CN115202022A - Scanning light field imaging system and method with isotropic resolution - Google Patents
Scanning light field imaging system and method with isotropic resolution Download PDFInfo
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- G02B21/00—Microscopes
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- G02B21/002—Scanning microscopes
- G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
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Abstract
The invention discloses a scanning light field imaging system and a scanning light field imaging method with isotropic resolution, wherein the scanning light field imaging system comprises a sample inclined module and a scanning light field acquisition module, the sample inclined module comprises an object stage and a plane mirror, an inclined surface is arranged on the object stage, a plane mirror is arranged on the inclined surface, the plane mirror surface of the plane mirror is parallel to the inclined surface, and a sample to be sampled is placed on the plane mirror; the scanning light field acquisition module comprises a microscope, a relay lens pair with a phase space scanning function, a micro lens array and an imaging sensor, wherein an objective lens of the microscope is positioned above a sample to be sampled. The system realizes the improvement of the axial resolution of the reconstructed body by adding the inclined mirror surface and the reconstruction process specially designed for the inclined mirror surface, and achieves the isotropic standard.
Description
Technical Field
The invention relates to the technical field of microscopic science, in particular to a scanning light field imaging system and a scanning light field imaging method with isotropic resolution.
Background
In recent years, microscopic science has been developed in a great deal, and no great effort has been made to improve the resolution of microscopes, from the first simple microscopes to the present electron microscopes. In order to observe biological samples more clearly, the resolution in four dimensions (lateral resolution (X direction and Y direction), axial resolution (Z direction) and temporal resolution (t)) which are most concerned by scientists are continuously increasing in the development of technology. However, limited by the influence of the existing imaging device and factors such as optically unavoidable phase difference and diffraction, it is difficult for a single imaging method to observe a biological sample by means of other computational imaging means in consideration of the resolution of the sample in the above four dimensions.
The light field imaging is a very popular topic in recent years, and by collecting angle information of light rays in a space, the light field imaging can completely recover three-dimensional information of a system. This is immeasurable help for scene refocusing, scene reconstruction and depth estimation. The light field is originally used in the field of reconstruction and refocusing of a macroscopic scene, however, through proper optical system design, the light field can also be easily used in the field of microscopy, and through acquisition of the light field system, the axial resolution of a sample can be more remarkably improved, and meanwhile, a three-dimensional model of a biological sample is constructed. In order to further improve the resolution of a sample, a scanning light field imaging system is provided, which can effectively improve the transverse resolution while ensuring the axial resolution of a light field by performing sub-pixel scanning on the sample.
However, the scanning light field imaging system is limited by the single direction acquisition NA and cannot provide sufficient axial resolution, and generally, the axial resolution of the sample reconstructed based on the original image acquired by the scanning light field is often much lower than the transverse resolution. In order to improve the axial resolution of the scanning light field system and realize isotropic resolution, it is necessary to sufficiently collect light of various angles emitted by a sample into a three-dimensional space.
Disclosure of Invention
It is an object of the present invention to provide a scanning light field imaging system and method with isotropic resolution to overcome the deficiencies of the prior art.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention discloses a scanning light field imaging system with isotropic resolution, which comprises a sample inclined module and a scanning light field acquisition module, wherein the sample inclined module comprises an object stage and a plane mirror, an inclined surface is arranged on the object stage, the plane mirror is arranged on the inclined surface, the mirror surface of the plane mirror is parallel to the inclined surface, and a sample to be sampled is placed on the plane mirror; the scanning light field acquisition module comprises a microscope, a relay lens pair with a phase space scanning function, a micro lens array and an imaging sensor, wherein the objective lens of the microscope is positioned above a sample to be sampled, and the image of the sample to be sampled is sequentially composed of the microscope, the relay lens pair with the phase space scanning function, the micro lens array and the imaging sensor.
Preferably, an installation groove is formed in the inclined plane, and the plane mirror is installed in the installation groove.
Preferably, the inclination angle of the inclined surface is not more than 45 degrees.
