CN112859315A - Multicolor dual-mode structured light illumination microscopic imaging system and imaging method thereof - Google Patents
Multicolor dual-mode structured light illumination microscopic imaging system and imaging method thereof Download PDFInfo
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Abstract
The invention discloses a multicolor dual-mode structured light illumination microscopic imaging system and an imaging method thereof. The invention adopts a digital micromirror array to realize a multicolor dual-mode structured light illumination microscopic imaging system, and a dichroic mirror and a reflector are combined to meet a blaze condition; compared with the traditional grating diffraction, the digital micromirror array greatly accelerates the imaging speed; according to the invention, the space filter is switched by the conversion rotating frame into a two-dimensional light-passing hole filter under a two-dimensional structure light illumination mode or into a three-dimensional light-passing hole filter under a three-dimensional structure light illumination mode, so that the switching of a two-dimensional and three-dimensional structure light illumination microscopic imaging mode can be realized rapidly; aiming at the characteristic of a digital micromirror array blazed grating, the invention provides a multi-angle illumination coupling mode, and can realize multi-color imaging.
Description
Technical Field
The invention relates to the field of optical microscopic imaging, in particular to a multicolor dual-mode structured light illumination microscopic imaging system based on a digital micromirror array and laser interference and an imaging method thereof.
Background
Fluorescence microscopic imaging can be used for researching structures and functions of cells and biomacromolecules and plays an important role in the fields of life science research and the like. However, the resolution of the conventional wide-field fluorescence microscopy imaging technology is limited by the diffraction limit, and the structural information with high resolution cannot be acquired. In addition, the traditional wide-field fluorescence microscopic imaging technology also has the problem of defocusing, and complete three-dimensional structural information cannot be obtained in principle. The structured light illumination microscopic imaging is taken as a novel fluorescence microscopic imaging technology, and is distinguished in the fluorescence microscopic imaging by the advantages of high resolution, high imaging speed, small phototoxicity, no need of special sample preparation, multiple imaging modes and the like. The two-dimensional structured light illumination microscopic imaging realized by the double-beam interference technology can acquire sample information which is twice as large as an optical diffraction limit in a two-dimensional plane, and becomes an effective tool for biological research of biologists. However, the two-dimensional structured light illumination can only obtain super-resolution imaging in a two-dimensional plane, and cannot obtain super-resolution imaging in a three-dimensional space.
In order to solve the above problems, one solution is to use a three-beam interference technique to form three-dimensional structured light illumination, so as to encode high-frequency information of a three-dimensional space into low-frequency information of an acquired image, and after acquiring original images of multiple phases in multiple directions, realize super-resolution imaging of the three-dimensional space through three-dimensional reconstruction of the image. Heretofore, three-dimensional structured light illumination has been achieved primarily by diffraction from a grating or liquid crystal spatial light modulator. The traditional three-dimensional structure light illumination microscopic imaging generates 0-level and +/-1-level linearly polarized light to interfere on the surface of a sample through grating diffraction, the structure illumination light with different angles in different directions is realized through the rotation and translation of the grating, the system is complex and the imaging speed is low, and the real-time dynamic imaging of living cells is difficult to carry out. In addition, when the liquid crystal spatial light modulator is used for three-dimensional structured light illumination microscopic imaging, because adjacent-order linearly polarized light generated by the liquid crystal spatial light modulator has mutually perpendicular polarization directions, 0-order and +/-1-order linearly polarized light is directly used for interference, and high-contrast illumination structured light cannot be formed, and complex polarization adjustment of diffracted light is required.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a multicolor dual-mode structured light illumination microscopic imaging system based on a digital micromirror array and laser interference and an imaging method thereof.
One objective of the present invention is to provide a multi-color dual-mode structured light illumination micro-imaging system based on digital micromirror array and laser interference.
