has a size of 550mm×300mm×350mm, which is not suitable for point-of-care testing (POCT). As a result, the SIM system cannot be widely promoted and applied.

Summary of the invention

Based on this, it is necessary to provide a structured light illumination microscopic imaging system and method to address the above technical issues.

The present application provides a structured light illumination microscopic imaging system, comprising:

An illumination system, used for emitting a light wave signal to a diffraction optical system;

A diffraction optical system is used to modulate the received light wave signal to generate a two-dimensional sinusoidal periodic lattice light spot, and use the two-dimensional sinusoidal periodic lattice light spot to scan the target sample;

A translation stage is used to move the target sample so that the two-dimensional sinusoidal periodic dot matrix light spot performs phase shift scanning on the target sample;

The microscopic imaging system is used to obtain a scanned image of the target sample after the translation stage moves the target sample each time, and perform super-resolution reconstruction based on the obtained multiple scanned images.

In one embodiment, the lighting system comprises:

An illumination component, used for emitting a light wave signal;

The collimating and beam expanding component is used to collimate and expand the light wave signal so that the spot size of the light wave signal adapts to the receiving surface size of the diffractive optical system.

In one embodiment, the lighting component is a laser or LED light emitter, or other incoherent light sources.

In one embodiment, a diffractive optical system comprises a diffractive optical element and an illumination objective, wherein:

The diffractive optical element is attached to the entrance pupil position of the illumination objective lens or is located at a preset position in front of the entrance pupil position of the illumination objective lens, and the aperture size of the diffractive optical element is adapted to the entrance pupil size of the illumination objective lens, and the target sample is located on the back focal plane of the illumination objective lens;

A diffractive optical element, used for modulating the light field distribution of the received light wave signal;

The illumination objective lens is used to cooperate with the diffractive optical element to generate a two-dimensional sinusoidal periodic dot-matrix light spot on the back focal plane of the illumination objective lens.

In one of the embodiments, the diffractive optical element is any one or any combination of a binary optical element, a holographic optical element, a micro-nano optical element, a metasurface and a spatial light modulator.

In one embodiment, the system further comprises a control system,

The control system is used to control the movement of the translation stage and control the microscopic imaging system to obtain a scanning image of the target sample.

In one embodiment, the microscopic imaging system comprises:

An imaging objective lens is used to image a target sample scanned by a two-dimensional sinusoidal periodic dot-matrix light spot to obtain an initial image;

A fluorescent color filter, used for filtering the initial image;

a tube lens for aberration correction and magnification matching of the initial image;

The image sensor is used to obtain a scanned image of a target sample and perform super-resolution reconstruction based on multiple scanned images.

In one embodiment, the two-dimensional sinusoidal periodic lattice light spot is a lattice light spot generated by adding or multiplying two or more one-dimensional sinusoidal distribution patterns in different directions in a plane.

In one embodiment, the mathematical model of the two-dimensional sinusoidal periodic dot-matrix light spot is:

or,

为二维结构光图案光强分布，n为结构光照明图案的序号，在两种不同的图案下，n通常为[1,5]与[1,9]区间的整数，

为空域内的位置矢量，

分别为二维结构光图案在x、y方向周期所对应的空间频率，φ_xn、φ_yn分别为第n帧原始图像在x、y方向的相位参数。in,

is the light intensity distribution of the two-dimensional structured light pattern, n is the serial number of the structured light illumination pattern, and in two different patterns, n is usually an integer between [1,5] and [1,9].

is the position vector in the airspace,

are the spatial frequencies corresponding to the periods of the two-dimensional structured light pattern in the x and y directions, respectively; φ _xn and φ _yn are the phase parameters of the original image of the nth frame in the x and y directions, respectively.

Second aspect:

The present application provides a structured light illumination microscopic imaging method, comprising:

A two-dimensional sinusoidal periodic lattice light spot is generated by using a diffractive optical element, and the two-dimensional sinusoidal periodic lattice light spot is imaged on a target sample of a structured light illumination microscope imaging system;

The target sample is phase-shifted scanned using a two-dimensional sinusoidal periodic dot-matrix light spot to obtain multiple scanned images, and super-resolution reconstruction is performed based on the multiple scanned images.

