CN106885796B - Super-resolution fluorescence digital holographic tomography microscopic imaging system and method - Google Patents
Super-resolution fluorescence digital holographic tomography microscopic imaging system and method Download PDFInfo
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Abstract
A super-resolution fluorescence digital holographic tomography microscopic imaging system and a method belong to the field of fluorescence digital holography and super-resolution imaging. The laser after amplitude modulation by the spatial light modulator loaded with a proper mask structure enters a microscope objective lens to generate three-dimensional structure excitation light on a sample to be imaged. The fluorescence of the excited sample is collected by the microscope objective, after passing through the dichroic mirror and the imaging system, the diffraction beam splitting and the phase shifting are carried out by the spatial light modulator, and the two beams of light interfere at the position of the image detector to form a hologram and are recorded. Reconstructing the recorded hologram in a computer through a numerical algorithm, wherein for a structure with a certain depth in a sample, a reconstructed image of super-resolution and optical fault can be obtained respectively; the super-resolution optical tomography can be realized by fusing the super-resolution and the reconstruction of the optical tomography by using a numerical algorithm; the reconstruction distance of the hologram is changed, so that super-resolution optical tomography three-dimensional imaging of structures at different inner depths of the sample can be realized.
Description
Technical Field
The invention discloses a super-resolution fluorescence digital holographic three-dimensional tomographic imaging system and method based on structural illumination and fluorescence digital holography, and belongs to the technical field of fluorescence digital holography and super-resolution imaging.
Background
Digital holography is used as an interference imaging technology, and can realize three-dimensional numerical reconstruction or quantitative phase contrast imaging of a coherent illumination sample image. Digital holographic microscopy has been widely used in specific research in the biomedical field. In practical imaging applications, digital holographic microscopy has the advantages of being fast, real-time, three-dimensional, high-precision, and the like, compared with conventional microscopic imaging techniques. The appearance of fluorescence digital holography solves the problem of the dependence of the traditional digital holography on the coherence of the illumination light source, and expands the digital holographic microscopy technology to the field of space incoherent imaging. By utilizing the spatial self-coherence characteristic of light, devices such as a spatial light modulator and the like are used for carrying out operations such as light splitting, phase shifting and the like on light waves emitted by a sample in the fluorescent digital holographic technology, then an image acquisition device is used for recording holograms, the holograms are processed through a numerical algorithm in a computer, and finally three-dimensional reconstruction of images at different depths of the fluorescent sample can be realized. While fluorescence digital holography has three-dimensional imaging capabilities, imaging results at certain depths within the sample are more susceptible to fluorescence from other depths of the sample, i.e., optical tomography is less capable. The fluorescent digital holographic technology is combined with confocal microscopy and other technologies, so that high-resolution optical tomography of the sample can be realized. However, in order to realize three-dimensional reconstruction of sample information, two-dimensional or three-dimensional scanning is still required for the sample in most of the existing similar researches, and the acquisition speed of the system is slow. And the focused light spot irradiated on the sample in the confocal technology has higher energy density, which is easy to cause photobleaching of fluorescent protein or dye.
Structure illumination microscopy is a super-resolution imaging technique that is mainly applied to the field of wide-field fluorescence microscopy. In conventional structure illumination microscopy, devices such as a laser illumination grating or a spatial light modulator are generally utilized to generate stripe-shaped intensity periodic distribution excitation light on a sample. The excited fluorescence has similar periodic intensity distribution, and then the fluorescence signal is detected by an imaging system and an image detector, and a super-resolution fluorescence sample image is reconstructed in a computer by combining a numerical algorithm. The super-resolution image obtained by the method is only two-dimensional, and the three-dimensional information of the sample is lacking. By means of a structural light tomography reconstruction algorithm, the optical tomography results of the sample can be reconstructed from the raw data acquired by the structural light microscopy. However, the resolution of the reconstructed optical tomographic result is at most equal to the diffraction limited resolution, i.e., super-resolution imaging cannot be achieved. In combination with the multi-beam interference technique, excitation light of three-dimensional structure can be generated to illuminate the sample. And scanning the sample in the depth direction by using the precision piezoelectric translation stage, recording fluorescent images at different depths of a series of samples, and performing three-dimensional numerical operation on the images to realize three-dimensional super-resolution reconstruction of fluorescent sample images. However, the scanning process of this method takes a long time, the recorded data volume is large, the numerical calculation amount involved is large, and the time is long. And thus are not suitable for dynamic sample imaging or observation of dynamic processes within the sample.