The invention discloses a multicolor dual-mode structured light illumination microscopic imaging system based on a digital micromirror array and laser interference, which comprises: the device comprises an illumination light source, a beam combining device, a wavelength selection device, a polarization control device, a collimation and beam expanding device, an incidence angle adjusting device, a digital micromirror array, a condensing lens, a spatial filter, a first dichroic mirror, a second dichroic mirror, a 4f system, a polarization correction device, an objective lens, a tube lens, a camera, a data acquisition card and a transformation rotating frame; the illumination light source comprises a plurality of single-wavelength lasers, each single-wavelength laser emits linearly polarized laser with one wavelength, and the plurality of lasers respectively emit linearly polarized laser with different wavelengths; the linear polarized lasers with different wavelengths are combined by the beam combining device and transmitted to the wavelength selection device; the wavelength of the passing linearly polarized laser is quickly selected through a wavelength selection device, the linearly polarized laser with the selected wavelength is subjected to polarization control of light through a polarization control device, and the polarization direction of the linearly polarized laser modulated by the polarization control device is consistent with the direction of the binary periodic fringes loaded on the digital micromirror array; after being expanded by the collimation and beam expansion device, the beam is incident to the digital micromirror array; in the structured light illumination microscopic imaging system, a digital micro-mirror array is equivalent to a diffraction grating, linearly polarized light of different orders formed by diffraction of the digital micro-mirror array is interfered on the surface of a sample to form illumination structured light, and incident linearly polarized laser needs to meet the blaze condition of the digital micro-mirror array, so that the illumination structured light with high contrast is generated, wherein the blaze condition is that incident light with different wavelengths has corresponding incident angles; the incident angle adjusting device adopts the combination of a dichroic mirror and a reflecting mirror, adjusts the routes of the linear polarized laser with different wavelengths according to the blaze condition, and adjusts the linear polarized laser with the selected wavelength to be incident to the digital micromirror array at a corresponding incident angle, so that the blaze condition is met; the plane of the digital micromirror array is vertical to the optical axis, and periodic binary periodic stripes which are alternate between black and white are loaded on the digital micromirror array and reflected by the digital micromirror array to form multi-level linearly polarized diffracted light; the multi-level linearly polarized diffraction light is focused by the condensing lens and then enters the spatial filter, the spatial filter is arranged on the conversion rotating frame, and the spatial filter is selected to be a three-dimensional light-passing hole filter under a three-dimensional structure light illumination mode or a two-dimensional light-passing hole filter under a two-dimensional structure light illumination mode through the conversion rotating frame;
under the three-dimensional structured light illumination mode, 0-order and +/-1-order polarized diffracted lights pass through the spatial filter; the 0-level and +/-1-level polarized diffraction light passes through the first dichroic mirror, then passes through the 4f system, then passes through the second dichroic mirror, is converged to the rear focal plane of the objective lens, and is incident on a sample plane after passing through the objective lens for interference, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective lens, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator through the 4f system; the 0-order and +/-1-order linearly polarized diffracted lights interfere on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective lens after returning, filtered by an emission filter after passing through a second dichroic mirror, and focused by a lens barrel lens; the s-axis and the p-axis of the first dichroic mirror and the second dichroic mirror are interchanged, so that polarization distortion caused by the fact that one dichroic mirror is used independently is eliminated; the fluorescence focused by the lens barrel is projected on a camera to realize digital imaging; acquiring fluorescence with corresponding wavelength by a camera under the selected wavelength to obtain an original image; keeping the angle of the binary periodic stripes unchanged, carrying out 2 pi/n phase shift on the binary periodic stripes loaded on the digital micromirror array to form new binary periodic stripes, acquiring a second original image through a camera, and repeating the process for n times to obtain n original images with different phases in the same direction to form an original image in the first direction; the binary periodic stripes rotate around a horizontal optical axis by an angle of pi/k, and the same data acquisition process is carried out in the first direction, so that original images in k directions are obtained, n original images are arranged in each direction, and k multiplied by n original images in the k directions form a group of original images; stepping a sample in a set step length along the direction of an optical axis, and still acquiring a group of original images in k directions and n phases in each direction; acquiring a plurality of groups of original images in a set range through multiple steps, thereby forming a plurality of layers of original images; performing three-dimensional reconstruction through a three-dimensional structured light illumination microscopic imaging algorithm to obtain a three-dimensional super-resolution image of the sample;
under the two-dimensional structure light illumination mode, selecting a spatial filter as a two-dimensional light-passing hole filter under the two-dimensional structure light illumination mode through changing a rotating frame, and allowing plus or minus 1-order polarized diffracted light passing through the spatial filter to pass through; the +/-1-order polarized diffraction light passes through the first dichroic mirror, then passes through the 4f system, then passes through the second dichroic mirror, is converged to the rear focal plane of the objective lens, passes through the objective lens and then hits a sample plane for interference, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective lens, and the rear focal plane of the objective lens is located on the Fourier plane of the spatial light modulator through the 4f system; the plus or minus 1-order linear polarization diffraction light interferes on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective lens after returning, filtered by an emission filter after passing through a second dichroic mirror, and focused by a lens barrel lens; the s-axis and the p-axis of the first dichroic mirror and the second dichroic mirror are interchanged, so that polarization distortion caused by the fact that one dichroic mirror is used independently is eliminated; the fluorescence focused by the lens barrel is projected on a camera to realize digital imaging; acquiring fluorescence with corresponding wavelength by a camera under the selected wavelength to obtain an original image; keeping the angle of the binary periodic stripes unchanged, carrying out phase shift of 2 pi/n ' on the binary periodic stripes loaded on the digital micromirror array to form new binary periodic stripes, acquiring a second original image through a camera, and repeating and summing n ' times in the process to obtain n ' original images with different phases in the same direction to form an original image in the first direction; the binary periodic stripes rotate around a horizontal optical axis by an angle of pi/k ', and the same data acquisition process is carried out in the first direction, so that original images in k' directions are obtained, n 'original images are arranged in each direction, and k' × n 'original images in the k' directions are formed into a group of original images; and reconstructing the sample by a two-dimensional structured light illumination microscopic imaging algorithm to obtain a two-dimensional super-resolution image of the sample.
