CN116047739A - A super-resolution microscopic imaging method and system based on structured light illumination - Google Patents

Abstract

The super-resolution microscopic imaging system comprises a light source, an objective table and a super-resolution imaging device positioned between the light source and the objective table, wherein the imaging device comprises a grating positioned in front of the light source, a collimating lens positioned in front of the grating and an infinity correcting objective positioned in front of the collimating lens; the imaging system further comprises an image acquisition device capable of carrying out imaging acquisition on the object stage. The super-resolution microscopic imaging method and the super-resolution microscopic imaging system for the structured light illumination can realize the obvious microscopic imaging of the structured light illumination without using equipment with huge volumes and high price such as SLM, DMD and the like by using the small and light grating combined with the lens and the objective lens.

Description

A super-resolution microscopic imaging method and system based on structured light illumination

technical field

The invention belongs to the field of optics and relates to imaging technology, in particular to a super-resolution microscopic imaging method and system for structured light illumination.

Background technique

Optical microscopy has the advantages of specific labeling and dynamic real-time imaging of living cells, so it is widely used in life science research. However, the spatial resolution of traditional microscopic imaging technology is limited by the diffraction limit, so that the resolution of optical microscopy is 200 to 300 nm in the horizontal direction and 500 to 700 nm in the vertical direction, which greatly limits the application of optical microscopy technology. .

Structure illumination microscopy (SIM) technology is one of the mainstream super-resolution optical microscopy imaging technologies. The SIM system can break through the limitation of the diffraction limit of traditional optical microscopes and can produce higher imaging resolution.

Structured light lighting is a lighting method that changes the spatial structure of lighting light. Usually, the structured light of lighting is a carrier frequency stripe. The sample is illuminated by specially modulated structured light, and then the information of the focal plane is extracted from the modulated image data through a specific algorithm, thereby breaking through the limitation of the diffraction limit. However, special light field modulators, such as digital micromirror devices (Digital Micromirror Devices, DMDs) and spatial light modulators (Spatial Light Modulators, SLMs), are usually required for special modulation of illumination light. However, the structure of the optical field modulator manufactured by the current technology is very complicated, which leads to the overall volume of the SIM system is too large, it is not easy to build the system, and the detection is very inconvenient, so that the SIM technology cannot be widely promoted and applied.

Contents of the invention

In order to overcome the defects in the prior art, the invention discloses a super-resolution microscopic imaging method and system under structured light illumination.

The super-resolution microscopic imaging system of structured light illumination according to the present invention includes a light source and an object stage, and is characterized in that it also includes an object stage illumination light generating device located between the light source and the object stage, and the object stage The illumination light generating device comprises a grating positioned in front of the light source, a collimator lens positioned in front of the grating, and an infinity corrected objective lens positioned in front of the collimator lens; the grating is positioned on a conjugate plane of the infinity corrected objective lens, and the stage Located at the focal plane of the infinity-corrected objective lens;

The imaging system also includes a microscope for capturing images of the stage and an image acquisition device connected to the microscope;

The light source, grating, microscope, collimator lens and infinity corrected objective lens meet the following conditions: F2/( d*F1)=λ/2NA

Where d is the grating constant, NA is the numerical aperture of the microscope, F1 and F2 are the focal lengths of the collimator lens and the infinity corrected objective lens respectively, and λ is the wavelength of light emitted by the light source.

Preferably: the image acquisition device includes a CMOS camera and a display connected thereto.

Preferably: the light source is an LED lamp.

The invention also discloses a super-resolution microscopic imaging method with structured light illumination, which is characterized in that it comprises the following steps:

S1. Calculate and adjust the grating constant according to the luminescence of the light source; or adjust the luminescence of the light source according to the grating constant, so that the grating constant of the grating and the light source meet the following conditions:

For the diffraction limit determined by the light source, the spatial frequency of the structured light obtained by the stage illumination light generating device is equal to the diffraction limit; that is, F2/( d*F1)=λ/2NA

Among them, d is the grating constant, NA is the numerical aperture of the microscope, F1 and F2 are the focal lengths of the collimator lens and the infinity corrected objective lens respectively, and λ is the wavelength of light emitted by the light source;

S2. Fix the direction of the grating, change the phase of the grating, and obtain the frequency domain information of each phase in a specific direction;

S3. Change the direction of the grating, repeat step S2, and obtain the frequency domain information of each phase in each direction;

S4. Concatenate all the frequency domain information obtained in step S3 to form a super-resolution image.

The super-resolution microscopic imaging method and system of the structured light illumination of the present invention, the present invention can not need to use bulky and expensive equipment such as SLM and DMD by using a small and light grating combined with a lens and an objective lens, that is, Structured illumination microscopic imaging can be realized.

Description of drawings

Fig. 1 is a schematic diagram of a specific embodiment of a super-resolution microscopic imaging system illuminated by structured light according to the present invention;

Fig. 2 is a schematic diagram of a super-resolution image obtained after changing the grating direction and splicing in a specific embodiment of the present invention; A1, A2, and A3 in Fig. 2 represent different structured light directions respectively.