In order to solve the above limitations, the invention combines three-dimensional structure illumination microscopy and fluorescence digital holography, and discloses a super-resolution fluorescence digital holographic tomography microscopic imaging system and method. Compared with the existing structure illumination microscopy or fluorescence digital holography, the system and the method provided by the invention can realize wide-field super-resolution optical tomographic three-dimensional imaging of a fluorescent sample.
Disclosure of Invention
In order to combine the fluorescent digital holographic technology in the existing structure illumination microscopy, realize the optical tomography of super-resolution and improve the imaging speed, the invention provides a super-resolution fluorescent digital holographic tomography microscopic imaging system and method.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the invention discloses a super-resolution fluorescent digital holographic tomography microscopic imaging system which is characterized by comprising a laser (1), an optical fiber (2), a collimating lens (3), an I spatial light modulator (4), a first lens (5), a dichroic mirror (6), a second lens (7), a third lens (8), a reflecting mirror (9), a microscope objective (10), a fourth lens (12), a sample (11), a beam splitting prism (13), an II spatial light modulator (14) and an image collector (15), wherein the laser is connected with an optical path; the method is characterized in that: the light emitted by the laser (1) is conducted by the optical fiber (2) and collimated by the collimating lens (3) to illuminate the I spatial light modulator (4), the light reflected by the I spatial light modulator (4) is converged by the first lens (5) and then reflected by the dichroic mirror (6), the light reflected by the dichroic mirror (6) is further reflected by the second lens (7) and the third lens (8) in sequence and then enters the micro-objective lens (10) after being reflected by the reflecting mirror (9), the light after passing through the micro-objective lens (10) irradiates on the sample (11), the excited fluorescence is collected by the micro-objective lens (10) and then reflected by the reflecting mirror (9), and then sequentially passes through the third lens (8), the second lens (7), the dichroic mirror (6) and the beam splitting prism (13) and then irradiates on the II spatial light modulator (14), and the light reflected by the II spatial light modulator (14) is reflected by the beam splitting prism (13) again and then irradiates on the image collector (15), and the light intensity distribution is recorded by the image collector (15); the I spatial light modulator (4), the II spatial light modulator (14) and the image collector (15) are all connected with a computer (16).
Wherein, in order to produce the illumination light of three-dimensional structure, it is further preferable to produce and display the binary grating on the I space light modulator (4) in the computer (16); in order to achieve the splitting and phase shifting and thus the recording of the holograms, a diffraction splitting phase mask pattern is also generated in a computer (16) and displayed on a II spatial light modulator (14).
The microscope objective (10) is an infinity focus objective; the I spatial light modulator (4) is positioned on the conjugate plane of the back focal plane of the micro objective lens (10), namely the axial distance from the I spatial light modulator (4) to the first lens (5) is the focal length of the first lens (5), the axial distance from the first lens (5) to the second lens (7) is the sum of the focal lengths of the first lens (5) and the second lens (7), the axial distance from the second lens (7) to the third lens (8) is the sum of the focal lengths of the second lens (7) to the third lens (8), and the axial distance from the third lens (8) to the back focal plane of the micro objective lens (10) is the focal length of the third lens (8).
The II spatial light modulator (14) is a pure phase spatial light modulator, and fluorescence incident on the surface of the II spatial light modulator is required to pass through a polaroid to form linearly polarized light; and the included angle between the polarization orientation of the polaroid and the polarization sensitive axis of the II spatial light modulator (14) is 45 degrees.