The invention utilizes the digital micro-mirror array to generate three beams of linearly polarized light, utilizes the polarization control device to carry out polarization control in front of the digital micro-mirror array, and then interferes the three beams of light after polarization modulation on the surface of a sample to realize structured light illumination microimaging. However, the digital micromirror array is used as a blazed grating, different wavelength light needs to meet different incidence angles through the diffraction of the digital micromirror array, and the invention enables the different wavelength light to be optically coupled with 0-level after the diffraction of the digital micromirror array through the special incidence angle design, thereby realizing the multicolor structured light illumination microscopic imaging based on the digital micromirror array. In addition, the invention can rapidly switch between two modes of three-dimensional multicolor structure light illumination microscopic imaging and two-dimensional multicolor structure light illumination microscopic imaging, and is suitable for different imaging requirements.
The diffraction problem of the digital micromirror array is reduced to a one-dimensional discussion. α is the incident angle, i.e. the angle between the incident light and the normal of the digital micromirror array plane, β is the corresponding reflection angle, so there is the grating equation:
where λ is the wavelength of the incident light, d is the pixel size of the digital micromirror array, and m is a factor that quantifies the flare condition. If m is an integer, the flare condition is satisfied, and if m is an integer plus 0.5, the flare condition is far from the flare condition. For better quantification of sparkle conditions, we define with μ:
therefore, when μ is 0.5, it means that the flare condition is satisfied, and when μ is 0, the flare condition is completely deviated.
The sample is placed on a three-dimensional stage. The wavelength selection device, the polarization control device, the digital micromirror array, the three-dimensional objective table and the camera are respectively connected to the data acquisition card, and the data acquisition card is used for realizing integral and synchronous control. The conversion rotating frame is connected to the data acquisition card, and the data acquisition card is used for realizing control switching.
The beam combining device adopts a dichroic mirror, linear polarization lasers with different wavelengths are respectively emitted by aiming at the plurality of lasers, and the linear polarization lasers with different wavelengths are combined by the dichroic mirror with corresponding wavelengths.
The illumination light source comprises N single-wavelength lasers, N is a natural number larger than or equal to 2, correspondingly, the beam combining device adopts N-1 dichroic mirrors, one or more reflectors are correspondingly arranged on each wavelength, and therefore the spatial orientation of the light path with the corresponding wavelength is adjusted, and beams of all wavelengths are combined. According to the blaze condition, incident lights with different wavelengths have corresponding incident angles, linear polarized lasers with N wavelengths have L groups of incident angles, the incident angle adjusting device correspondingly adopts the combination of L-1 groups of dichroic mirrors and reflecting mirrors, the light with corresponding wavelengths is reflected or transmitted by the dichroic mirrors respectively, the angles are adjusted, and then the light is reflected by the corresponding reflecting mirrors to be further adjusted to the incident angles meeting the blaze condition.
Under the three-dimensional structure light illumination mode, the spatial filter is a three-dimensional light-passing hole filter; under the two-dimensional structure light illumination mode, the spatial filter is a two-dimensional light-passing hole filter; the three-dimensional light-passing hole filter is provided with a 0-level through hole positioned in the center and N pairs of first-level through holes which are symmetrical about the center, and each pair of first-level through holes are respectively positioned on a straight line passing through the center; the two-dimensional light-passing hole filter has N pairs of first-level through holes which are symmetrical about a center, and each pair of first-level through holes are respectively positioned on a straight line passing through the center.
The wavelength selection device adopts an acousto-optic tunable filter or N acousto-optic modulators, N is the number of single-wavelength lasers, and N is a natural number more than or equal to 2; the polarization control device adopts an electro-optical modulator, an ultrafast ferroelectric liquid crystal polarization rotator or a nematic liquid crystal phase retarder.
The sample plane is the front focal plane of the objective lens; the 0-level light and the positive-negative first-level light are focused on a back focal plane of the objective lens, are emitted in parallel after passing through the objective lens, and interfere on a sample plane to generate structured light.
Another objective of the present invention is to provide a method for multicolor dual-mode structured light illumination microscopy based on digital micromirror array and laser interference.
