CN116047739A - Super-resolution microscopic imaging method and system with structured light illumination - Google Patents
Super-resolution microscopic imaging method and system with structured light illumination Download PDFInfo
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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
Technical Field
The invention belongs to the field of optics, relates to imaging technology, and particularly relates to a super-resolution microscopic imaging method and system for illumination of structured light.
Background
The optical microscope has the advantages of specific marking and dynamic real-time imaging of living cells, so that the optical microscope is widely applied to life science research. However, the spatial resolution of conventional microscopic imaging techniques is limited by diffraction limits, such that the resolution of the optical microscope is 200 to 300nm in the lateral direction and 500 to 700nm in the longitudinal direction, greatly limiting the application of optical microscopy.
Structured light obvious micro imaging (Structure illumination microscopy, SIM) technology is one of the currently mainstream super-resolution optical micro imaging technologies. The SIM system can break through the limit of diffraction limit of the conventional optical microscope and can generate higher imaging resolution.
Structured light illumination is an illumination mode by changing the spatial structure of illumination light, and usually the illuminated structured light is a carrier frequency stripe. And (3) illuminating the sample by using the specially modulated structured light, and extracting focal plane information from the modulated image data by using a specific algorithm, thereby breaking through the limit of diffraction limit. However, when the illumination light is specially modulated, it is often necessary to resort to specific light field modulators, e.g. based on digital micromirror array devices (Digital Micromirror Device, DMD), spatial light modulators (Spatial Light Modulator, SLM), etc. However, the structure of the optical field modulator manufactured by the current technology is very complex, so that the whole volume of the SIM system is too large, the system is not easy to build, and the detection is very inconvenient, so that the SIM technology cannot be widely popularized and applied.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention discloses a super-resolution microscopic imaging method and system for structured light illumination.
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 generating device positioned between the light source and the objective table, wherein the objective table illumination light generating 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.
Preferably: the image acquisition device comprises a CMOS camera and a display connected with the CMOS camera.
Preferably: the light source is an LED lamp.
The invention also discloses a super-resolution microscopic imaging method of the structured light illumination, which is characterized by comprising the following steps:
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.
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.
Drawings
FIG. 1 is a schematic diagram of one embodiment of a structured light illuminated super-resolution microscopy imaging system according to the present invention;
FIG. 2 is a schematic diagram of a super-resolution image obtained after the direction of the grating is changed and spliced in an embodiment of the present invention; in fig. 2, A1, A2, A3 each represent a different structured light direction.
FIG. 3 is a schematic diagram showing the distribution of high frequency information obtained by changing the direction and phase of structured light according to an embodiment of the present invention;
FIG. 4 is a schematic diagram showing the distribution of high frequency information obtained by changing the direction and phase of structured light according to another embodiment of the present invention;
the abscissa in fig. 2 to 4 is the x-direction and the y-direction of the frequency domain space, respectively;
in the figure: 10. a light source; 20. a grating; 30. a collimating lens; 40. an infinity corrected objective; 50. an objective table; 51. a sample; 60. a CMOS camera; 70. a display; 80. and (3) a microscope.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The super-resolution microscopic imaging system with structured light illumination comprises a light source and an objective table, and is characterized by further comprising an objective table illumination light generating device arranged between the light source 10 and the objective table, wherein the objective table illumination light generating device comprises a grating 20 arranged in front of the light source, a collimating lens 30 arranged in front of the grating, and an infinity correcting objective 40 arranged in front of the collimating lens.
In one embodiment, as shown in fig. 1, an LED lamp is used as a light source 10 to emit light, a grating 20 is located on the light emitting side of the LED lamp 10, the light is modulated into periodically alternating light and dark stripes by the grating 20, the diffracted light containing ±1 st order is generated, and the diffracted light emitted by the grating is configured into parallel light by a collimating lens 30.
The collimated light passes through an infinity corrected objective 40 to micro-project the grating fringes onto a sample such as an animal cell, plant tissue, etc. on a stage 50. The light interferes on the surface of the sample to produce 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 on the focal plane of the infinity corrected objective. The fringe spacing x=d×f1/F2 seen in the microscope system, where d is the grating constant and F1 and F2 are the focal lengths of the collimating lens and the infinity corrected objective, respectively.
Assuming, for example, a grating constant of 3 microns, a collimator lens focal length of 3 cm, and an infinity corrected objective focal length of 2 cm, the fringe spacing seen in the microscope system can be reduced from 3 microns to 3 microns x (2 cm/3 cm) approximately equal to 2 microns, with the smaller fringe spacing resulting in a greater spatial frequency of structured light. The better the super-resolution of the experimental results.