Preferably, the sum of the observation angle a3 corresponding to the objective lens NA of the microscope and the objective lens chamfer a1 of the microscope is 90 degrees or less, and the inclination angle a2 of the inclined surface is equal to or less than the objective lens chamfer a1 of the microscope.
Preferably, the object stage is of a front-back closed structure, a culture solution is added into the object stage, and the sample to be sampled is located in the culture solution.
The invention also discloses a scanning light field imaging method with isotropic resolution, which specifically comprises the following steps:
s1, acquiring a sample light field original image, and performing standard scanning light field reconstruction to obtain a primary reconstructed body;
s2, carrying out mirror surface estimation on the preliminary reconstructed body, and determining the only optimal mirror surface position;
s3, obtaining a mirror-based symmetric body of the primary reconstructed body after determining the only optimal mirror position;
s4, performing combined secondary reconstruction on the sample by using the symmetric body of the S3, the primary reconstruction body of the S1 and the original image of the sample light field; and obtaining a final reconstruction result.
Preferably, the S1 specifically includes the following substeps:
s11, acquiring a sample light field original image under phase space scanning through a scanning light field imaging system with isotropic resolution;
and S12, performing standard scanning light field reconstruction through a light field deconvolution algorithm to obtain a primary three-dimensional reconstruction result.
Preferably, the S2 specifically includes the following substeps:
s21, carrying out parametric modeling on the mirror surface position to obtain a parametric model of the mirror surface position;
s22, obtaining the parametric modeling of the mirror symmetric body of the preliminary three-dimensional reconstruction result based on the parametric model of the mirror position and the preliminary three-dimensional reconstruction result to obtain the parametric model of the mirror symmetric body;
and S23, taking the coincidence degree of the parameterized model of the mirror symmetry body and the preliminary three-dimensional reconstruction result as an optimization function, and obtaining parameters corresponding to the optimal mirror position through an optimization method.
Preferably, the step S4 specifically includes the following substeps:
s41, constructing an initialized three-dimensional reconstruction body combined with secondary reconstruction based on the weighted average value of the symmetric body of S3 and the primary reconstruction body of S1;
and S42, combining the initialized three-dimensional reconstruction body of the secondary reconstruction, the original image of the sample light field and the fixed constraint of the optimal mirror surface position, and carrying out scanning light field reconstruction containing the fixed constraint to obtain a final three-dimensional reconstruction result with isotropic resolution.
The invention has the beneficial effects that:
the invention aims to realize isotropic reconstruction based on scanning light field imaging, and improve the axial resolution of a reconstruction result to reach a resolution level close to or the same as the transverse resolution level. Before the method, a large difference exists between the transverse resolution and the axial resolution based on the acquisition and reconstruction result of the scanning light field, so that the effect of a reconstructed sample on three-dimensional display is greatly reduced, and the difficulty of sample analysis is improved. The system realizes the improvement of the axial resolution of the reconstructed body by adding the inclined mirror surface and the reconstruction process specially designed for the inclined mirror surface, and achieves the isotropic standard.
The acquisition process of the system can be realized after the scanning light field system is simply modified, and the reconstruction process of the system can be realized on a common PC machine with a display card.
The features and advantages of the present invention will be described in detail by embodiments in conjunction with the accompanying drawings.
Drawings
FIG. 1 is a schematic diagram of a sample tilt module according to the present invention;
FIG. 2 is a schematic diagram of a scanning lightfield imaging system of isotropic resolution according to the present invention;
FIG. 3 is a schematic view of the objective lens and the inclined surface of the microscope of the present invention;
FIG. 4 is a schematic flow chart of a scanning light field imaging method of isotropic resolution in accordance with the present invention;
FIG. 5 shows the general light field reconstruction result and the mirror enhanced isotropic light field reconstruction result of a group of Chlamydomonas;
FIG. 6 shows the general light field reconstruction and the specular enhanced isotropic light field reconstruction of a cell;
in the figure: 1-an objective table, 2-an inclined plane, 3-a plane mirror, 4-a microscope, 5-a relay lens pair with a phase space scanning function, 6-a micro lens array and 7-an imaging sensor.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood, however, that the detailed description herein of specific embodiments is intended to illustrate the invention and not to limit the scope of the invention. Moreover, in the following description, descriptions of well-known structures and techniques are omitted so as to not unnecessarily obscure the concepts of the present invention.