The structured light illumination microscopic imaging system and method provided in the embodiments of the present application have the advantages of simple structure, small size, fast speed, and good imaging quality. In the structured light illumination microscopic imaging system, the illumination system is used to emit a light wave signal to the diffraction optical system, and the diffraction optical system is used to modulate the received light wave signal to generate a two-dimensional sinusoidal periodic lattice spot, and use the two-dimensional sinusoidal periodic lattice spot to scan the target sample. The displacement stage is used to move the target sample so that the two-dimensional sinusoidal periodic lattice spot performs phase shift scanning on the target sample. The microscopic imaging system is used to obtain a scanned image of the target sample after the displacement stage moves the target sample each time, and perform super-resolution reconstruction based on the obtained multiple scanned images. The illumination optical path of the structured light illumination microscopic imaging system provided in the embodiments of the present application is relatively simple, and it can be achieved by simply adding a diffraction optical element to the illumination optical path, or by imaging the diffraction optical element at or near the entrance pupil of the illumination objective through the imaging system; the structural complexity of the structured light illumination microscopic imaging system is simplified, and the cost of the structured light illumination microscopic imaging system is reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic diagram of the structure of a structured light illumination microscopic imaging system provided in an embodiment of the present application;

FIG2 is a schematic diagram of a lighting system provided in an embodiment of the present application;

FIG3 is a schematic diagram of a diffractive optical system provided in an embodiment of the present application;

FIG4 is a schematic diagram of a microscopic imaging system provided in an embodiment of the present application;

FIG5 is a schematic diagram of a control system provided in an embodiment of the present application;

FIG6 is a basic flow chart of a diffractive optical element design provided in an embodiment of the present application;

FIG7 is a schematic diagram of the distribution of a two-dimensional sinusoidal periodic lattice light spot provided in an embodiment of the present application;

FIG8 is an original image of a two-dimensional sinusoidal periodic dot-matrix light spot irradiated onto a target sample;

FIG9 is a schematic diagram of experimental results of imaging microspheres using a structured light illumination microscopic imaging system provided in an embodiment of the present application;

FIG. 10 is a schematic diagram of the imaging results of cell microtubules by the structured light illumination microscopy imaging system provided in an embodiment of the present application.

DETAILED DESCRIPTION

In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific implementation disclosed below.

Structured illumination microscopy (SIM) is a super-resolution technology that uses periodic structured light patterns to illuminate samples to improve imaging resolution.

Generally, microscope imaging can be regarded as a low-pass filtering process of the sample's spatial spectrum. The high-frequency spectrum representing the sample details will be lost in the filtering process, thus limiting the sample image resolution.

In the prior art, structured light illumination microscopy (SIM) uses periodic structured light patterns in the spatial domain to illuminate the sample, shifting the super-resolution spectrum information that the original optical imaging system cannot transmit into the detectable area of the system, and aliasing it in the spatial spectrum of the original image for detection. Subsequently, it is only necessary to calculate and separate these super-resolution spectra to expand the range of the sample's spatial spectrum, thereby improving the resolution of the corresponding spatial domain image.

In practical applications, since structured light patterns need to be generated through an objective lens, their spatial frequency is also limited by the diffraction limit, so the spatial spectrum range (diameter) can usually be expanded by 1 times, that is, the imaging resolution is increased by 1 times. The imaging resolution of structured light illumination microscopes in the visible light band is generally around 100nm. Since SIM super-resolution imaging requires very few original images to be collected, it has a significant imaging speed advantage over other technologies. Even though its resolution is not as good as that of single molecule localization super-resolution microscopy (SMLM) and stimulated emission dissipation (STED) microscopy, SIM has still become one of the several super-resolution technologies widely used today.

At present, the demand for an application called Point-of-care testing (POCT) is growing rapidly in the field of modern medical research. In the past, patients usually had to go to large medical institutions in person for precise medical tests or take samples from patients and then send them to large medical institutions for precise medical tests. The emergence of POCT allows patients to take samples and test them in the community or at home, significantly shortening the interval from sampling to sample testing, avoiding sample changes caused by long-term transportation or environmental changes, and affecting the test results. The characteristics of point-of-care testing itself, which can be used anytime, anywhere and widely, determine that the testing equipment used must be compact, portable, simple and flexible to use, and low-cost.