The invention discloses a multicolor dual-mode structured light illumination microimaging method based on a digital micromirror array and laser interference, which comprises a three-dimensional structured light illumination mode and a two-dimensional structured light illumination mode:
three-dimensional structured light illumination mode
1) The illumination light source comprises a plurality of single-wavelength lasers, each single-wavelength laser emits linearly polarized laser with one wavelength, and the plurality of lasers respectively emit linearly polarized laser with different wavelengths;
2) the linear polarized lasers with different wavelengths are combined by the beam combining device and transmitted to the wavelength selection device;
3) the wavelength of the passing linearly polarized laser is quickly selected through a wavelength selection device, the linearly polarized laser with the selected wavelength is subjected to polarization control of light through a polarization control device, and the polarization direction of the linearly polarized laser modulated by the polarization control device is consistent with the direction of the binary periodic fringes loaded on the digital micromirror array;
4) after being expanded by the collimation and beam expansion device, the beam is incident to the digital micromirror array;
5) in the structured light illumination microscopic imaging system, a digital micro-mirror array is equivalent to a diffraction grating, linearly polarized light of different orders formed by diffraction of the digital micro-mirror array is interfered on the surface of a sample to form illumination structured light, and incident linearly polarized laser needs to meet the blaze condition of the digital micro-mirror array, so that the illumination structured light with high contrast is generated, wherein the blaze condition is that incident light with different wavelengths has corresponding incident angles; the incident angle adjusting device adopts the combination of a dichroic mirror and a reflecting mirror, adjusts the routes of the linear polarized laser with different wavelengths according to the blaze condition, and adjusts the linear polarized laser with the selected wavelength to be incident to the digital micromirror array at a corresponding incident angle, so that the blaze condition is met; the plane of the digital micromirror array is vertical to the optical axis, and periodic binary periodic stripes which are alternate between black and white are loaded on the digital micromirror array and reflected by the digital micromirror array to form multi-level linearly polarized diffracted light; the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter;
6) selecting a spatial filter as a three-dimensional light-passing hole filter under a three-dimensional structure light illumination mode by changing a rotating frame; the 0-order and +/-1-order polarized diffracted lights pass through the spatial filter;
7) the 0-level and +/-1-level polarized diffraction light passes through the first dichroic mirror, then passes through the 4f system, then passes through the second dichroic mirror, is converged to the rear focal plane of the objective lens, passes through the objective lens and then hits a sample plane for interference, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective lens, and namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator through the 4f system;
8) the 0-order and +/-1-order linearly polarized diffracted lights interfere on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective lens after returning, filtered by an emission filter after passing through a second dichroic mirror, and focused by a lens barrel lens; the s-axis and the p-axis of the first dichroic mirror and the second dichroic mirror are interchanged, so that polarization distortion caused by the fact that one dichroic mirror is used independently is eliminated;
9) the fluorescence focused by the lens barrel is projected on a camera to realize digital imaging;
10) acquiring fluorescence with corresponding wavelength by a camera under the selected wavelength to obtain an original image;
11) keeping the angle of the binary periodic stripes unchanged, carrying out phase shift of 2 pi/n on the binary periodic stripes loaded on the digital micromirror array to form new binary periodic stripes, repeating the steps 1) -10) to obtain a second original image, and repeating the steps for n times to obtain n original images with different phases in the same direction to form an original image in the first direction;
12) the binary periodic stripes rotate around a horizontal optical axis by an angle of pi/k, and the steps 1) to 11) are repeated, so that original images in k directions are obtained, n original images are arranged in each direction, k multiplied by n original images in the k directions form a group of original images, and k and n are natural numbers more than or equal to 2;
13) controlling a three-dimensional stage for placing a sample to move along the optical axis direction, so that the sample is stepped by a set step length along the optical axis direction, and still acquiring a group of original images in k directions and n phases in each direction; acquiring a plurality of groups of original images in a set range through multiple steps, thereby forming a plurality of layers of original images;
14) performing three-dimensional reconstruction through a three-dimensional structured light illumination microscopic imaging algorithm to obtain a three-dimensional super-resolution image of the sample;
two-dimensional structured light illumination mode
1) The illumination light source comprises a plurality of single-wavelength lasers, each single-wavelength laser emits linearly polarized laser with one wavelength, and the plurality of lasers respectively emit linearly polarized laser with different wavelengths;
2) the linear polarized lasers with different wavelengths are combined by the beam combining device and transmitted to the wavelength selection device;
3) the wavelength of the passing linearly polarized laser is quickly selected through a wavelength selection device, the linearly polarized laser with the selected wavelength is subjected to polarization control of light through a polarization control device, and the polarization direction of the linearly polarized laser modulated by the polarization control device is consistent with the direction of the binary periodic fringes loaded on the digital micromirror array;
4) after being expanded by the collimation and beam expansion device, the beam is incident to the digital micromirror array;