By selecting a grating with a proper grating constant, cosine structure illumination light with spatial frequency close to diffraction limit can be obtained; the purpose of generating cosine structured illumination light is to achieve high frequency information transfer.
The optical imaging system may be regarded as a low-pass filter, and the spatial frequency modulation function is also called an Optical Transfer Function (OTF), where the size of the optical transfer function determines the spatial frequency range that the optical imaging system can pass through, and the larger the spatial frequency range, the more detectable high-frequency information is meant, the higher the spatial resolution of the optical imaging system is determined by the diffraction limit, however, the higher the high-frequency information outside the diffraction limit cannot enter into the OTF due to the existence of the diffraction limit.
The Abbe theory proves that the highest spatial frequency of an optical imaging system with fixed numerical aperture NA and wavelength lambda is 2 NA/lambda, namely Abbe diffraction limit; the impulse response function (delta function) can enable the function convolved with the impulse response function to generate space translation, namely the transfer characteristic, in an optical imaging system, the object images exactly meet the convolution relation, so that the possibility of building the relation exists in theory; because the frequency spectrum distribution of the cosine function is three impulse response functions, the object is loaded with the structured light meeting the cosine function distribution in the airspace, and the high-frequency information beyond the diffraction limit can be carried.
The invention generates cosine structure illumination light, can transfer high-frequency information outside the diffraction limit into the diffraction limit, namely, the high-frequency information is transferred downwards to low-frequency information, so that the frequency domain information of a sample can be acquired through an optical imaging system such as a microscope system, and then the transferred high-frequency information is restored to the original position through a specific algorithm, thereby achieving super-resolution imaging with resolution breaking through the diffraction limit.
By changing the direction of the grating, the direction of cosine fringes in the illumination light with a cosine structure can be changed, and then the direction of the illumination light with the cosine structure is changed, as shown in fig. 2, one specific embodiment assumes that the three structure light directions of a1=0°, a2=60°, a3=120° can be adjusted by changing the direction of the grating, and then the phase is changed twice in each direction, 3 frequency domain information in the direction is obtained by changing the phase each time, namely, a center solid circle in fig. 2 and two dotted circles in the corresponding direction, thereby obtaining nine frequency domain information, and after splicing, an approximately isotropic two-dimensional super-resolution image can be obtained, and the more the direction and the phase change amount are, the more the finally spliced image is close to a complete real image.
The diffraction limit refers to the maximum frequency range of single object point imaging due to the limitation of diffraction phenomenon, and the diffraction limit is determined by the original light parameters emitted by the light source; the grating constant d is the gap that produces diffracted light, further defining the range of spatial frequencies that can be acquired by the microscopy system.
The relationship between structured light frequency and diffraction limit is discussed below:
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 microscope 80 in the imaging system.
The inverse of the diffraction limit k0 thus determined is represented in fig. 2 to 4 as the radius of each OTF circle.
The frequency of the structured light received on the stage is the inverse of the fringe spacing x=d×f1/F2, where d is the grating constant and F1 and F2 are the focal lengths of the collimating lens and the infinity corrected objective, respectively.
The structured light frequency is made equal to the diffraction limit, 1/x=λ/2NA,
that is, when the expression F2/(d×f1) =λ/2NA is satisfied, the frequency of the structured light received at 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 dashed circle is within the OTF circle, and at this time, if the structured light direction is changed, for example, a1=0°, a2=60°, a3=120° is called out, the three structured light directions are changed in each direction by two times, as shown in fig. 3, when the spatial frequency of the structured light is within the OTF circle, the high-frequency information obtained by changing the directions and phases is the full dashed circle coverage area of the right half in fig. 3, and the overlapping portion is not repeatedly calculated.
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 dashed circle is on the OTF circle, and the direction and the twice phases of the structured light are changed as described in the foregoing, as shown in fig. 4, when the spatial frequency of the structured light is on the OTF circle, the high-frequency information obtained by changing the direction and the phase is the full dashed circle coverage area of the right half part in fig. 4.
Comparing fig. 3 and fig. 4, it can be seen that the total dashed circle coverage area of fig. 4 is larger than that of fig. 3, i.e. more high frequency information is obtained when the spatial frequency of the structured light reaches the diffraction limit.
The CMOS camera 60 is positioned on the light-emitting side of the stage 50 and is configured to receive fluorescent signals generated by the sample. The computer 70 is connected to the CMOS camera 60 for processing the relevant data to produce super-resolution imaging.
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.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in 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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