As one of the most suitable technologies for in vivo imaging in super-resolution technology, SIM must be miniaturized to meet the application needs of future POCT. However, the current SIM is a bulky and expensive microscopy system, which is far from meeting the requirements of POCT applications for equipment portability and cost reduction. The fundamental reason is that the current SIM usually needs to generate periodic structured light to illuminate the sample by interference or projection. The periodic modulation of the light field, spatial filtering, polarization control, and coupling with the microscope optical path in the system make the SIM system bulky (the size of the current commercial Nikon microscope SIM system is: 550mm×300mm×350mm). The spatial light modulator (SLM) and digital micromirror device (DMD) used are also expensive, which brings many obstacles to the further promotion and application of SIM.

The embodiment of the present application provides a structured light illumination super-resolution microscopic imaging system based on a diffractive optical element 1021, which can reduce the size of a SIM and reduce the cost of a SIM.

Please refer to Figure 1, which is a structural schematic diagram of a structured light illumination microscopic imaging system provided in an embodiment of the present application. The structured light illumination microscopic imaging system includes an illumination system 101, a diffraction optical system 102, a translation stage (not shown in the figure) and a microscopic imaging system 103.

The illumination system 101 is used to emit a light wave signal to the diffraction optical system 102. Optionally, in the embodiment of the present application, the light wave signal may be a collimated wave, a spherical wave, a Gaussian beam, or an illumination light wave of arbitrary distribution.

The diffraction optical system 102 is used to modulate the received light wave signal to generate a two-dimensional sinusoidal periodic lattice light spot, and use the two-dimensional sinusoidal periodic lattice light spot to scan the target sample.

The translation stage is used to move the target sample so that the two-dimensional sinusoidal periodic dot matrix light spot performs phase shift scanning on the target sample.

The microscopic imaging system 103 is used to obtain a scanned image of the target sample each time the translation stage moves the target sample, and perform super-resolution reconstruction based on the obtained multiple scanned images.

Optionally, in the structured illumination microscopy imaging system provided by the present application, due to the feedback control of the displacement stage itself, after each phase shift scan, it is necessary to wait for a certain stabilization time of the displacement stage. Generally, it is necessary to wait for the displacement stage to stabilize before starting to shoot after each displacement. In actual use, in order to simplify the control process, the trigger signal of the displacement stage can be used to directly control the image sensor to shoot, and the sample image can be shot immediately after the displacement stage moves.

It can be understood that in the embodiment of the present invention, the illumination system 101 is used to emit a light wave signal to the diffraction optical system 102, and the diffraction optical system 102 modulates the light wave signal to generate a two-dimensional sinusoidal periodic lattice light spot in the airspace as structured light, and uses the structured light (two-dimensional sinusoidal periodic lattice light spot) to illuminate the target sample, and the target sample is moved by the displacement stage to achieve phase shift detection and reconstruct super-resolution information. The microscopic imaging system 103 is used to record the sample information of the target sample at different positions and perform super-resolution reconstruction. The characteristic of this method is that the diffraction optical system 102 is used to generate a two-dimensional sinusoidal periodic lattice (array) light spot as structured light illumination, and it can be realized by cooperating with an ordinary computer for data processing, and has the advantages of simple structure, small size, fast speed, good imaging quality, etc., and is suitable for medical and biological application needs for instant detection.

The structured light illumination microscopic imaging system provided in the embodiment of the present application combines the advantages of super-resolution of structured light illumination microscopes and the advantages of miniaturization of diffractive optical elements, and has the advantages of simple structure, small size, fast speed, good imaging quality, etc., which provides possibilities for application needs such as instant detection; further, based on the diffractive optical system, a diffraction method is used to realize the illumination of the target sample by a two-dimensional sinusoidal periodic lattice light spot, thereby improving the imaging efficiency and energy utilization; the illumination optical path of the structured light illumination microscopic imaging system provided in the embodiment of the present application is relatively simple, and it only needs to add a diffractive optical element to the illumination optical path, or to image the diffractive optical element at or near the entrance pupil of the illumination objective through an imaging system; the structural complexity of the structured light illumination microscopic imaging system is simplified, and the cost of the structured light illumination microscopic imaging system is reduced.

