CN111458318B - Super-resolution imaging method and system utilizing square lattice structure light illumination - Google Patents

Super-resolution imaging method and system utilizing square lattice structure light illumination Download PDF

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CN111458318B
CN111458318B CN202010397842.0A CN202010397842A CN111458318B CN 111458318 B CN111458318 B CN 111458318B CN 202010397842 A CN202010397842 A CN 202010397842A CN 111458318 B CN111458318 B CN 111458318B
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汪召军
雷铭
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Xian Jiaotong University
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Abstract

The invention discloses a super-resolution imaging method and a super-resolution imaging system illuminated by square lattice structure light, which mainly comprise the following steps: forming a lattice structure light field on a sample plane, and performing phase shift by controlling a light field modulation device; acquiring a fluorescent image illuminated by the lattice structure light; obtaining a super-resolution image by using a frequency domain reconstruction algorithm; compared with the common stripe structure light, the tetragonal lattice structure light has lower light dose under the same peak intensity, the phototoxicity of the system can be greatly reduced, living cells can be observed for a longer time, in addition, only phase shift needs to be carried out on illumination patterns in the imaging process, and rotation is not needed, so that the polarization control in the imaging process is greatly simplified, the complexity of the system can be reduced, and the construction cost of the system is effectively reduced.

Description

Super-resolution imaging method and system utilizing square lattice structure light illumination
Technical Field
The invention belongs to the technical field of optics, and particularly relates to a super-resolution imaging method and system utilizing square lattice structured light to illuminate, which can be widely applied to the research in the fields of biology, medicine, microelectronics, material science and the like.
Background
The spatial resolution of the traditional optical microscope is limited by the diffraction limit of light, can only reach half of the wavelength order of light, and greatly limits the optical microscopeThe range of application of (1). How to realize imaging with higher spatial resolution has been one of the important research topics in the field of optical microscopy. At the end of the twentieth century, the super-resolution fluorescence microscopic imaging technology breaks the limitation of optical diffraction, so that the human can spy on the microscopic biological world at the nanometer level, and an unprecedented tool is provided for mysterious exploration of human life. Overcoming the diffraction limit is essential in enabling the system to distinguish between fluorescent molecules in the diffraction limited region and can generally be achieved by two types of methods. The first category of methods achieves the goal of distinguishing between different fluorescent molecules by randomly activating and localizing a Single fluorescent Molecule within a diffraction limited region at different time points, including Photoactivated Localization Microscopy (PALM) and random Optical Reconstruction Microscopy (STORM), both collectively referred to as Single Molecule Localization Microscopy (SMLM). The second category is the differentiation of fluorescent molecules in diffraction-limited regions by modulating their Emission signal with a special illumination field, including Stimulated Emission Depletion (STED) [5 ]]And Super-resolution Structured Illumination Microscopy (SR-SIM). The highest resolution of the current super-resolution fluorescence microscopic imaging technology is close to the resolution level of an electron microscope, a powerful tool is provided for modern biomedicine, and related researches are pushed to a new depth. Among many super-resolution imaging methods, the SIM has the highest imaging rate and the lowest excitation power density (-1W/cm)2) Therefore, the dynamic super-resolution observation can be carried out for a long time. In addition, the linear SIM is compatible with the traditional fluorescent molecules and fluorescent dyes, special optical switch dyes or proteins are not needed, and the application range of super-resolution imaging is greatly expanded. These advantages make SIM attractive for dynamic behavior observation of organelles, biological macromolecules and assemblies thereof. However, the existing SIM generally uses the stripe structure light, and this illumination method makes a lot of redundant illumination in the imaging process, thereby greatly increasing the phototoxicity of the system, and being very disadvantageous to the living body imaging. In addition, SIM based on stripe structure light illumination is in the imaging processThe method not only needs to translate and rotate the stripes, but also needs to quickly and precisely control the polarization state of the light beam in the switching process, so that the construction cost of the system is greatly increased.
Disclosure of Invention
Aiming at the problems of high phototoxicity, complex system, high construction cost and the like of the current fringe structure light illumination super-resolution microscopy method, the invention provides a super-resolution imaging method (square lattice SIM method for short) utilizing square lattice structure light illumination, the phototoxicity of the super-resolution imaging method is reduced by one time compared with the traditional method, rotation and polarization control are not needed in the imaging process, and the construction cost and the construction difficulty of the system can be greatly reduced.
Meanwhile, the invention also provides a system and a computer readable storage medium for realizing the super-resolution imaging method by utilizing the square lattice structured light illumination.