The diffraction spectral phase mask loaded on the II spatial light modulator (14) is generated through numerical operation in a computer (16), and the phase distribution of the positive lens with certain size is correspondingly determined for a certain determined focal length; the diffraction spectral phase mask is generally composed of three phase distribution pictures with a defined phase shift relative to each other.
The placement position of the image collector (15) should be specially selected to optimize the quality of the recorded holograms; when the system (shown in figure 1) is used for experiments, one position of the collector (15) is fixed, the image collector (15) is moved forwards and backwards along the axial direction near the position of the image collector (15), and the clear images of the fluorescent sample can be obtained at two axial positions before and after the position of the image collector (15); the axial distance from these two axial positions to the II spatial light modulator (14) is denoted d 1 And d 2 The method comprises the steps of carrying out a first treatment on the surface of the The specific placement position of the image collector (15) should be selected as:the axial distance from the II spatial light modulator 14 is: 2d 1 d 2 /(d 1 +d 2 )。
The super-resolution fluorescence digital holographic tomography microscopic imaging method is characterized in that an image acquired by using a super-resolution fluorescence digital holographic tomography microscopic imaging system is processed through a numerical calculation process so as to reconstruct a super-resolution optical tomography image of a fluorescence sample; the method comprises the following specific steps:
(1) Displaying a binary grating which is generated in advance on the spatial light modulator I, sequentially displaying phase masks with different phase shift values on the spatial light modulator II, and respectively recording corresponding pictures by the image collector;
(2) Changing the grating phase value and generating a new binary grating, and repeating the step (1);
(3) Changing the grating orientation, and repeating the steps (1) and (2);
(4) Calculating in a computer a sample reconstruction image of the structural illumination by a phase shift algorithm and a scalar diffraction numerical algorithm for the hologram obtained in step (1); changing the reproduction distance in the scalar diffraction algorithm, a sample reproduction image of the structure illumination at different depths can be obtained;
(5) Repeating the step (4) for the images recorded in the steps (2) and (3) to obtain sample reproduction images of the structure illumination at different grating phases and different grating orientations; changing the reproduction distance in the scalar diffraction algorithm, a sample reproduction image of the structure illumination at different depths can be obtained;
(6) Aiming at a certain identical depth, namely structural illumination reproduction images with identical reproduction distance, synthesizing all the images obtained in the step (5) through a structural illumination super-resolution reconstruction algorithm, and finally obtaining a super-resolution reconstruction image of a sample at the depth;
(7) Combining all the images obtained in the step (5) through a structural illumination tomographic reconstruction algorithm aiming at a structural illumination reconstruction image with the same depth, namely the same reconstruction distance, and finally obtaining an optical tomographic reconstruction image of a sample at the depth;
(8) Carrying out frequency domain filtering and fusion on the super-resolution reconstructed image and the optical tomographic reconstructed image of the sample at a certain depth obtained in the steps (6) and (7) to obtain the super-resolution optical tomographic reconstructed image at the depth;
(7) Repeating steps (6) - (8) for all possible reproduction distances to obtain super-resolution optical tomographic images at different depths within the sample.
The depth of the invention is the distance between the sample (11) and the microscope objective (10).
The main advantages of the present system and method are: the three-dimensional reconstruction of structures at different depths in a sample can be realized by combining the advantages of the fluorescent digital holographic technology in the existing structure illumination microscopic imaging technology; meanwhile, super-resolution optical tomography can be realized, and the acquisition speed is high; an I spatial light modulator (4) is used as an element for generating structural illumination, so that the structural illumination has a large degree of freedom of selection in specific parameters such as the period, the form, the modulation degree and the like of the structure. The system and the method provided by the invention are used in the field of super-resolution observation application of three-dimensional dynamic fluorescent samples or three-dimensional dynamic processes in fluorescent samples.