Fig. 3 is a schematic diagram of the distribution of high-frequency information obtained by changing the direction and phase of structured light in a specific embodiment of the present invention;

Fig. 4 is a schematic diagram of the distribution of high-frequency information obtained by changing the direction and phase of structured light in another specific embodiment of the present invention;

In Fig. 2 to Fig. 4, the abscissa and ordinate are respectively the x direction and the y direction of the frequency domain space;

In the figure: 10, light source; 20, grating; 30, collimating lens; 40, infinity corrected objective lens; 50, stage; 51, sample; 60, CMOS camera; 70, display; 80, microscope.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

The super-resolution microscopic imaging system of structured light illumination according to the present invention includes a light source and an object stage, and is characterized in that it also includes an object stage illumination light generating device located between the light source 10 and the object stage, and the object The table illumination light generating device includes a grating 20 in front of the light source, a collimator lens 30 in front of the grating, and an infinity correcting objective lens 40 in front of the collimator lens.

As shown in Fig. 1, in a specific embodiment, an LED lamp is used as a light source 10 to emit light, and a grating 20 is located on the light emitting side of the LED lamp 10, and the light is modulated into periodic light and dark stripes through the grating 20, generating a pattern with ±1 levels. The diffracted light, the diffracted light emitted from the grating is then configured as parallel light through the collimating lens 30 .

The parallel light passes through the infinity-corrected objective lens 40 to micro-project the grating stripes onto the samples such as animal cells and plant tissues on the stage 50 . The light interferes on the surface of the sample, producing cosine structured illumination to illuminate the sample.

The grating should be placed on the conjugate plane of the infinity-corrected objective and the sample placed in the focal plane of the infinity-corrected objective. Then the fringe interval seen in the microscopic system is X=d*F1/F2, where d is the grating constant, F1 and F2 are the focal lengths of the collimator lens and the infinity corrected objective lens, respectively.

For example, assuming that the grating constant is 3 microns, the focal length of the collimator lens is 3 cm, and the focal length of the infinity corrected objective lens is 2 cm, the fringe interval seen in the microscopic system can be reduced from 3 microns to 3 microns × (2 cm/3 cm) is approximately equal to 2 microns, the smaller the fringe interval, the greater the spatial frequency of the structured light. The better the super-resolution of the experimental results is.

By choosing a grating with a suitable grating constant, cosine structured illumination light with a spatial frequency close to the diffraction limit can be obtained; the purpose of generating cosine structured illumination light is to realize high frequency information transfer.

The optical imaging system can be regarded as a low-pass filter, and its spatial frequency modulation function is also called the optical transfer function (OTF). The size of the optical transfer function determines the spatial frequency range that the optical imaging system can pass. The larger the spatial frequency range, the more The more high-frequency information that can be detected, the boundary of the optical transfer function is determined by the diffraction limit, and the spatial resolution of the optical imaging system is also higher. However, due to the existence of the diffraction limit, high-frequency information outside the diffraction limit cannot enter the OTF. .

Abbe's theory proves that the highest spatial frequency of an optical imaging system with fixed numerical aperture NA and wavelength λ is 2NA/λ, which is the Abbe diffraction limit. High-frequency information can be transported to the range of the diffraction limit in a certain way, and the detection Then analyze and restore it; the impulse response function (δ function) can cause the function convoluted with it to be translated in space, that is, it has the characteristics of transportation. In the optical imaging system, the object image just satisfies the convolution relationship, so theoretically there is It is possible to build a relationship. However, the transfer process occurs in the frequency domain, while the imaging process is in the air domain. It is necessary to solve how to operate in the air domain to realize the transfer in the frequency domain. Since the spectrum distribution of the cosine function is three impulse response functions, therefore, in the air domain Loading structured light that satisfies the cosine function distribution on the object can realize the transfer of high-frequency information outside the diffraction limit.

The generation of cosine structured illumination light in the present invention can transfer the high-frequency information outside the diffraction limit to the diffraction limit, that is, the high-frequency information is moved down to the low-frequency information, so that the frequency domain information of the sample can be collected through an optical imaging system such as a microscope system , and then restore the transferred high-frequency information to its original position through a specific algorithm, thus achieving super-resolution imaging with a resolution that breaks through the diffraction limit.

By changing the direction of the grating, the direction of the cosine stripes in the cosine structured illumination light can be changed, and then the direction of the cosine structured light can be changed. A specific embodiment is shown in Figure 2. Assuming that by changing the direction of the grating, A1=0°, A2 can be adjusted =60°, A3=120° in three structured light directions, and then change the phase twice in each direction, and change the phase each time to get 3 frequency domain information in this direction, that is, the central solid circle in Figure 2 and the corresponding Two dotted circles in the direction, so as to obtain nine frequency domain information. After splicing, an approximately isotropic two-dimensional super-resolution image can be obtained. The more the direction and phase changes, the closer the final spliced image is to the complete real image.