The technical solution of the invention is as follows:
a super-resolution imaging method utilizing tetragonal lattice structured light illumination comprises the following steps under the condition of linear excitation response:
step 1) generation and phase shift of lattice structure light field:
the light source is used for lighting, and after being modulated by the light field modulation device, a square lattice structured light field is formed on the sample plane; the intensity of the square lattice structured light field can be expressed in the form of multiplication of sinusoidal fringes in two orthogonal directions, and the distribution of formula (1) is satisfied:
Figure BDA0002488333030000021
wherein r is the coordinate of a two-dimensional plane, I0Representing the average intensity of the lattice structured light field, m being the degree of modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure BDA0002488333030000031
and
Figure BDA0002488333030000032
the phases of the light field in two orthogonal directions respectively;
setting the initial phase of the light field to
Figure BDA0002488333030000033
The light field modulation device is controlled to carry out phase shift, so that the light field of the square lattice structure moves in two orthogonal directions in a sample plane, each direction is three-step phase shift, the phase shift amount can be selected randomly, and 3 multiplied by 3 light fields of the lattice structure with different phase shifts are generated in total and are used for illuminating and exciting a sample to generate a fluorescence signal;
step 2) acquiring a fluorescence image by an area array digital camera:
after a sample is excited by a plurality of lattice structure illuminating light fields with different phase shifts, the fluorescence images formed on an imaging surface are sequentially recorded by a digital camera sCMOS or EMCCD, and 9 lattice structure illuminating fluorescence images are obtained;
step 3) obtaining a super-resolution image by using a frequency domain reconstruction algorithm:
fourier transform is respectively carried out on the 9 fluorescence images shot in the step 2) to obtain respective frequency spectrums thereof, and then the light intensity average value I of the structured light field is used0Degree of modulation m, spatial frequency k0And (3) obtaining a frequency spectrum by separation, assuming that the optical transfer function of the system is H (k), and translating and superposing the frequency spectrum components obtained by separation to obtain a spread spectrum
Figure BDA0002488333030000034
Then, the optical transfer function of the system is H (k) to carry out translation and superposition to obtain an expanded optical transfer function
Figure BDA0002488333030000035
Thereafter using the obtained spread spectrum
Figure BDA0002488333030000036
And extended optical transfer function
Figure BDA0002488333030000037
And (3) completing wiener deconvolution operation to obtain a final super-resolution image:
Figure BDA0002488333030000038
wherein A (k) is an apodization function, k represents a two-dimensional coordinate of the spectrum space, and α is a wiener filter parameter, assuming kdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is expressed as
Figure BDA0002488333030000039
Further limiting, the step (3) is specifically:
step 3.1) Fourier transform is respectively carried out on the 9 fluorescence images shot in the step 2) to obtain respective frequency spectrums thereof, and the frequency spectrums are respectively recorded as
Figure BDA0002488333030000041
Figure BDA0002488333030000042
Wherein k represents a two-dimensional coordinate of the spectrum space;
step 3.2) light intensity average value I according to the structured light field0Degree of modulation m, spatial frequency k0The frequency spectrum shown in formula (2) can be obtained by separation, and is respectively recorded as:
Figure BDA0002488333030000043
Figure BDA0002488333030000044
Figure BDA0002488333030000045
wherein
Figure BDA0002488333030000046
The matrix P in equation (2) is a 9 x 9 matrix, each row is similar in form, only the phase terms are different,
Figure BDA0002488333030000047
sequentially taking values according to the method in the step 1), wherein the total number of the values is 9 different values which can be recorded as
Figure BDA0002488333030000048
Figure BDA0002488333030000049
Step 3.3) assuming that the optical transfer function of the system is H (k), translating and superposing the 9 spectrum components obtained in the step 3.2) to obtain a spread spectrum
Figure BDA00024883330300000410
Expressed as:
Figure BDA00024883330300000411
Figure BDA0002488333030000051
step 3.4) translating and superposing the optical transfer function of the system to obtain an expanded optical transfer function
Figure BDA0002488333030000052
Expressed as:
Figure BDA0002488333030000053
step 3.5) of using the spread spectrum obtained in step 3.3) and step 3.4)
Figure BDA0002488333030000054
And extended optical transfer function
Figure BDA0002488333030000055
And (3) completing wiener deconvolution operation to obtain a final super-resolution image:
Figure BDA0002488333030000056
where α is the wiener filter parameter, A (k) is the apodization function, assuming k isdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is then expressed as
Figure BDA0002488333030000057
Further limiting, the phase shift is performed by controlling the optical field modulation device in the step (1), specifically: the lattice-like grating accomplishes the translation operation by using a moving high-precision two-dimensional piezoelectric translation stage.
Further limiting, the step (1) is specifically as follows:
(1.1) illuminating by a laser light source or an LED, modulating by a Spatial Light Modulator (SLM) or a Digital Micromirror Device (DMD) by utilizing a four-beam interference or projection method, and then mutually interfering at a focal plane to form a square lattice structured light field, wherein the intensity of the square lattice structured light field can be expressed in a form of multiplication of sinusoidal fringes in two orthogonal directions, and the distribution of the formula (1) is satisfied:
Figure BDA0002488333030000058
wherein r is the coordinate of a two-dimensional plane, I0Representing the average intensity of the structured light field in the form of a lattice, m being the degree of modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure BDA0002488333030000061
and
Figure BDA0002488333030000062
the phases of the light field in two orthogonal directions respectively;
(1.2) performing equal-interval phase shift or non-equal-interval phase shift by controlling a Spatial Light Modulator (SLM) or a digital micro-mirror device (DMD).
The application also provides a super-resolution imaging system illuminated by the square lattice structure light, which comprises a processor and a memory, wherein the memory stores a computer program, and the computer program executes the method in the step 3) in the super-resolution imaging method illuminated by the square lattice structure light when running in the processor.
The present application further provides a computer-readable storage medium storing a computer program, which when executed, implements the method of step 3) in the above super-resolution imaging method using tetragonal lattice structured light illumination.
Compared with the prior art, the invention has the advantages that:
compared with the common stripe structure light, the tetragonal structure light has lower light dose under the same peak intensity, so that the phototoxicity of the system can be greatly reduced, and living cells can be observed for a longer time. In addition, only phase shift is needed to be carried out on the illumination pattern in the imaging process, and rotation is not needed, so that polarization control in the imaging process is greatly simplified, the complexity of the system can be reduced, and the construction cost of the system is effectively reduced.
Drawings
FIG. 1 is a light path diagram of an interferometric lattice SIM super-resolution microscope system based on spatial light modulator SLM modulation and laser illumination;
the reference numbers in the figures are: the system comprises a 1-laser illumination source, a 2-polarizing beam splitter, a 3-half wave plate, a 4-spatial light modulator SLM, a 5-quarter wave plate, 6, 8 and 9-lenses, 7-specially designed spatial filter, 10-dichroic mirror, 11-reflector, 12-objective lens, 13-sample and objective table, 14-emission filter, 15-barrel lens and 16-area array digital camera.