In one embodiment, the embodiment of the present application also provides a structured light illumination microscopy imaging method, which is applied to the structured light illumination microscopy imaging system provided in the embodiment of the present application. The method includes using a diffraction optical element to generate a two-dimensional sinusoidal periodic lattice light spot, and imaging the two-dimensional sinusoidal periodic lattice light spot on a target sample of the structured light illumination microscope imaging system; using the two-dimensional sinusoidal periodic lattice light spot to perform phase shifting scanning on the target sample to obtain multiple scanned images, and performing super-resolution reconstruction based on the multiple scanned images.

The light wave signal passes through the illumination system and the diffractive optical system in sequence, and generates a two-dimensional sinusoidal periodic lattice light spot on the back focal plane of the illumination objective lens of the diffractive optical system, that is, a two-dimensional sinusoidal periodic lattice light spot is generated on the sample surface to illuminate the target sample. The target sample is detected by multi-step phase shifting using a translation stage; the information of the target sample passes through the imaging objective lens, fluorescent filter, tube lens and image sensor in sequence, and the image sensor processes the scanned images of the target sample at different positions to reconstruct a super-resolution image.

In one embodiment, as shown in FIG. 2 , the illumination system 101 includes an illumination component 1011 and a collimating and beam expanding component 1012 .

The lighting assembly 1011 is used to emit the light wave signal. In the embodiment of the present application, the light wave signal can be a monochromatic laser signal, an LED light signal, or an incoherent light signal, and the light source can be a laser, an LED light emitter, or other incoherent light sources.

The collimating and beam expanding assembly 1012 is used to collimate and expand the light wave signal so that the spot size of the light wave signal adapts to the size of the receiving surface of the diffractive optical system 102 .

The collimation and beam expansion assembly 1012 includes a collimator and a beam expander, wherein the collimator is used to convert the light waves emitted by the light source into collimated light beams, and the beam expander is used to expand the light wave signals and expand the aperture of the light source. After collimation and beam expansion, a uniform light wave of a preset size can be obtained. The preset size is the receiving surface size of the diffractive optical system 102.

The spot size of the light wave signal after collimation and beam expansion adapts to the size of the receiving surface of the diffractive optical system 102, which may mean that the spot size of the light wave signal after collimation and beam expansion is equal to the size of the receiving surface of the diffractive optical system 102. It may also mean that the spot of the light wave signal after collimation and beam expansion can be completely irradiated onto the receiving surface of the diffractive optical system 102.

By making the spot size of the light wave signal adapt to the size of the receiving surface of the diffractive optical system 102, the light wave signal can be incident into the diffractive optical system 102 to the maximum extent, thereby improving the resolution of the microscopic imaging system.

In one embodiment, as shown in FIG3 , the diffractive optical system 102 includes a diffractive optical element 1021 (English: Diffractive Optical Element, referred to as: DOE) and an illumination objective lens 1022, wherein: the diffractive optical element 1021 can be attached to the entrance pupil position of the illumination objective lens 1022, or can be located at a preset position in front of the entrance pupil position of the illumination objective lens 1022, wherein the preset position in front refers to a position at a preset distance from the entrance pupil plane of the illumination objective lens 1022. The aperture size of the diffractive optical element 1021 is adapted to the entrance pupil size of the illumination objective lens 1022, and the target sample is located on the back focal plane of the illumination objective lens 1022.

In practical applications, if the aperture size of the diffractive optical element 1021 is larger than the entrance pupil size of the illumination objective lens 1022, the formed two-dimensional sinusoidal periodic lattice spot will be deformed, affecting the super-resolution reconstruction effect. If the aperture size of the diffractive optical element 1021 is smaller than the entrance pupil size of the illumination objective lens 1022, the resolution of the microscopic imaging system will be reduced. If the aperture size of the diffractive optical element 1021 is equal to the entrance pupil size of the illumination objective lens 1022, the best resolution effect can be achieved.

The diffractive optical element 1021 is used to modulate the light field distribution of the received light wave signal.

The illumination objective lens 1022 is used to cooperate with the diffractive optical element 1021 to generate a two-dimensional sinusoidal periodic dot-matrix light spot on the back focal plane of the illumination objective lens 1022 .

Optionally, the diffractive optical element 1021 may be a binary optical element, or may be one or a combination of various elements such as a holographic optical element, a micro-nano optical element, a metasurface, and a spatial light modulator that realize phase modulation and/or amplitude modulation of a light field.