Drawings
FIG. 1 is a schematic diagram of the constitution of a super-resolution fluorescence digital holographic tomography system of the present invention;
the system comprises a laser 1, an optical fiber 2, a collimation lens 3, a spatial light modulator 4I, a first lens 5, a dichroic mirror 6, a second lens 7, a third lens 8, a reflecting mirror 9, a microscope objective lens 10, a sample 11, a fourth lens 12, a beam splitting prism 13, a spatial light modulator 14II, an image collector 15 and a computer 16.
FIG. 2 is a diagram showing the structure of two samples used in the numerical simulation of the present invention;
FIG. 3 is an image (b) finally reconstructed by the system and method of the present invention in a numerical simulation referred to in the description of a specific real-time mode of the present invention; and an image (a) obtained by a conventional fluorescence microscopy imaging system under the same optical parameters.
Detailed Description
The present invention will be further illustrated with reference to the following examples, but the present invention is not limited to the following examples.
Example 1
The imaging process and the subsequent numerical processing method of the super-resolution fluorescence digital holographic tomography system are simulated in a computer by using numerical simulation software, the adopted super-resolution optical tomography fluorescence digital holographic tomography system is shown in figure 1, two samples with different structures shown in figure 2 are selected, and the samples are simultaneously placed at different depths in a space of the sample 11. The imaging process of the system is simulated, and after recording all the holograms required, the reconstruction algorithm is used to reconstruct the results shown in fig. 3 (b). Comparing the results obtained with the conventional fluorescence microscopy imaging system given in fig. 3 (a), it can be seen that the crosstalk between two object images at different depths within the sample space is smaller in fig. 3 (b); also in fig. 3 (b), the details of the sample constituted by the structures such as numerals and horizontal lines can be more clearly distinguished. Thus, the super-resolution optical tomography characteristic of the method proposed by the invention is demonstrated.
Claims (3)
1. The super-resolution fluorescence digital holographic tomography microscopic imaging system is characterized by comprising a laser (1), an optical fiber (2), a collimating lens (3), an I spatial light modulator (4), a first lens (5), a dichroic mirror (6), a second lens (7), a third lens (8), a reflecting mirror (9), a microscope objective (10), a fourth lens (12), a sample (11), a beam splitting prism (13), an II spatial light modulator (14) and an image collector (15), wherein the laser is connected with the optical path; the light emitted by the laser (1) is conducted by the optical fiber (2) and collimated by the collimating lens (3) to illuminate the I spatial light modulator (4), the light reflected by the I spatial light modulator (4) is converged by the first lens (5) and then reflected by the dichroic mirror (6), the light reflected by the dichroic mirror (6) is further reflected by the second lens (7) and the third lens (8) in sequence and then enters the micro-objective lens (10) after being reflected by the reflecting mirror (9), the light after passing through the micro-objective lens (10) irradiates on the sample (11), the excited fluorescence is collected by the micro-objective lens (10) and then reflected by the reflecting mirror (9), and then sequentially passes through the third lens (8), the second lens (7), the dichroic mirror (6) and the beam splitting prism (13) and then irradiates on the II spatial light modulator (14), and the light reflected by the II spatial light modulator (14) is reflected by the beam splitting prism (13) again and then irradiates on the image collector (15), and the light intensity distribution is recorded by the image collector (15); the I spatial light modulator (4), the II spatial light modulator (14) and the image collector (15) are connected with a computer (16);
generating and displaying a binary grating on an I spatial light modulator (4) in a computer (16); in order to achieve the light splitting and phase shifting and thus the recording of the hologram, a diffraction splitting phase mask pattern is also generated in a computer (16) and displayed on a II spatial light modulator (14);
the microscope objective (10) is an infinity focus objective; the I spatial light modulator (4) is positioned on the conjugate plane of the back focal plane of the micro objective lens (10), namely the axial distance from the I spatial light modulator (4) to the first lens (5) is the focal length of the first lens (5), the axial distance from the first lens (5) to the second lens (7) is the sum of the focal lengths of the first lens (5) and the second lens (7), the axial distance from the second lens (7) to the third lens (8) is the sum of the focal lengths of the second lens (7) to the third lens (8), and the axial distance from the third lens (8) to the back focal plane of the micro objective lens (10) is the focal length of the third lens (8);