FIG. 2 is a light path diagram of an interferometric lattice SIM super-resolution microscope system based on lattice point diffraction grating and laser illumination;
the reference numbers in the figures are: 1-laser illumination source, 2-lattice point diffraction grating, 3-quarter wave plate, 4, 6, 7-lens, 5-specially designed spatial filter, 8-dichroic mirror, 9-reflector, 10-objective lens, 11-sample and objective table, 12-emission filter, 13-tube lens and 14-area array digital camera.
FIG. 3 is a light path diagram of a projection type lattice SIM super-resolution microscope system based on digital micromirror DMD modulation and LED illumination;
the reference numbers in the figures are: the system comprises a light source 1-an LED illumination light source, a 2-condenser, a 3-total internal reflection beam splitter prism, a 4-digital micromirror device, a 5-collimating lens, a 6-exciting light filter, a 7-dichroic mirror, an 8-reflector, a 9-microscope objective, a 10-sample and objective table, an 11-emitting light filter, a 12-tube lens and a 13-area array digital camera.
FIG. 4 is a light path diagram of a projection type lattice SIM super-resolution microscope system based on lattice point diffraction grating and LED illumination;
the reference numbers in the figures are: 1-LED lighting source, 2-condenser, 3-lattice diffraction grating, 4-collimating lens, 5-exciting light filter, 6-dichroic mirror, 7-reflector, 8-microscope objective, 9-sample and objective table, 10-emitting light filter, 11-tube lens and 12-area array digital camera.
FIG. 5 is a graph comparing the intensity distribution of a fringe structure light field and a square lattice structure light field; (a) the intensity distribution of the fringe light field; (b) intensity distribution of a tetragonal lattice light field.
FIG. 6 is an original image and spread spectrum under illumination with a square lattice structure; (a) original images collected under illumination of a square lattice structure; (b) the frequency spectrum corresponding to the original image; (c) the spread spectrum range obtained after spectrum separation, shift and splicing.
FIG. 7 is a wide field illumination image and a square lattice SIM super-resolution image contrast image taken by the same system; (a) a wide field illumination image; (b) and obtaining a super-resolution image by using the square lattice SIM.
Detailed Description
The invention is further described with reference to the following figures and specific embodiments.
The method can be implemented after the mainstream SIM super-resolution microscope system is improved, and comprises an interference type square lattice SIM super-resolution microscope system based on SLM modulation and laser illumination, a projection type square lattice SIM super-resolution microscope system based on DMD modulation and LED illumination, a projection type square lattice SIM super-resolution microscope system based on lattice diffraction grating and LED illumination and the like.
An interferometric lattice illumination SIM super-resolution microscope system based on spatial light modulator SLM modulation and laser illumination is shown in figure 1: the device comprises a laser illumination light source 1 after beam expansion and collimation, a beam splitter 2 arranged after beam expansion and collimation laser beams, a half-wave plate 3 and a spatial light modulator 4 which are sequentially arranged on a transmission light path of the polarization beam splitter 2, a quarter-wave plate 5 and a lens 6 which are arranged on a reflection light path of the polarization beam splitter 2, a spatial filter 7 arranged at the rear end of the lens 6, a telescope system which is arranged behind the spatial filter 7 and consists of a lens 8 and a lens 9, a dichroic mirror 10 arranged behind the telescope system, a microscope objective 12 and a sample and objective table 13 which are arranged on a transmission light path of the dichroic mirror 10, a transmitting optical filter 14 and a barrel lens 15 which are arranged on the reflection light path of the dichroic mirror 10, and a planar array digital camera 16 arranged behind the barrel lens 15. The laser light source can be a continuous laser or a pulse laser; can be a single wavelength laser, a multi-wavelength laser, a supercontinuum laser, etc. The spatial light modulator 6 is a reflective ferroelectric liquid crystal spatial light modulator. The light path is formed by improving a main stream of a lighting microscope based on a stripe structure, and the main difference from the traditional lighting microscope based on the stripe structure is as follows: the spatial light modulator is loaded with a dot matrix pattern instead of a stripe pattern; the switching of the polarization state is not needed in the imaging process; stripe rotation is not needed in the imaging process; the distribution of apertures in the spatial filter is different (as shown in the inset in figure 1). The great advantage of not requiring polarization switching and not requiring fringe rotation provides the possibility of further simplification of the optical path.
As an optional light path configuration, the interferometric lattice SIM super-resolution microscope system based on the lattice diffraction grating and the laser illumination shown in FIG. 2 can greatly reduce the complexity and the construction cost of the system, and does not significantly sacrifice the imaging speed and the imaging effect of the system. An interferometric lattice SIM super-resolution microscope system based on lattice point diffraction grating and laser illumination is shown in figure 2: the device comprises a laser illumination light source 1 after beam expansion and collimation, a two-dimensional lattice point grating 2 arranged after beam expansion and collimation laser beams, a quarter wave plate 3 and a lens 4 which are sequentially arranged behind the lattice point grating 2, a spatial filter 5 arranged at the rear end of the lens 4, a telescope system which is arranged behind the spatial filter 5 and consists of a lens 6 and a lens 7, a dichroic mirror 8 arranged behind the telescope system, a silver mirror 9 micro-objective lens 10 and a sample and objective table 11 which are arranged on a transmission light path of the dichroic mirror 8, a transmitting optical filter 12 and a barrel lens 13 which are arranged on a reflection light path of the dichroic mirror 8, and a planar array digital camera 14 arranged behind the barrel lens 13. The two-dimensional lattice-point grating 2 may be an amplitude grating or a phase grating, and its amplitude or phase distribution is shown in the inset in fig. 2. The laser light source can be a continuous laser or a pulse laser; can be a single wavelength laser, a multi-wavelength laser, a supercontinuum laser, etc.