Optionally, in the embodiment of the present application, the diffractive optical element 1021 can be used to adjust factors such as the period and number of the two-dimensional sinusoidal periodic dot lattice light spots on the rear focal plane of the illumination objective lens 1022.

Optionally, the two-dimensional sinusoidal periodic lattice light spot may be a lattice light spot generated by adding or multiplying two or more one-dimensional sinusoidal distribution patterns in different directions in a plane.

It should be noted that the collimation and expansion assembly is used to collimate and expand the light wave signal so that the spot size of the light wave signal adapts to the receiving surface size of the diffractive optical system 102 . Its essence is to adapt the spot size of the light wave signal to the receiving surface size of the diffractive optical element 1021 .

It is understandable that the diffractive optical element 1021 can modulate the light field distribution of the collimated and expanded light wave signal, so that a specific light spot pattern is formed in the airspace after modulation, that is, a two-dimensional sinusoidal periodic lattice light spot. Among them, the illumination objective lens 1022 is used to cooperate with the diffractive optical element 1021 to present the two-dimensional sinusoidal periodic lattice light spot on the back focal plane of the illumination objective lens 1022. Among them, the target sample is located on the back focal plane of the illumination objective lens 1022, so that the two-dimensional sinusoidal periodic lattice light spot can just irradiate the target sample, thereby achieving the purpose of scanning the target sample.

In the embodiment of the present application, a two-dimensional sinusoidal periodic lattice light spot is generated by the diffraction optical system 102, and the two-dimensional sinusoidal periodic lattice light spot is used to scan the target sample, so that more information on the target sample can be obtained.

In one embodiment, as shown in FIG. 4 , the microscopic imaging system 103 includes an imaging objective lens 1031 , a fluorescent color filter 1032 , a tube lens 1033 , and an image sensor 1034 , wherein:

The imaging objective lens 1031 is used to image the target sample scanned by the two-dimensional sinusoidal periodic dot-matrix spot to obtain an initial image. The fluorescent color filter 1032 is used to filter the initial image. The tube lens 1033 is used to perform aberration correction and magnification matching on the initial image. The image sensor 1034 is used to obtain a scanned image of the target sample and perform super-resolution reconstruction based on multiple scanned images.

Optionally, in an embodiment of the present application, the image sensor 1034 includes a data processing module, which can process scanned images of target samples at different positions to reconstruct super-resolution images.

It can be understood that the imaging objective 1031 images the target sample illuminated by the two-dimensional sinusoidal periodic dot matrix light spot; the fluorescent filter 1032 can improve the signal-to-noise ratio of the microscopic imaging system, enhance the fluorescence signal in the excitation band, and minimize unnecessary radiation; the tube lens 1033 is used to match and correct the magnification and aberration of the microscopic imaging system; and the image sensor 1034 is used to obtain information of the target sample under the illumination of the two-dimensional sinusoidal periodic dot matrix light spot.

Optionally, the image sensor can be various devices for acquiring image information, such as CMOS (Complementary Metal Oxide Semiconductor), sCMOS (Science Complementary Metal Oxide Semiconductor), CCD (Charge-coupled Device), and EMCCD (Electron-Multiplying Charge-coupled Device).

In one embodiment, as shown in FIG. 5 , the structured light illumination microscopic imaging system provided in the embodiment of the present application further includes a control system 105 , and the control system 105 is used to control the movement of the translation stage 104 and control the microscopic imaging system 103 to obtain a scanning image of the target sample.

Optionally, the control system can also be connected to the illumination system 101 and the diffraction optical system 102 to control the illumination system 101 and the diffraction optical system 102 to start, wherein after the illumination system 101 and the diffraction optical system 102 are started, the illumination system 101 emits a light wave signal to the diffraction optical element, and the diffraction optical element modulates the light wave signal to form a two-dimensional sinusoidal periodic dot matrix spot on the back focal plane of the illumination objective lens. At the same time, the control system controls the movement of the translation stage 104, and after each movement, controls the microscopic imaging system 103 to obtain a scanning image of the target sample, thereby obtaining multiple scanning images, and then performing super-resolution reconstruction based on the multiple scanning images.

It is understandable that from the principle of structured light microscopy, the spatial frequency of the two-dimensional structured light illumination stripes is the main factor that determines the resolution improvement of the structured light microscopy system. As long as the sample is illuminated with a two-dimensional structured light pattern that meets the requirements, the structured light microscope can achieve the corresponding linear/nonlinear super-resolution imaging. Therefore, accurately generating the required two-dimensional structured light illumination pattern is crucial for super-resolution microscopy.