the II spatial light modulator (14) is a pure phase spatial light modulator, and fluorescence incident on the surface of the II spatial light modulator is required to pass through a polaroid to form linearly polarized light; the included angle between the polarization orientation of the polaroid and the polarization sensitive axis of the II spatial light modulator (14) is 45 degrees;
the placement of the image collector (15) should be specifically selected to optimize the quality of the recorded holograms; when an experiment is carried out, one position of the collector (15) is fixed, the image collector (15) is moved forwards and backwards along the axial direction near the position of the image collector (15), and the clear images of the fluorescent sample can be obtained at two axial positions before and after the image collector (15); the axial distance of these two axial positions to the II spatial light modulator (14)Respectively marked asd 1 Andd 2 the method comprises the steps of carrying out a first treatment on the surface of the The specific placement position of the image collector (15) should be selected as: the axial distance from the II spatial light modulator (14) is as follows: 2d 1 d 2 /( d 1 + d 2 )。
2. A super-resolution fluorescence digital holographic tomography system as claimed in claim 1, in which the diffraction spectral phase mask loaded on the II spatial light modulator (14) is generated by numerical operations in a computer (16) corresponding to the phase distribution of positive lenses of determined size for a certain determined focal length; the diffraction spectral phase mask is composed of three phase distribution pictures with a certain phase shift between them.
3. A method for performing super-resolution fluorescence digital holographic tomography by using the system as claimed in any one of claims 1-2, wherein the image obtained by using the super-resolution fluorescence digital holographic tomography system is processed by a numerical calculation process to reconstruct a super-resolution optical tomographic image of the fluorescent sample; the method comprises the following specific steps:
(1) Displaying a binary grating which is generated in advance on the spatial light modulator I, sequentially displaying phase masks with different phase shift values on the spatial light modulator II, and respectively recording corresponding pictures by the image collector;
(2) Changing the grating phase value and generating a new binary grating, and repeating the step (1);
(3) Changing the grating orientation, and repeating the steps (1) and (2);
(4) Calculating in a computer a sample reconstruction image of the structural illumination by a phase shift algorithm and a scalar diffraction numerical algorithm for the hologram obtained in step (1); changing the reproduction distance in the scalar diffraction algorithm, a sample reproduction image of the structure illumination at different depths can be obtained;
(5) Repeating the step (4) for the images recorded in the steps (2) and (3) to obtain sample reproduction images of the structure illumination at different grating phases and different grating orientations; changing the reproduction distance in the scalar diffraction algorithm, a sample reproduction image of the structure illumination at different depths can be obtained;
(6) Aiming at a certain identical depth, namely structural illumination reproduction images with identical reproduction distance, synthesizing all the images obtained in the step (5) through a structural illumination super-resolution reconstruction algorithm, and finally obtaining a super-resolution reconstruction image of a sample at the depth;
(7) Combining all the images obtained in the step (5) through a structural illumination tomographic reconstruction algorithm aiming at a structural illumination reconstruction image with the same depth, namely the same reconstruction distance, and finally obtaining an optical tomographic reconstruction image of a sample at the depth;
(8) Carrying out frequency domain filtering and fusion on the super-resolution reconstructed image and the optical tomographic reconstructed image of the sample at a certain depth obtained in the steps (6) and (7) to obtain the super-resolution optical tomographic reconstructed image at the depth;
(9) Repeating steps (6) - (8) for all possible reproduction distances to obtain super-resolution optical tomographic images at different depths within the sample.
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| CN111736334B (en) * | 2020-06-22 | 2022-03-29 | 武汉市艾玻睿光电科技有限公司 | Device for realizing complex amplitude light field regulation and control based on spatial light modulator |
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