In addition, there are two optical path configurations for generating a lattice illumination light field based on projection, including: a projection type lattice SIM super-resolution microscope system based on digital micromirror DMD modulation and LED illumination (shown in FIG. 3) and a projection type lattice SIM super-resolution microscope system based on lattice point diffraction grating and LED illumination (shown in FIG. 4).
Wherein, the projection type lattice SIM super-resolution microscope system based on digital micromirror device DMD modulation and LED illumination is shown in FIG. 3: the device comprises an LED light source 1, a condenser 2 arranged behind the light source, a structured light generation module which is arranged behind the condenser 2 and consists of a total internal reflection beam splitter prism 3 and a digital micromirror device 4, a collimating lens 5 and an excitation light filter 6 which are sequentially arranged on a transmission light path of the structured light generation module, a dichroic mirror 7 arranged behind the excitation light filter 5, a reflecting mirror 8 arranged on a transmission light path of the dichroic mirror 7, a microscope objective 9 and a sample and objective table 10 which are arranged behind the reflecting mirror 8, a transmission light filter 11 and a barrel lens 12 which are sequentially arranged on a reflection light path of the dichroic mirror 7, and an area array digital camera 13 arranged behind the barrel lens 12. The LED light source 1 may be a single-band or multi-band LED light source, and the band of the filter set composed of the excitation light filter 6, the dichroic mirror 7, and the emission light filter 11 needs to be matched with the band of the LED light source 1. In addition, by removing the excitation-light filter 6 and the emission-light filter 11 and replacing the dichroic mirror 7 with a beam splitter, reflective imaging can be performed.
The light path of the projection type lattice SIM super-resolution microscope system based on the lattice point diffraction grating and LED illumination is shown in figure 4: the device comprises an LED light source 1, a condenser lens 2 arranged behind the light source, a lattice point diffraction grating 3 arranged behind the condenser lens 2, a collimating lens 4 and an excitation light filter 5 which are sequentially arranged behind the lattice point diffraction grating 3, a dichroic mirror 6 arranged behind the excitation light filter 5, a reflecting mirror 7 arranged on a transmission light path of the dichroic mirror 6, a microscope objective 8 and a sample and objective table 9 which are arranged behind the reflecting mirror 7, a transmitting light filter 10 and a barrel lens 11 which are sequentially arranged on a reflection light path of the dichroic mirror 6, and an area array digital camera 12 arranged behind the barrel lens 11. The LED light source 1 may be a single-band or multi-band LED light source, and the band of the filter set composed of the excitation light filter 5, the dichroic mirror 6, and the emission light filter 10 needs to be matched with the band of the LED light source 1. In addition, by removing the excitation light filter 5 and the emission light filter 10 and replacing the dichroic mirror 6 with a beam splitter, reflective imaging can be performed.
In addition, it is proved by experiments that under the same illumination peak value, the illumination dose of the lattice illumination light field is half of that of the traditional stripe structure illumination, and the intensity distribution of the lattice illumination light field is shown in fig. 5. By contrast, conventional stripe-structured lighting is uniformly illuminated along the stripes, whereas lattice-structured lighting suffers from bright-dark fluctuations, so that the total light dose is significantly lower than that of stripe lighting. For the case of a modulation depth of 1, the light field distributions of the stripe structure illumination and the lattice structure illumination, respectively, can be represented as follows:
Figure BDA0002488333030000111
Figure BDA0002488333030000112
both have illumination peaks of I0. Integrating the two equations on the focal plane can obtain the ratio of the two equations as 2: 1. that is, under the same illumination peak, the illumination dose of the lattice structure illumination is half of that of the stripe structure illumination, so that compared with the common stripe structure light, the tetragonal structure light of the present application has a lower light dose under the same peak intensity, can greatly reduce the phototoxicity of the system, can observe living cells for a longer time, and can be widely applied to the intensive research in the fields of biology, medicine, microelectronics, material science, and the like.
Example 1
The embodiment of the method for acquiring the super-resolution image by the interferometric lattice illumination SIM super-resolution microscope system based on the SLM modulation and the laser illumination is realized by the following steps:
step 1, using the interferometric lattice SIM super-resolution microscope system based on spatial light modulator SLM modulation and laser illumination shown in fig. 1, making 488nm wavelength laser after collimation and beam expansion incident to a polarization beam splitter 2, penetrating a half-wave plate, vertically irradiating a spatial light modulator SLM4 and returning in an original path, and generating a vertically linear polarization diffraction beam on a reflection light path of the polarization beam splitter. The vertically polarized light beam is converted into circularly polarized light by the quarter-wave plate 5 and converged by the lens 6, the converged multi-order diffracted zero-order and other high-order diffracted light beams are blocked by the customized spatial filter 7 (as shown in the inset of fig. 1), and only ± 1-order diffracted light beams are transmitted, including four light beams. Subsequently, these four beams of light are relayed by the telescopic system to the objective lens back pupil and enter the microscope objective lens 12, and the four beams of oblique circularly polarized light interfere with each other at the focal plane and form a square lattice-like illumination light field for illuminating the sample.
The intensity of the lattice structure light field can be expressed in the form of multiplication of sinusoidal fringes in two orthogonal directions, and the distribution of formula (1) is satisfied:
Figure BDA0002488333030000121
wherein r is the coordinate of a two-dimensional plane, I0Representing the average intensity of the lattice structured light field, m being the degree of modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure BDA0002488333030000122
and
Figure BDA0002488333030000123
the phases of the light field in two orthogonal directions, respectively.
Step 2, placing the Hela cell sample on an objective table and adjusting the Hela cell sample to an objective focal plane, and illuminating by using lattice structure light to excite the marked microtube to emit fluorescence; the fluorescence is collected by the objective lens, transmitted through the emission optical filter 14, the tube lens 15, and finally imaged on the sensor of the area-array digital camera 16.