In the structured light illumination microscopic imaging system provided in the embodiment of the present application, the two-dimensional structured light illumination pattern is a two-dimensional sinusoidal periodic dot matrix light spot. The process of designing the two-dimensional sinusoidal periodic dot matrix light spot in the present application is described below:

In the embodiment of the present application, the mathematical model of the two-dimensional sinusoidal periodic dot-matrix light spot includes the following two types:

and,

为二维正弦周期性点阵光斑的光强分布，n为二维正弦周期性点阵光斑的序号，在两种不同的图案下，n通常为[1,5]与[1,9]区间的整数，

为空域内的位置矢量，

分别为二维正弦周期性点阵光斑在x、y方向周期所对应的空间频率，φ_xn、φ_yn分别为第n帧原始图像在x、y方向的相位参数。in,

is the light intensity distribution of the two-dimensional sinusoidal periodic lattice light spot, n is the serial number of the two-dimensional sinusoidal periodic lattice light spot, and in two different patterns, n is usually an integer between [1,5] and [1,9].

is the position vector in the airspace,

are the spatial frequencies corresponding to the periods of the two-dimensional sinusoidal periodic dot matrix light spots in the x and y directions, respectively; φ _xn and φ _yn are the phase parameters of the original image of the nth frame in the x and y directions, respectively.

分别为

二维正弦周期性点阵光斑的空间频谱，

为空间频域内的频率矢量，δ为狄拉克脉冲函数。in,

They are

The spatial spectrum of a two-dimensional sinusoidal periodic dot-matrix light spot.

is the frequency vector in the spatial frequency domain, and δ is the Dirac pulse function.

Under plane wave incidence, the transmittance function of the diffractive optical element DOE is in a Fourier transform relationship with the Fraunhofer diffraction field of the objective focal plane (i.e., the sample plane). At this time, how to design the phase distribution of the DOE so that the intensity distribution of the diffraction field is a two-dimensional sinusoidal periodic lattice spot with a certain spatial frequency is the core issue. For a given target diffraction field distribution, the phase of the DOE can be designed using an iterative Fourier transform algorithm (IFTA). As shown in Figure 6, the main steps include:

1. Assign the initial equal-amplitude phase field to the DOE plane:

为DOE平面内光场的振幅分布，

为DOE平面内光场的相位分布。Where i is the number of iterations,

is the amplitude distribution of the light field in the DOE plane,

is the phase distribution of the light field in the DOE plane.

2. Calculate the diffraction field distribution of the sample plane through Fourier transform:

Where τ ⁽ⁱ⁾ (x) is the amplitude distribution at the rear focal plane of the objective (i.e., the sample plane), and ψ ⁽ⁱ⁾ (x) is the phase distribution.

3. In the sample plane, the amplitude distribution of the target diffraction field (two-dimensional structured light illumination pattern) is used to replace the amplitude distribution of the sample plane, and the phase distribution remains unchanged to obtain a new light field:

4. Calculate the DOE plane light field distribution through inverse Fourier transform:

重新置为均匀振幅分布。5. Distribute the light field amplitude on the DOE plane

Reset to uniform amplitude distribution.

将收敛为近似的均匀分布，而样品平面光场的振幅分布τ⁽ⁱ⁾(x)则收敛到目标衍射场(二维结构光照明图案)的振幅分布。此时DOE平面的相位分布

即为所需设计的DOE相位，通过衍射可生成所需的二维正弦周期性点阵光斑。Repeat steps 2-5 iteratively. Light field amplitude distribution at the DOE plane

The phase distribution of the DOE plane is ^:

That is the desired designed DOE phase, and the desired two-dimensional sinusoidal periodic lattice spot can be generated by diffraction.

The performance of the structured light illumination microscopic imaging system provided in the present application is described below with reference to examples.

In an embodiment of the present application, a 488nm laser can be used to generate wide-aperture collimated light through a collimating and expanding assembly, which is modulated by a diffractive optical element DOE installed at or near the entrance pupil of the illumination objective lens, and incident on the illumination objective lens to generate a designed two-dimensional structured light illumination pattern on the rear focal plane of the illumination objective lens, that is, a two-dimensional sinusoidal periodic dot matrix spot.