5) in the structured light illumination microscopic imaging system, a digital micro-mirror array is equivalent to a diffraction grating, linearly polarized light of different orders formed by diffraction of the digital micro-mirror array is interfered on the surface of a sample to form illumination structured light, and incident linearly polarized laser needs to meet the blaze condition of the digital micro-mirror array, so that the illumination structured light with high contrast is generated, wherein the blaze condition is that incident light with different wavelengths has corresponding incident angles; the incident angle adjusting device adopts the combination of a dichroic mirror and a reflecting mirror, adjusts the routes of the linear polarized laser with different wavelengths according to the blaze condition, and adjusts the linear polarized laser with the selected wavelength to be incident to the digital micromirror array at a corresponding incident angle, so that the blaze condition is met; the plane of the digital micromirror array is vertical to the optical axis, and periodic binary periodic stripes which are alternate between black and white are loaded on the digital micromirror array and reflected by the digital micromirror array to form multi-level linearly polarized diffracted light; the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter;
6) selecting a spatial filter as a two-dimensional light-passing hole filter under a three-dimensional structure light illumination mode by changing a rotating frame; the plus or minus 1 order polarized diffracted light passes through the spatial filter;
7) the +/-1-order polarized diffraction light passes through the first dichroic mirror, then passes through the 4f system, then passes through the second dichroic mirror, is converged to the rear focal plane of the objective lens, passes through the objective lens and then hits a sample plane for interference, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective lens, and the rear focal plane of the objective lens is located on the Fourier plane of the spatial light modulator through the 4f system;
8) the plus or minus 1-order linear polarization diffraction light interferes on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective lens after returning, filtered by an emission filter after passing through a second dichroic mirror, and focused by a lens barrel lens; the s-axis and the p-axis of the first dichroic mirror and the second dichroic mirror are interchanged, so that polarization distortion caused by the fact that one dichroic mirror is used independently is eliminated;
9) the fluorescence focused by the lens barrel is projected on a camera to realize digital imaging;
10) acquiring fluorescence with corresponding wavelength by a camera under the selected wavelength to obtain an original image;
11) keeping the angle of the binary periodic stripes unchanged, carrying out phase shift of 2 pi/n ' on the binary periodic stripes loaded on the digital micromirror array to form new binary periodic stripes, repeating the steps 1) -10) n ' times to obtain n ' original images with different phases in the same direction to form an original image in the first direction;
12) the binary periodic stripes rotate around a horizontal optical axis by an angle of pi/k ', the steps 1) -11) are repeated for k ' times, so that original images in k ' directions are obtained, n ' original images are arranged in each direction, k ' directions are totally k ' × n ' original images to form a group of original images, and k ' and n ' are natural numbers more than or equal to 2;
13) and reconstructing the sample by a two-dimensional structured light illumination microscopic imaging algorithm to obtain a two-dimensional super-resolution image of the sample.
Wherein, in the step 13) of the three-dimensional structured light illumination mode, the set step length is 100-300 nm.
The invention has the advantages that:
the invention adopts the digital micromirror array to realize the multicolor dual-mode structured light illumination microscopic imaging system, and has the following advantages: 1. compared with the traditional grating diffraction, the digital micromirror array greatly accelerates the imaging speed; 2. the system can realize the switching of the rapid two-dimensional and three-dimensional structured light illumination microscopic imaging modes; 3. aiming at the characteristic of a digital micromirror array blazed grating, the invention provides a multi-angle illumination coupling mode, and can realize multi-color imaging.
Drawings
FIG. 1 is a diagram showing the relationship between the contrast of interference fringes and the difference between positive and negative first-order light intensities, (b) a diagram showing the relationship between the blaze condition and the incident angle of the illumination light and the wavelength of the illumination light, and (c) a diagram showing the multi-color imaging realized by the switch state of the digital micromirror array;
FIG. 2 is a schematic diagram of one embodiment of a multi-color dual-mode structured light illuminated microimaging system based on a digital micromirror array and laser interference of the present invention.
Detailed Description
The invention will be further elucidated by means of specific embodiments in the following with reference to the drawing.
As shown in fig. 1, the multi-color dual-mode structured light illumination micro-imaging system based on digital micromirror array and laser interference of the present embodiment includes: the device comprises an illumination light source, a beam combining device, an acousto-optic tunable filter AOTF, an electro-optic modulator EOM, a collimation beam expanding device, an incidence angle adjusting device, a digital micromirror array DMD, a condenser lens L3, a spatial filter SM, a first dichroic mirror DM1, a second dichroic mirror DM2, a 4f system, a polarization correcting device, an objective OBJ, a tube lens L6, a camera sCMOS, a data acquisition card and a transformation rotating frame; the illumination light source comprises four single-wavelength lasers L1-L4, each single-wavelength laser emits linearly polarized laser with one wavelength, and the lasers emit linearly polarized laser with different wavelengths respectively at 370nm, 488nm, 561nm and 640 nm; the beam combining device adopts fourth to sixth dichroic mirrors DM 4-DM 6, linear polarized laser light emitted by a first single-wavelength laser L1 is reflected by a first reflecting mirror M1 and a second reflecting mirror M2, and linear polarized laser light emitted by second to fourth single-wavelength lasers L2-L4 is respectively reflected by third to fifth reflecting mirrors M3-M5, then is respectively combined by fourth to sixth dichroic mirrors DM 4-DM 6, and is transmitted to an acousto-optic tunable filter AOTF; the wavelength of the passing linear polarization laser is rapidly selected through an acousto-optic tunable filter AOTF, the linear polarization laser with the selected wavelength is electrified with an optical modulator EOM to carry out optical polarization control, and the polarization direction of the linear polarization laser modulated by the electrified optical modulator EOM is consistent with the direction of a binary periodic stripe loaded on a digital micromirror array DMD; the collimation and beam expansion device adopts a first lens L1 and a second lens L2, after being reflected by a sixth reflector M6, the collimation and beam expansion device expands the beam, and after being reflected by a seventh reflector M7, the incidence angle is adjusted by an incidence angle adjusting device and the beam is incident to the digital micromirror array