Assuming an initial phase of the light field of
Figure BDA0002488333030000124
Controlling a Spatial Light Modulator (SLM) to move the lattice structure light field in two orthogonal directions in a sample plane by loading and refreshing the translated 9 lattice illumination patterns in square distribution, wherein each direction is three-step phase shift, and for equal-interval phase shift, the movement amount is recorded as
Figure BDA0002488333030000125
The phase shift amounts of the 9 illumination patterns are sequentially set to (0,0), (2 pi/3, 0), (4 pi/3, 0), (0,2 pi/3), (2 pi/3 ), (4 pi/3, 2 pi/3), (0,4 pi/3), (2 pi/3, 4 pi/3), (4 pi/3 ) and (4 pi/3, 4 pi/3), and are used for illuminating and exciting the sample to generate a fluorescence signal.
Step 3, respectively collecting corresponding 9 fluorescence by the area array digital cameraImage, noted D11，D12，D13，D21，D22，D23，D31，D32，D33As shown in fig. 6(a), the phase shift coordinates (3 × 3) are arranged in two orthogonal directions, each having 3 different phases. Assuming that the magnification ratio between the object space and the image space is 1, the images taken by the camera can be written as: d11(r)，D12(r)，D13(r)、D21(r)，D22(r)，D23(r)、D31(r)，D32(r)，D33(r); these images are stored in computer memory, hard disk, or floppy disk.
Step 4, firstly, Fourier transform is carried out on the 9 shot original fluorescence images to obtain the frequency spectrum frequency spectrums corresponding to the original images
Figure BDA0002488333030000131
Figure BDA0002488333030000132
Wherein k represents a two-dimensional coordinate of the spectrum space; the distribution is shown in FIG. 6 (b). Secondly, the average light intensity I of the light field is determined according to the lattice structure0Degree of modulation m, spatial frequency k in two orthogonal directions1And k2Initial phase in two orthogonal directions
Figure BDA0002488333030000133
And phase shift amount 2 pi/3, and 9 space frequency spectrums shown in formula (2) are obtained through calculation and are respectively recorded as:
Figure BDA0002488333030000134
Figure BDA0002488333030000135
Figure BDA0002488333030000136
wherein
Figure BDA0002488333030000137
The matrix P in equation (2) is a 9 × 9 matrix, each row is similar in form, and only the phase terms are different.
Figure BDA0002488333030000138
Sequentially taking values according to the method in the step 1), wherein the total number of the values is 9 different values which can be recorded as
Figure BDA0002488333030000139
Figure BDA00024883330300001310
For a phase shift that is equally spaced apart,
Figure BDA00024883330300001311
Figure BDA00024883330300001312
next, assuming that the optical transfer function of the system is h (k), the 9 spectral components obtained in the formula (1) are translated and superimposed to obtain a spread spectrum in the form of
Figure BDA0002488333030000141
Expressed as:
Figure BDA0002488333030000142
the spread spectrum corresponds to a spectral range as shown in fig. 6 (c). Then, the optical transfer function is translated and superposed to obtain an expanded optical transfer function
Figure BDA0002488333030000143
Prepare for wiener deconvolution:
Figure BDA0002488333030000144
finally, the spread spectrum obtained previously is utilized
Figure BDA0002488333030000145
And extended optical transfer function
Figure BDA0002488333030000146
And (3) completing the wiener deconvolution operation to obtain a super-resolution image of the microtube sample:
Figure BDA0002488333030000147
where α is the wiener filter parameter, which depends on the signal-to-noise ratio of the image, and A (k) is the apodization function, assuming kdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is then expressed as
Figure BDA0002488333030000148
Fig. 7 is a wide field illumination image and a tetragonal SIM super-resolution image contrast image of microtubules in hela cells obtained by an interferometric lattice illumination SIM super-resolution microscopy system based on spatial light modulator SLM modulation and laser illumination. The experiment used a 100 × microscope objective with a numerical aperture NA of 1.49. Fig. 7(a) is a general wide-field fluorescence image, and fig. 7(b) is a lattice-structured light illumination super-resolution image obtained using the method of the present invention. By comparison, the resolution of the image obtained by the method of the invention is obviously higher than that of the common wide-field image.
Example 2
The embodiment is a method for acquiring a super-resolution image based on an interferometric lattice SIM super-resolution microscope system with a lattice point diffraction grating and laser illumination, which is specifically realized by the following steps:
step 1, using the interferometric lattice SIM super-resolution microscope system based on the lattice point diffraction grating and laser illumination shown in figure 2. The 488nm wavelength laser after collimation and expansion is incident to the two-dimensional lattice-shaped grating 2, the grating divides the incident laser into four beams, the vertically polarized light beam is converted into circularly polarized light by the quarter-wave plate 3 and is converged by the lens 4, the converged multi-order diffracted zero-order and other high-order diffracted light beams are blocked by the customized spatial filter 5 (as shown in an illustration in figure 2), only +/-1-order diffracted light beams can penetrate through the spatial filter, and the four beams comprise four beams of light. Subsequently, these four beams of light are relayed by the telescopic system to the objective lens back pupil and enter the microscope objective lens 10, and the four oblique circularly polarized lights interfere with each other at the focal plane and form a square lattice-like illumination light field for illuminating the sample.
The intensity of the lattice structure light field can be expressed in the form of multiplication of sinusoidal fringes in two orthogonal directions, and the distribution of formula (1) is satisfied:
Figure BDA0002488333030000151
wherein r is the coordinate of a two-dimensional plane, I0Representing the average intensity of the lattice structured light field, m being the degree of modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure BDA0002488333030000152
and
Figure BDA0002488333030000153
the phases of the light field in two orthogonal directions, respectively.