Among them, the diffractive optical element DOE and the two-dimensional sinusoidal periodic lattice light spot generated by its diffraction are shown in Figure 7. In Figure 7, (a) represents the phase distribution of the diffractive optical element, (b) represents the light intensity distribution of the two-dimensional sinusoidal periodic lattice light spot, (c) and (b) represent the light intensity curves at the horizontal line position, and (d) and (b) represent the light intensity curves at the vertical line position.

After the target sample is illuminated by the two-dimensional sinusoidal periodic lattice light spot, the corresponding image is imaged by the imaging objective lens and collected by the image sensor, as shown in Figure 8. The target sample is phase-shifted and scanned using a translation stage to capture original images of the target sample at different positions. In this way, the structured light illumination microscopy imaging system provided in the embodiment of the present application has two major elements that SIM must meet for super-resolution imaging: two-dimensional sinusoidal periodic lattice light spot illumination of the target sample and phase-shift detection of the two-dimensional sinusoidal periodic lattice light spot.

In the embodiment of the present application, according to the different illumination objective lenses and the design of the diffractive optical element, the generated two-dimensional sinusoidal periodic lattice spot changes accordingly, and the maximum spatial frequency is about twice the spatial frequency corresponding to the diffraction limit resolution determined by the illumination objective lens 1022. The phase-shift scanning step of the translation stage is set to about one-third of the illumination pattern period, so that the phase-shift scanning can achieve relatively uniform sampling within one period of two or more directions of the two-dimensional illumination pattern, so as to ensure that the reconstruction result obtains a uniform resolution improvement effect in the entire field of view. Further iterative reconstruction can improve the quality of super-resolution microscopic imaging of the sample.

As shown in Figure 9, it is the imaging result of fluorescent microspheres by the structured light illumination super-resolution microscopy system based on diffractive optical elements. Figure 9 (a) shows the imaging result of the microspheres under wide-field illumination. (b) shows the super-resolution result under illumination of a two-dimensional sinusoidal periodic dot-matrix light spot generated by modulation of the diffractive optical element. (c) shows the light intensity curves of the wide-field image and the super-resolution image at the position selected by the line. It can be seen intuitively from Figure 9 that the resolution of the super-resolution image is significantly improved compared to the wide-field image. Among them, the local enlarged images of (a) and (b) show that the adjacent microspheres that were originally indistinguishable in the wide-field image can be distinguished in the image of the structured light illumination super-resolution microscopy system. The light intensity curve at the line position is taken for analysis as shown in (c). After fitting, the center distance between the two microspheres is 567nm, which has exceeded the theoretical diffraction limit resolution of 990nm.

Further, in order to verify the biological imaging capability of the structured light illumination microscopic imaging system provided in the embodiment of the present application, an imaging experiment was performed on a fixed cell sample of A549 cells, wherein A549 cell adenocarcinoma human alveolar basal epithelial cells are samples commonly used to study the type II lung epithelial cell model in drug metabolism. Among them, the cell microtubules are labeled with dyes, and the experimental results are shown in Figure 10. Among them, Figure 10 (a) shows the wide-field results, local magnification results (lower right) and spatial spectrum of wide-field imaging (upper right) of cell microtubules. (b) shows the super-resolution results, local magnification results (lower right) and spatial spectrum of super-resolution imaging (upper right) of cell microtubules under two-dimensional sinusoidal periodic dot matrix illumination modulated and generated by the diffractive optical element 1021. (c) shows the light intensity curves of the wide-field image and the super-resolution image at the position selected by the line. The comparison between the wide-field image and the super-resolution image of cell microtubules also shows a significant resolution improvement effect. A lot of evidence of resolution improvement can be found in the field of view, as shown in (a) and (b). In the locally enlarged area, the cell microtubules observed in the wide-field image are blurred and the microtubule structure cannot be distinguished, while the super-resolution image shows a clear microtubule structure. In addition, the spatial spectrum of the super-resolution image has also been significantly expanded compared to the wide-field image, which can also prove that the imaging resolution of the system has exceeded the diffraction limit. The light intensity curves of the wide-field image and the super-resolution image are compared at the line position. As shown in (c), in the wide-field image, two close cell microtubules cannot be distinguished, but they can be successfully distinguished in the super-resolution image. After data fitting, the centers of the two microtubules are about 419nm apart, which is less than the diffraction limit of 625nm corresponding to the fluorescence wavelength, achieving super-resolution imaging.