DMD; the incident angle adjusting device adopts the combination of a third dichroic mirror DM3 and an eighth reflecting mirror M8, adjusts the routes of linear polarized laser with different wavelengths according to the blaze condition, and adjusts the linear polarized laser with the selected wavelength to be incident to the digital micromirror array DMD at a corresponding incident angle, so that the blaze condition is met; the plane of the digital micromirror array DMD is vertical to the optical axis, and periodic binary periodic stripes which are alternate between black and white are loaded on the digital micromirror array DMD and reflected by the digital micromirror array DMD to form multi-level linear polarization diffraction light; the multistage linearly polarized diffracted light is focused by a condenser lens L3 and then passes through a ninth reflector M9 to a spatial filter SM, the spatial filter SM is arranged on a transformation rotating frame, the transformation rotating frame adopts an electric fast transformation rotating frame, and the spatial filter SM is selected to be a three-dimensional light-passing hole filter under a three-dimensional structure light illumination mode or a two-dimensional light-passing hole filter under a two-dimensional structure light illumination mode through the transformation rotating frame; under the three-dimensional structured light illumination mode, 0-order and +/-1-order polarized diffracted lights pass through the spatial filter SM; the 0-level and +/-1-level polarized diffraction light passes through a 4f system after passing through a first dichroic mirror DM1, the 4f system adopts a fourth lens L4 and a fifth lens L5, is reflected by a fourth lens through a tenth reflecting mirror M10 and then to a fifth lens L5, is reflected by an eleventh reflecting mirror M11 and then passes through a second dichroic mirror DM2 to be converged at the rear focal plane of an objective OBJ, and is incident on a sample plane for interference after passing through the objective OBJ, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective OBJ, namely the rear focal plane of the objective OBJ is positioned on the Fourier plane of the spatial light modulator through the 4f system; the 0-order and +/-1-order linearly polarized diffracted lights interfere on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective OBJ after returning, is reflected by a second dichroic mirror DM2, passes through an emission filter EF for filtering through a tenth reflecting mirror M12, and is focused by a tube lens L6; the s-axis and p-axis of first dichroic mirror DM1 and second dichroic mirror DM2 are interchanged, thereby eliminating polarization distortion introduced by using one dichroic mirror alone; the fluorescence focused by the tube lens L6 is projected on the sCMOS camera to realize digital imaging.
Digital micromirror arrays are widely used in industry as spatial light modulators, and consist of an array of micromirrors on a CMOS memory cell, which can modulate the light wave front by the turning/switching state of each micromirror. The digital micro-mirror array plays a role as a diffraction grating in a structured light illumination micro-imaging system, and different orders of linearly polarized light formed by diffraction of the digital micro-mirror array form illumination structured light through interference on the surface of a sample. The key of the application of the digital micromirror array to the structured light illumination microscopic imaging is to ensure that incident light meets the blaze condition of the digital micromirror array DMD, so that high-contrast illumination structured light is generated. The satisfaction of the blaze condition requires matching of the incident light wavelength, the grating constant and the incident angle. The required grating constant is given for achieving a specific resolution, so polychromatic structured light illumination microscopy based on digital micromirror array DMD requires the selection of the appropriate laser wavelength and angle of incidence. The square micro-mirror on the digital micro-mirror array DMD rotates around the diagonal line of the micro-mirror by +/-12 degrees to realize the switching of the switch state, and the diffraction effects of the digital micro-mirror array DMD along the X and Y directions of the side length of the micro-mirror are the same, so that the diffraction problem of the digital micro-mirror array DMD can be simplified into one-dimensional discussion. If α is the angle between the incident light and the normal of the DMD plane, and β is the corresponding reflection angle, then there is a grating equation:
where λ is the wavelength of the incident light, d is the pixel size of the DMD, and m is a factor that quantifies the flare condition. If m is an integer, the flare condition is satisfied, and if m is an integer plus 0.5, the flare condition is far from the flare condition. For better quantification of sparkle conditions, we define with μ:
therefore, when μ is 0.5, it means that the flare condition is satisfied, and when μ is 0, the flare condition is completely deviated. There are multiple incident angles a satisfying the condition for a given incident light wavelength λ, and fig. 1(b) is a graph of the incident linearly polarized light angle, wavelength, and flare condition, where the intensity values in the graph indicate the value of the flare condition μ, and a value of μ higher than 0.43 is experimentally considered preferable. In the present embodiment, for wavelengths 370nm, 488nm, 561nm, and 640nm, two sets of incident angles satisfying the blaze condition are found: the incident angles corresponding to the wavelengths of 561nm and 640nm are 48 degrees, and the incident angles correspond to the on state of the digital micromirror array DMD; the incident angles corresponding to 370nm and 488nm are 24 °, and corresponding to the off state of the DMD, as shown by the star mark value in fig. 1(b) and fig. 1(c), the four beams of light are emitted in the same direction after being reflected by the DMD. In structured light illumination microscopic imaging, binary periodic fringes are loaded on a digital micromirror array (DMD), and incident light is reflected by the digital micromirror array (DMD) to form +/-1-level linearly polarized diffracted light. When the incident condition meets the blaze condition, the generated plus or minus 1-order linearly polarized diffracted light has uniform light intensity, and then can form a high-contrast structured light illumination bright fringe on the surface of the sample through interference, as shown in fig. 1(a), so as to reconstruct a high-quality structured light illumination super-resolution image. Based on the relationship of fig. 1(b), different laser wavelengths can be incident to the DMD array at corresponding incident angles to satisfy the blaze condition. The invention provides a scheme for naturally coupling different wavelength diffraction light in a light path by analyzing the relationship between the wavelength and the incidence and diffraction angles, and realizes the multi-color structured light illumination microscopic imaging.