Step 2, placing the sample on an objective table, adjusting the sample to an objective focal plane, and illuminating by using lattice structure light to excite the marked microtube to emit fluorescence; the fluorescence is collected by the objective lens, passes through the emission optical filter 12 and the tube lens 13, and is finally imaged on the sensor of the area-array digital camera 14. Assuming an initial phase of the light field of
Figure BDA0002488333030000161
Controlling a piezoelectric precision translation stage to move the lattice-shaped grating so that the lattice-structured light field moves along two orthogonal directions in a sample plane, wherein each direction is three-step phase shift,for equally spaced phase shifts, the amount of shift is noted
Figure BDA0002488333030000162
The 9 phase shift amounts are sequentially set to be (0,0), (2 pi/3, 0), (4 pi/3, 0), (0,2 pi/3), (2 pi/3 ), (4 pi/3, 2 pi/3), (0,4 pi/3), (2 pi/3, 4 pi/3), (4 pi/3 ) and (4 pi/3, 4 pi/3) for illuminating and exciting the sample to generate a fluorescence signal. Assuming that the period of the lattice pattern loaded on the spatial light modulator SLM or the digital micromirror device DMD is 6 pixels, the lattice pattern is moved in two directions by 2 steps, respectively, by the number of pixels being (0,0), (2, 0), (4, 0), (0, 2), (2, 2), (4, 2), (0, 4), (2, 4), (4, 4), in this order.
Step 3, respectively collecting corresponding 9 fluorescence images by the area array digital camera, and recording the images as D11，D12，D13，D21，D22，D23，D31，D32，D33As shown in fig. 6(a), the phase shift coordinates (3 × 3) are arranged in two orthogonal directions, each having 3 different phases. Assuming that the magnification ratio between the object space and the image space is 1, the images taken by the camera can be written as: d11(r)，D12(r)，D13(r)、D21(r)，D22(r)，D23(r)、D31(r)，D32(r)，D33(r); these images are stored in computer memory, hard disk, or floppy disk.
Step 4, firstly, Fourier transform is carried out on the 9 shot original fluorescence images to obtain the frequency spectrum frequency spectrums corresponding to the original images
Figure BDA0002488333030000163
Figure BDA0002488333030000164
Wherein k represents a two-dimensional coordinate of the spectrum space; the distribution is shown in FIG. 6 (b). Secondly, the average light intensity I of the light field is determined according to the lattice structure0Degree of modulation m, spatial frequency k in two orthogonal directions1And k2Initial phase in two orthogonal directions
Figure BDA0002488333030000165
And phase shift amount 2 pi/3, and 9 space frequency spectrums shown in formula (2) are obtained through calculation and are respectively recorded as:
Figure BDA0002488333030000166
Figure BDA0002488333030000167
Figure BDA0002488333030000171
wherein
Figure BDA0002488333030000172
The matrix P in equation (2) is a 9 × 9 matrix, each row is similar in form, and only the phase terms are different.
Figure BDA00024883330300001711
Sequentially taking values according to the method in the step 1), wherein the total number of the values is 9 different values which can be recorded as
Figure BDA0002488333030000173
Figure BDA0002488333030000174
For a phase shift that is equally spaced apart,
Figure BDA0002488333030000175
Figure BDA0002488333030000176
next, assuming that the optical transfer function of the system is h (k), the 9 spectral components obtained in the formula (1) are translated and superimposed to obtain a spread spectrum in the form of
Figure BDA0002488333030000177
Expressed as:
Figure BDA0002488333030000178
the spread spectrum corresponds to a spectral range as shown in fig. 6 (c). Then, the optical transfer function is translated and superposed to obtain an expanded optical transfer function
Figure BDA0002488333030000179
Prepare for wiener deconvolution:
Figure BDA00024883330300001710
Figure BDA0002488333030000181
finally, the spread spectrum obtained previously is utilized
Figure BDA0002488333030000182
And extended optical transfer function
Figure BDA0002488333030000183
And (3) completing the wiener deconvolution operation to obtain a super-resolution image of the microtube sample:
Figure BDA0002488333030000184
where α is the wiener filter parameter, which depends on the signal-to-noise ratio of the image, and A (k) is the apodization function, assuming kdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is then expressed as
Figure BDA0002488333030000185
Example 3
The embodiment is a projection type lattice SIM super-resolution microscope system based on a digital micro-mirror device and LED illumination. In addition, the embodiment will explain the implementation of non-equidistant phase shift and the corresponding method for acquiring super-resolution image, which is specifically realized by the following steps:
step 1, using a projection type lattice SIM super-resolution microscope system based on digital micromirror device DMD modulation and LED illumination shown in figure 3, light emitted by an LED is focused by a condenser lens 2, enters a structured light generation module consisting of a total internal reflection beam splitter prism 3 and a digital micromirror device 4, is collimated by a collimating lens 5, is filtered by an excitation light filter 6, penetrates through a dichroic mirror 7, and enters a back pupil of a microscope objective lens 9. The collimator lens 5 and the microscope objective 9 form a projection system, and the pattern loaded on the DMD is projected to the focal plane of the objective to form a square lattice-shaped illumination light field for illuminating the sample.
The intensity of the lattice structure light field can be expressed in the form of multiplication of sinusoidal fringes in two orthogonal directions, and the distribution of formula (1) is satisfied:
Figure BDA0002488333030000186
wherein r is the coordinate of a two-dimensional plane, I0Representing the average intensity of the lattice structured light field, m being the degree of modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure BDA0002488333030000191
and
Figure BDA0002488333030000192
the phases of the light field in two orthogonal directions, respectively.
Step 2, placing the Hela cell sample on an objective table and adjusting the Hela cell sample to an objective focal plane, and illuminating by using lattice structure light to excite the marked microtube to emit fluorescence; the fluorescence is collected by the objective lens, passes through the emission optical filter 11 and the tube lens 12, and is finally imaged on the sensor of the area-array digital camera 13.