The technical features of the above embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims.

Claims (9)

1. A structured light illuminated microscopy imaging system, comprising:

an illumination system for emitting a lightwave signal onto the diffractive optical system;

the diffraction optical system is used for modulating the received light wave signal to generate a two-dimensional sinusoidal periodic lattice light spot and scanning a target sample by using the two-dimensional sinusoidal periodic lattice light spot;

the diffraction optical system comprises a diffraction optical element and an illumination objective lens, wherein the diffraction optical element is used for modulating the light field distribution of the received light wave signal and adjusting the period and the number of the two-dimensional sinusoidal periodic lattice light spots on the back focal plane of the illumination objective lens; the illumination objective lens is used for being matched with the diffraction optical element to generate the two-dimensional sine periodic dot matrix light spot on the back focal plane of the illumination objective lens;

the displacement platform is used for moving the target sample so that the two-dimensional sinusoidal periodic lattice light spot performs phase-shifting scanning on the target sample;

the microscopic imaging system is used for acquiring a scanning image of the target sample after the displacement table moves the target sample every time and performing super-resolution reconstruction on the basis of the acquired multiple scanning images;

the two-dimensional sinusoidal periodic lattice light spots are lattice light spots generated by adding or multiplying two or more one-dimensional sinusoidal distribution patterns in different directions in a plane.

2. The structured light illuminated microscopy imaging system of claim 1, wherein the illumination system comprises:

an illumination assembly for emitting the lightwave signal;

and the collimation and beam expansion assembly is used for collimating and expanding the light wave signals so as to adapt the spot size of the light wave signals to the size of a receiving surface of the diffraction optical system.

3. The structured light illuminated microscopy imaging system of claim 2, wherein the illumination assembly is a laser or an LED illuminator.

4. The structured light illuminated microscopy imaging system as claimed in claim 1, wherein the diffractive optical element is attached to or located at a predetermined position in front of the entrance pupil position of the illumination objective, and the aperture size of the diffractive optical element is adapted to the entrance pupil size of the illumination objective, and the target sample is located on the back focal plane of the illumination objective.

5. The structured light illuminated microscopy imaging system of claim 1, wherein the diffractive optical element is any one or any combination of a binary optical element, a holographic optical element, a micro-nano optical element, a super-structured surface and a spatial light modulator.

6. The structured light illuminated microscopy imaging system of claim 1, wherein the system further comprises a control system,

and the control system is used for controlling the displacement table to move and controlling the microscopic imaging system to acquire the scanning image of the target sample.

7. The structured light illuminated microscopy imaging system of claim 1, wherein the microscopy imaging system comprises:

the imaging objective lens is used for imaging the target sample scanned by the two-dimensional sinusoidal periodic dot matrix light spots to obtain an initial image;

the fluorescence color filter is used for filtering the initial image;

a tube lens for performing aberration correction and magnification matching on the initial image;

and the image sensor is used for acquiring the scanning images of the target sample and performing super-resolution reconstruction according to the plurality of scanning images.

8. The structured light illuminated microscopy imaging system of claim 1, wherein the mathematical model of the two-dimensional sinusoidal periodic lattice spot is:

or ,

wherein ,

for a two-dimensional structured light pattern intensity distribution, n is the number of the structured light illumination pattern, and n is usually [1,9 ] in two different patterns]The integer number of the interval (a) is,

for the position vector in the null field,

the spatial frequency phi corresponding to the period of the two-dimensional structured light pattern in the x and y directions _xn 、φ _yn The phase parameters of the n frame original image in the x and y directions are respectively.

9. A structured light illuminated microscopy imaging method applied to a structured light illuminated microscopy imaging system according to any one of claims 1 to 8, the method comprising:

generating a two-dimensional sinusoidal periodic lattice light spot by using a diffractive optical element, wherein the two-dimensional sinusoidal periodic lattice light spot is imaged on a target sample of a structured light illumination microscope imaging system;

and performing phase-shift scanning on a target sample by using the two-dimensional sinusoidal periodic dot matrix light spot to obtain a plurality of scanning images, and performing super-resolution reconstruction according to the plurality of scanning images.

Images