Referring to fig. 1, the scanning light field imaging system with isotropic resolution of the present invention comprises a sample tilting module and a scanning light field acquisition module, wherein the sample tilting module comprises an object stage 1 and a plane mirror 3, the object stage is provided with a tilting surface 2, the tilting surface 2 is provided with a plane mirror 3, the plane mirror 3 is parallel to the tilting surface 2, and a sample to be sampled is placed on the plane mirror 3; the scanning light field acquisition module comprises a microscope 4, a relay lens pair (comprising a 4f lens group and a high-speed two-dimensional galvanometer) 5 with a phase space scanning function, a micro lens array 6 and an imaging sensor 7, wherein an objective lens of the microscope 4 is positioned above a sample to be sampled, and an image of the sample to be sampled sequentially comprises the microscope 4, the relay lens pair 5 with the phase space scanning function, the micro lens array 6 and the imaging sensor 7.
In a possible embodiment, a mounting groove is arranged on the inclined surface, and the plane mirror is mounted in the mounting groove.
In a possible embodiment, the inclination angle of the inclined surface is less than or equal to 45 degrees.
In a possible embodiment, the sum of the objective chamfer a1 of the microscope 4 and the corresponding observation angle a3 of the objective NA of the microscope 4 is less than or equal to 90 degrees, and the inclination angle a2 of the inclined surface 2 is less than or equal to the objective chamfer a1 of the microscope 4.
In a possible embodiment, the stage is a front-back closed structure, the stage 1 is filled with a culture solution, and the sample to be sampled is located in the culture solution.
The invention discloses a scanning light field imaging method with isotropic resolution, which specifically comprises the following steps:
s1, obtaining a sample light field original image, and performing standard scanning light field reconstruction to obtain a primary reconstructed body;
s2, carrying out mirror surface estimation on the primary reconstructed body, and determining the only optimal mirror surface position;
s3, determining the only optimal mirror surface position to obtain a mirror surface-based symmetric body of the primary reconstructed body;
s4, performing combined secondary reconstruction on the sample by using the symmetric body of the S3, the primary reconstruction body of the S1 and the original image of the sample light field; and obtaining a final reconstruction result.
In a possible embodiment, the S1 specifically includes the following sub-steps:
s11, acquiring a sample light field original image under phase space scanning through a scanning light field imaging system with isotropic resolution;
and S12, performing standard scanning light field reconstruction through a light field deconvolution algorithm to obtain a primary three-dimensional reconstruction result.
In a possible embodiment, the S2 specifically includes the following sub-steps:
s21, carrying out parametric modeling on the mirror surface position to obtain a parametric model of the mirror surface position;
s22, obtaining the parametric modeling of the mirror symmetric body of the preliminary three-dimensional reconstruction result based on the parametric model of the mirror position and the preliminary three-dimensional reconstruction result to obtain the parametric model of the mirror symmetric body;
and S23, taking the coincidence degree of the parameterized model of the mirror symmetry body and the preliminary three-dimensional reconstruction result as an optimization function, and obtaining parameters corresponding to the optimal mirror position through an optimization method.
In a possible embodiment, the S4 specifically includes the following sub-steps:
s41, constructing an initialized three-dimensional reconstruction body combined with secondary reconstruction based on the weighted average value of the symmetric body of S3 and the primary reconstruction body of S1;
and S42, combining the initialized three-dimensional reconstruction body of the secondary reconstruction, the original image of the sample light field and the fixed constraint of the optimal mirror surface position, and performing scanning light field reconstruction containing the fixed constraint to obtain a final three-dimensional reconstruction result with isotropic resolution.