Assuming an initial phase of the light field of
Figure BDA0002488333030000193
Controlling a Digital Micromirror Device (DMD) to move the lattice structure light field in two orthogonal directions in a sample plane by loading and refreshing the translated 9 lattice illumination patterns in square distribution, wherein each direction is three-step phase shift, and the movement amount is recorded as the non-equidistant phase shift
Figure BDA0002488333030000194
The phase shift amount of the 9 illumination patterns is sequentially set to be (0,0), (pi/2, 0), (pi, 0), (0, pi/2), (pi/2 ), (pi, pi/2), (0, pi), (pi/2, pi) and (pi, pi) for illuminating and exciting the sample to generate a fluorescence signal. Assuming that the period of the lattice pattern loaded on the spatial light modulator SLM or the digital micromirror device DMD is 4 pixels, the lattice pattern is shifted in two directions by 1 step, respectively, by the number of pixels in turn (0,0), (1, 0), (2, 0), (0, 1), (1, 1), (2, 1), (0, 2), (1, 2), (2, 2).
Step 3, respectively collecting corresponding 9 fluorescence images by the area array digital camera, and recording the images as D11，D12，D13，D21，D22，D23，D31，D32，D33As shown in fig. 6(a), the phase shift coordinates (3 × 3) are arranged in two orthogonal directions, each having 3 different phases. Assuming that the magnification ratio between the object space and the image space is 1, the images taken by the camera can be written as: d11(r)，D12(r)，D13(r)、D21(r)，D22(r)，D23(r)、D31(r)，D32(r)，D33(r); these images are stored in computer memory, hard disk, or floppy disk.
Step 4, firstly, Fourier transform is carried out on the 9 shot original fluorescence images to obtain the frequency spectrum frequency spectrums corresponding to the original images
Figure BDA0002488333030000201
Figure BDA0002488333030000202
Wherein k represents a two-dimensional coordinate of the spectrum space; the distribution is shown in FIG. 6 (b). Secondly, the average light intensity I of the light field is determined according to the lattice structure0Degree of modulation m, spatial frequency k in two orthogonal directions1And k2Initial phase in two orthogonal directions
Figure BDA0002488333030000203
And phase shift quantity, 9 space frequency spectrums shown in the formula (2) are obtained through calculation and are respectively recorded as:
Figure BDA0002488333030000204
Figure BDA0002488333030000205
Figure BDA0002488333030000206
wherein
Figure BDA0002488333030000207
The matrix P in equation (2) is a 9 × 9 matrix, each row is similar in form, and only the phase terms are different.
Figure BDA0002488333030000208
Sequentially taking values according to the method in the step 1), wherein the total number of the values is 9 different values which can be recorded as
Figure BDA0002488333030000209
Figure BDA00024883330300002010
For non-equidistant phase shifts, the amount of phase shift may be taken in multiple waysValues, in 1: 1: the phase shift interval of 2 is taken as an example,
Figure BDA00024883330300002011
Figure BDA00024883330300002012
next, assuming that the optical transfer function of the system is h (k), the 9 spectral components obtained in the formula (1) are translated and superimposed to obtain a spread spectrum in the form of
Figure BDA00024883330300002013
Expressed as:
Figure BDA0002488333030000211
the spread spectrum corresponds to a spectral range as shown in fig. 6 (c). Then, the optical transfer function is translated and superposed to obtain an expanded optical transfer function
Figure BDA0002488333030000212
Prepare for wiener deconvolution:
Figure BDA0002488333030000213
finally, the spread spectrum obtained previously is utilized
Figure BDA0002488333030000214
And extended optical transfer function
Figure BDA0002488333030000215
And (3) completing the wiener deconvolution operation to obtain a super-resolution image of the microtube sample:
Figure BDA0002488333030000216
wherein alpha is wiener filteringParameters, dependent on the signal-to-noise ratio of the image, A (k) is the apodization function, assuming kdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is then expressed as
Figure BDA0002488333030000217
The application also provides a super-resolution imaging system illuminated by the square lattice structure light, which comprises a processor and a memory, wherein a computer program is stored in the memory, and when the computer program runs in the processor, the super-resolution image reconstruction processing method in the step 4 is executed.
The present application also provides a computer-readable storage medium storing a program which, when executed, implements the above-described super-resolution image reconstruction processing method. In some possible embodiments, the invention may also be implemented in the form of a program product comprising program code means for causing a terminal device to carry out the steps according to various exemplary embodiments of the invention described in the method part of the description above, when said program product is run on the terminal device.
A program product for implementing the above method, which may employ a portable compact disc read only memory (CD-ROM) and include program code, may be run on a terminal device, such as a personal computer. However, the program product of the present invention is not limited thereto, and in the present invention, the computer readable storage medium may be any tangible medium that can contain, or store a program for use by or in connection with an instruction execution system, apparatus, or device.
The program product may employ any combination of one or more readable media. The readable medium may be a readable signal medium or a readable storage medium. A readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, or semiconductor system, apparatus, or device, or any combination of the foregoing. More specific examples (a non-exhaustive list) of the readable storage medium include: an electrical connection having one or more wires, a portable disk, a hard disk, a Random Access Memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or flash memory), an optical fiber, a portable compact disc read-only memory (CD-ROM), an optical storage device, a magnetic storage device, or any suitable combination of the foregoing.