The four wavelengths are obtained according to the blaze condition and correspond to two groups of incident angles, the incident angle adjusting device correspondingly adopts the combination of a group of dichroic mirrors and reflecting mirrors, the light with the corresponding wavelengths is reflected or transmitted by the dichroic mirrors respectively, the angles are adjusted, and then the light is further adjusted to the incident angle meeting the blaze condition through the reflection of the corresponding reflecting mirrors; wherein, the wavelengths of 561nm and 640nm are reflected by a third dichroic mirror DM3 and are incident to the digital micro-mirror array at an incident angle of 48 degrees; 370nm and 488nm are transmitted by the third dichroic mirror DM3, reflected by the eighth reflecting mirror and incident to the digital micro-mirror array at an incident angle of 24 degrees.
Example one
In the embodiment, a three-dimensional structured light illumination mode is adopted, and the multicolor dual-mode structured light illumination microimaging method comprises the following steps:
1) the illumination light source comprises four single-wavelength lasers, each single-wavelength laser emits linearly polarized laser with one wavelength, and the multiple lasers respectively emit linearly polarized laser with different wavelengths, namely 370nm, 488nm, 561nm and 640 nm;
2) the linear polarized lasers with different wavelengths are combined by the beam combining device and transmitted to the wavelength selection device;
3) the wavelength of the passing linear polarization laser is quickly selected through a wavelength selection device, the linear polarization laser with the selected wavelength is subjected to polarization control of light through a polarization control device, and the polarization direction of the linear polarization laser modulated through the polarization control device is consistent with the direction of the binary periodic fringes loaded on the digital micromirror array DMD;
4) after being expanded by the collimation and beam expansion device, the beam is incident to the digital micromirror array DMD;
5) in the structured light illumination microscopic imaging system, a digital micromirror array (DMD) is equivalent to a diffraction grating, linearly polarized light of different orders formed by diffraction of the DMD is interfered on the surface of a sample to form illumination structured light, and incident linearly polarized laser needs to meet the blaze condition of the DMD, so that high-contrast illumination structured light is generated, wherein the blaze condition is that incident light of different wavelengths has corresponding incident angles; the incident angle adjusting device adopts the combination of a dichroic mirror and a reflecting mirror, adjusts the routes of the linear polarized lasers with different wavelengths according to the blaze condition, and adjusts the linear polarized lasers with the selected wavelengths to be incident to the digital micromirror array DMD at corresponding incident angles, so that the blaze condition is met; the plane of the digital micromirror array DMD is vertical to the optical axis, and periodic binary periodic stripes which are alternate between black and white are loaded on the digital micromirror array DMD and reflected by the digital micromirror array DMD to form multi-level linear polarization diffraction light; the multi-stage linearly polarized diffraction light is focused by a condenser lens L3 and then reaches a spatial filter SM;
6) selecting a spatial filter SM as a three-dimensional light-passing hole filter in a three-dimensional structure light illumination mode by changing a rotating frame; passing through the spatial filter SM and then passing through the 0-order and +/-1-order polarized diffracted lights;
7) the 0-order and +/-1-order polarized diffraction light passes through the first dichroic mirror DM1, then passes through the 4f system, then passes through the second dichroic mirror DM2, is converged to the rear focal plane of the objective OBJ, passes through the objective OBJ and then strikes a sample plane for interference, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective OBJ, and namely the rear focal plane of the objective OBJ is located on the Fourier plane of the spatial light modulator through the 4f system;
8) the 0-order and +/-1-order linearly polarized diffracted lights interfere on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective OBJ after returning, filtered by an emission filter after passing through a second dichroic mirror DM2, and focused by a tube lens L6; the s-axis and p-axis of first dichroic mirror DM1 and second dichroic mirror DM2 are interchanged, thereby eliminating polarization distortion introduced by using one dichroic mirror alone;
9) the fluorescence focused by the tube lens L6 is projected on the sCMOS camera to realize digital imaging;
10) under the selected wavelength, a phase machine sCMOS acquires fluorescence with corresponding wavelength to obtain an original image;
11) keeping the angle of the binary periodic stripes unchanged, carrying out phase shift of 2 pi/5 on the binary periodic stripes loaded on the digital micromirror array DMD to form new binary periodic stripes, repeating the steps 1) -10) to obtain a second original image, and repeating the process for five times to obtain five original images with different phases in the same direction to form an original image in the first direction;
12) the binary periodic stripes are rotated by an angle of 60 degrees around a horizontal optical axis, and the steps 1) to 11) are repeated, so that original images in three directions are obtained, five original images are arranged in each direction, and 15 original images in the three directions form a group of original images;
13) controlling a three-dimensional stage for placing a sample to move along the optical axis direction, so that the sample is stepped along the optical axis direction by a step length of 125nm, and still acquiring a group of original images in three directions and five phases in each direction; after a plurality of steps are carried out,
collecting a plurality of groups of original images in a set range to form a plurality of layers of original images;
14) and performing three-dimensional reconstruction by a three-dimensional structured light illumination microscopic imaging algorithm to obtain a three-dimensional super-resolution image of the sample.