Diffraction limit refers to the maximum frequency range of a single object point imaging due to the limitation of diffraction phenomenon. The diffraction limit is determined by the original light parameters emitted by the light source; the grating constant d is the gap that generates diffracted light, and further determines the space that the microscopic system can collect range of frequencies.

The relationship between structured light frequency and diffraction limit is discussed as follows:

The diffraction limit k0 = λ/2NA is the theoretical resolution limit of the optical microscope,

Where λ is the wavelength of light, and NA is the numerical aperture of the microscope 80 in the imaging system.

The reciprocal of the diffraction limit k0 thus determined is shown in Figures 2 to 4 as the radii of the respective OTF circles.

The frequency of the structured light received on the stage is the reciprocal of the fringe interval, fringe interval X=d*F1/F2, where d is the grating constant, F1 and F2 are the focal lengths of the collimator lens and the infinity-corrected objective lens, respectively.

Let the structured light frequency equal to the diffraction limit, 1/X=λ/2NA,

That is, when the F2/( d*F1)=λ/2NA formula is established, the frequency of the structured light received on the stage is equal to the diffraction limit.

Assuming that the diffraction limit is k0, when the spatial frequency of the structured light does not reach the diffraction limit k0, the spatial frequency of the structured light is within the OTF circle, that is, the center of the dotted circle is within the OTF circle. At this time, if the direction of the structured light is changed, for example, call out A1=0°, A2=60°, A3=120° three structured light directions, and then change the phase twice in each direction, as shown in Figure 3, when the spatial frequency of the structured light is within the OTF circle, change the direction The high-frequency information obtained by summing and phase is the area covered by all the dotted circles in the right half of Figure 3, and the overlapping parts are not counted repeatedly.

When the spatial frequency of the structured light reaches the diffraction limit, the spatial frequency of the structured light is on the OTF circle, that is, the center of the dotted circle is on the OTF circle, and the direction of the structured light and the phase of the two times are changed as described in the previous figure, as shown in Figure 4 As shown, when the spatial frequency of the structured light is on the OTF circle, the high-frequency information obtained by changing the direction and phase is the area covered by the dotted circle in the right half of Figure 4.

Comparing Figure 3 and Figure 4, it can be seen that the coverage area of all the dotted circles in Figure 4 is larger than that in Figure 3, that is, more high-frequency information is obtained when the spatial frequency of structured light reaches the diffraction limit.

The CMOS camera 60 is located on the light emitting side of the stage 50 and is configured to receive fluorescence signals generated by the sample. The computer 70 is connected with the CMOS camera 60, and is used for processing related data, so as to generate super-resolution imaging.

The super-resolution microscopic imaging method and system of the structured light illumination of the present invention, the present invention can not need to use bulky and expensive equipment such as SLM and DMD by using a small and light grating combined with a lens and an objective lens, that is, Structured illumination microscopic imaging can be realized.

Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.

Claims (4)

1. The super-resolution microscopic imaging system with the structured light illumination comprises a light source and an objective table, and is characterized by further comprising an objective table illumination light generation device positioned between the light source and the objective table, wherein the objective table illumination light generation device comprises a grating positioned in front of the light source, a collimating lens positioned in front of the grating, and an infinity correcting objective positioned in front of the collimating lens; the grating is positioned on the conjugate plane of the infinity corrected objective, and the objective table is positioned on the focal plane of the infinity corrected objective;

the imaging system also comprises a microscope for capturing the image of the object stage and an image acquisition device connected with the microscope;

the light source, the grating, the microscope, the collimating lens and the infinity corrected objective meet the following conditions: f2/(d×f1) =λ/2NA

Where d is the grating constant, NA is the numerical aperture of the microscope, F1 and F2 are the focal lengths of the collimating lens and the infinity corrected objective, respectively, and λ is the wavelength of light emitted by the light source.

2. The structured-light illuminated super-resolution microscopy imaging system of claim 1, wherein: the image acquisition device comprises a CMOS camera and a display connected with the CMOS camera.

3. The structured-light illuminated super-resolution microscopy imaging system of claim 1, wherein: the light source is an LED lamp.

4. The super-resolution microscopic imaging method with structured light illumination is characterized by comprising the following steps of:

s1, calculating and adjusting a grating constant according to the light emitting condition of a light source; or according to the grating constant, adjusting the light source to emit light, so that the grating constant and the light source of the grating meet the following conditions:

for a diffraction limit determined by light emission of the light source, a spatial frequency of the structured light obtained by the stage illumination light generating device is equal to the diffraction limit; i.e. F2/(d.f1) =λ/2NA

Wherein d is a grating constant, NA is the numerical aperture F1 and F2 of the microscope, the focal lengths of the collimating lens and the infinity corrected objective lens are respectively shown, and lambda is the wavelength of light emitted by the light source;

s2, fixing the grating direction, changing the grating phase, and obtaining frequency domain information of each phase in a specific direction;

s3, changing the grating direction, and repeating the step S2 to obtain frequency domain information of each phase in each direction;

s4, splicing all the frequency domain information obtained in the step S3 to form a super-resolution image.

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