Claims (5)

1. A super-resolution imaging method utilizing square lattice structured light illumination is characterized by comprising the following steps of:
step 1) generation and phase shift of a square lattice structure light field:
the light source is used for lighting, and after being modulated by the light field modulation device, a square lattice structure light field is formed on the sample plane; the intensity of the square lattice structure light field can be expressed in a form of multiplication of sine stripes in two orthogonal directions, and the distribution of formula 1 is satisfied:
Figure FDA0002967509520000011
wherein r is the coordinate of a two-dimensional plane, I (r) represents the intensity distribution of the square lattice structure light field, I0Representing the average intensity of the lattice structured light field, m being the degree of modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure FDA0002967509520000012
and
Figure FDA0002967509520000013
the phases of the light field in two orthogonal directions respectively;
setting the initial phase of the light field to
Figure FDA0002967509520000014
The optical field modulation device is controlled to carry out phase shift, so that the optical field with the square lattice structure moves along two orthogonal directions in a sample plane, each direction is three-step phase shift, and the phase shift amount can be randomSelecting to generate 3 × 3 lattice-shaped structure light fields with different phase shifts for illuminating and exciting the sample to generate a fluorescence signal;
step 2) acquiring a fluorescence image by an area array digital camera:
after a sample is excited by a plurality of square lattice structure light fields with different phase shifts, the fluorescence images formed on an imaging surface are sequentially recorded by a digital camera sCMOS or EMCCD, and 9 fluorescence images of square lattice structure light field illumination are obtained in total;
step 3) obtaining a super-resolution image by using a frequency domain reconstruction algorithm:
fourier transform is respectively carried out on the 9 fluorescence images shot in the step 2) to obtain respective frequency spectrums thereof, and then the light intensity average value I of the structured light field is used0Degree of modulation m, spatial frequency k0And (3) obtaining a frequency spectrum by separation, assuming that the optical transfer function of the system is H (k), and translating and superposing the frequency spectrum components obtained by separation to obtain a spread spectrum
Figure FDA0002967509520000021
Then, the optical transfer function of the system is H (k) to carry out translation and superposition to obtain an expanded optical transfer function
Figure FDA0002967509520000022
Thereafter using the obtained spread spectrum
Figure FDA0002967509520000023
And extended optical transfer function
Figure FDA0002967509520000024
And (3) completing wiener deconvolution operation to obtain a final super-resolution image:
Figure FDA0002967509520000025
wherein, the ImageSRRepresenting a super-resolution image obtained by a frequency domain reconstruction algorithm, ift representing an inverse Fourier transform operationAs follows, A (k) is an apodization function, k represents a two-dimensional coordinate of the spectral space, α is a wiener filter parameter, assuming k isdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is expressed as
Figure FDA0002967509520000026
The step 3) is specifically as follows:
step 3.1) Fourier transform is respectively carried out on the 9 fluorescence images shot in the step 2) to obtain respective frequency spectrums thereof, and the frequency spectrums are respectively recorded as
Figure FDA0002967509520000027
Figure FDA0002967509520000028
Wherein k represents a two-dimensional coordinate of the spectrum space;
step 3.2) light intensity average value I according to the structured light field0Degree of modulation m, spatial frequency k0The frequency spectrums shown in formula 2 can be obtained by separation, and are respectively recorded as:
Figure FDA0002967509520000029
Figure FDA00029675095200000210
Figure FDA00029675095200000211
wherein
Figure FDA0002967509520000031
The matrix P in equation 2 is a 9 x 9 matrix, each row is similar in form, only the phase terms are different,
Figure FDA0002967509520000032
sequentially taking values according to the method in the step 1), wherein the total number of the values is 9 different values which can be recorded as
Figure FDA0002967509520000033
Figure FDA0002967509520000034
Step 3.3) assuming that the optical transfer function of the system is H (k), translating and superposing the 9 spectrum components obtained in the step 3.2) to obtain a spread spectrum
Figure FDA0002967509520000035
Expressed as:
Figure FDA0002967509520000036
step 3.4) translating and superposing the optical transfer function of the system to obtain an expanded optical transfer function
Figure FDA0002967509520000037
Expressed as:
Figure FDA0002967509520000038
step 3.5) of using the spread spectrum obtained in step 3.3) and step 3.4)
Figure FDA0002967509520000039
And extended optical transfer function
Figure FDA00029675095200000310
And (3) completing wiener deconvolution operation to obtain a final super-resolution image:
Figure FDA00029675095200000311
where α is the wiener filter parameter, A (k) is the apodization function, assuming k isdFor frequencies corresponding to the maximum range of the spread spectrum, the apodization function is then expressed as
Figure FDA0002967509520000041
2. The super-resolution imaging method using square lattice structured light for illumination according to claim 1, wherein the phase shift is performed by controlling a light field modulation device in step 1), specifically: the lattice-like grating accomplishes the translation operation by using a moving high-precision two-dimensional piezoelectric translation stage.
3. The super-resolution imaging method illuminated by square lattice structured light according to claim 1, wherein the step 1) is specifically as follows:
step 1.1) is illuminated by a laser light source or an LED, and after being modulated by a Spatial Light Modulator (SLM) or a Digital Micromirror Device (DMD) by utilizing a four-beam interference or projection method, the light beams interfere with each other at a focal plane to form a square lattice structure light field, wherein the intensity of the square lattice structure light field can be expressed in a form of multiplication of sine fringes in two orthogonal directions, and the distribution of a formula 1 is satisfied:
Figure FDA0002967509520000042
wherein r is the coordinate of a two-dimensional plane, I0Representing the average intensity of a light field with a square lattice structure, m being the modulation in a single direction, k1And k is2Respectively representing wave vectors in two orthogonal directions,
Figure FDA0002967509520000043
and
Figure FDA0002967509520000044
the phases of the light field in two orthogonal directions respectively;
step 1.2) performing equal-interval phase shift or non-equal-interval phase shift by controlling a Spatial Light Modulator (SLM) or a Digital Micromirror Device (DMD).
4. The utility model provides an utilize super resolution imaging system of square lattice structure light illumination which characterized in that: including processor and memory, its characterized in that: the memory stores a computer program which, when executed on the processor, performs the method of step 3) of the method of claim 1 for super-resolution imaging illuminated with tetragonal lattice structure light.
5. A computer-readable storage medium characterized by: a computer program is stored which, when executed, implements the method of step 3) in the super-resolution imaging method using square lattice structured light illumination of claim 1.
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