Example two
In the embodiment, a two-dimensional structured light illumination mode is adopted, and the multicolor dual-mode structured light illumination microimaging method comprises the following steps:
1) the illumination source comprises a plurality of single-wavelength lasers, each emitting linearly polarized laser light of one wavelength,
the multiple lasers respectively emit linear polarized lasers with different wavelengths;
2) the linear polarized lasers with different wavelengths are combined by the beam combining device and transmitted to the wavelength selection device;
3) the wavelength of the passing linear polarization laser is quickly selected through a wavelength selection device, the linear polarization laser with the selected wavelength is subjected to polarization control of light through a polarization control device, and the polarization direction of the linear polarization laser modulated through the polarization control device is consistent with the direction of the binary periodic fringes loaded on the digital micromirror array DMD;
4) after being expanded by the collimation and beam expansion device, the beam is incident to the digital micromirror array DMD;
5) in the structured light illumination microscopic imaging system, a digital micromirror array (DMD) is equivalent to a diffraction grating, linearly polarized light of different orders formed by diffraction of the DMD is interfered on the surface of a sample to form illumination structured light, and incident linearly polarized laser needs to meet the blaze condition of the DMD, so that high-contrast illumination structured light is generated, wherein the blaze condition is that incident light of different wavelengths has corresponding incident angles; the incident angle adjusting device adopts the combination of a dichroic mirror and a reflecting mirror, adjusts the routes of the linear polarized lasers with different wavelengths according to the blaze condition, and adjusts the linear polarized lasers with the selected wavelengths to be incident to the digital micromirror array DMD at corresponding incident angles, so that the blaze condition is met; the plane of the digital micromirror array DMD is vertical to the optical axis, and periodic binary periodic stripes which are alternate between black and white are loaded on the digital micromirror array DMD and reflected by the digital micromirror array DMD to form multi-level linear polarization diffraction light; the multi-stage linearly polarized diffraction light is focused by a condenser lens L3 and then reaches a spatial filter SM;
6) selecting a space filter SM as a two-dimensional light-passing hole filter in a three-dimensional structure light illumination mode by changing a rotating frame;
after passing through a spatial filter SM, the plus or minus 1-order polarized diffracted light passes through;
7) the +/-1-order polarized diffraction light passes through the first dichroic mirror DM1, then passes through the 4f system, then passes through the second dichroic mirror DM2, is converged to the rear focal plane of the objective OBJ, passes through the objective OBJ and then hits a sample plane for interference, the 4f system delays the Fourier plane of the spatial light modulator to the rear focal plane of the objective OBJ, and namely the rear focal plane of the objective OBJ is located on the Fourier plane of the spatial light modulator through the 4f system;
8) the plus or minus 1-level linear polarization diffraction light interferes on a sample plane to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by an objective OBJ after returning, filtered by an emission optical filter after passing through a second dichroic mirror DM2, and focused by a tube lens L6; the s-axis and p-axis of first dichroic mirror DM1 and second dichroic mirror DM2 are interchanged, thereby eliminating polarization distortion introduced by using one dichroic mirror alone;
9) the fluorescence focused by the tube lens L6 is projected on the sCMOS camera to realize digital imaging;
10) under the selected wavelength, a phase machine sCMOS acquires fluorescence with corresponding wavelength to obtain an original image;
11) keeping the angle of the binary periodic stripes unchanged, carrying out phase shift of 2 pi/3 on the binary periodic stripes loaded on the digital micromirror array DMD to form new binary periodic stripes, repeating the steps 1) -10) three times to obtain three original images with different phases in the same direction to form an original image in the first direction;
12) the binary periodic stripes are rotated around a horizontal optical axis by an angle of 60 degrees, and the steps 1) to 11) are repeated three times, so that original images in three directions are obtained, three original images are arranged in each direction, and nine original images in the three directions form a group of original images;
13) and reconstructing the sample by a two-dimensional structured light illumination microscopic imaging algorithm to obtain a two-dimensional super-resolution image of the sample.
Finally, it is noted that the disclosed embodiments are intended to aid in further understanding of the invention, but those skilled in the art will appreciate that: various substitutions and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the invention should not be limited to the embodiments disclosed, but the scope of the invention is defined by the appended claims.
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