Example (b):
as shown in fig. 1, the structure of the sample tilting module specially designed for this system is schematically shown in the present invention: the replaceable small mirror can be placed in the small groove on the right inclined plane, the sample is placed on the mirror surface, or the sample is embedded and fixed on the surface of the mirror by agarose gel and the like and then placed in the small groove on the right inclined plane, and then the placement of the sample is completed. The structural part mainly comprises the following key points:
the choice of the inclination of the bevels, the inclination of the bevel for placing the mirror surface (right bevel) should be kept matched to the working distance and NA of the objective lens, the size of the objective lens: specifically, the objective lens chamfer angle a1, the included angle a2 of the inclined plane of the placed mirror surface, and the observation angle a3 (as marked in fig. 3) corresponding to the objective lens NA satisfy a1+ a2 is less than or equal to 90 degrees, and a2 is less than or equal to a1; when an objective lens with large NA and short working distance is selected, the inclination angle of the mirror surface should be reduced; in contrast, with a small NA, long working distance objective, the tilt angle can be increased. The maximum angle of the tilt angle is forty-five degrees, and when the working distance of the objective lens is sufficiently long, the tilt angle can be fixed at 45 degrees. The inclination angle of the left inclined plane is consistent with that of the right inclined plane.
Selection of two-side seals: samples (such as living cells and algae) requiring a culture solution can be observed by filling the culture solution and using a water lens objective lens by sealing both sides of the triangular groove.
Selecting a plane mirror: the plane mirror should be chosen to fit the size of the groove, and in addition, due to the strict angle requirements of the algorithm, the bottom surface of the small groove should be guaranteed to be flat by using a fine machining mode, and in addition, the small mirror should be chosen to be a more flat mirror surface to obtain a better reconstruction effect.
A scanning light field system for imaging. As shown in fig. 2, the system is composed of a stage 1, a standard microscope 4, a relay lens pair (composed of a 4f lens group and a high-speed two-dimensional galvanometer) 5 including a phase space scanning function, a micro lens array 6, an imaging sensor 7 and other modules. The objective table is placed below the objective lens, and after a sample is placed, imaging can be performed through a standard scanning light field imaging system.
As shown in fig. 4, a dedicated algorithm for reconstruction. Because the shot sample simultaneously contains the mixed result of the image on the mirror surface and the image under the mirror surface, the traditional light field deconvolution algorithm cannot be used for reconstruction, and a special reconstruction algorithm is further required to be used. The mirror reconstruction algorithm mainly comprises the following key steps:
first, a conventional light field deconvolution of the sample is required to solve for the preliminary reconstruction. Before the traditional light field deconvolution, modeling and simulating a system optical Point Spread Function (PSF) are needed; furthermore, if the system includes aberration, in order to get rid of the influence of system aberration, the aberration of the system is estimated by actually acquiring the fluorescent ball image, and the full-depth simulated PSF including aberration is calculated as the real system PSF for three-dimensional reconstruction. After obtaining the PSF, the raw data is rearranged according to the phase space image while selecting the region of interest. The selected region is then deconvoluted with the light field using the following algorithm:
wherein
Is a complete iteration k and a frequency iteration u per round j The reconstructed volume of the lower part of the column,
are weights for balancing different shot noise of different spatial frequency components, <' > denotes a point multiplication process, BP () denotes a back propagation process, FP () denotes a forward propagation process, M (x, u, etc.) j ) Is the frequency u after spatial rearrangement of the acquired data phase j A corresponding image. It should be noted that the reconstructed object at this time includes both the image above the mirror and the image below the mirror.
And secondly, carrying out mirror surface estimation on the primary reconstruction result obtained in the step. Before the mirror estimation, the mirror is first modeled:
however, since the mirror surface is never perpendicular to the x-y plane, a 4 Not equal to 0, obtaining:
the expression for a plane can be written as:
P(b 1 ,b 2 ,b 3 )→z＝b 1 +b 2 x+b 3 y (2.5)
wherein
Are parameters for describing the position of the mirror in three-dimensional space. In addition, because the reconstructed three-dimensional distribution needs to be mirror-symmetric for multiple times, a mirror-symmetric matrix is usually generated based on the mirror position when the algorithm is implemented, and the position of each voxel after symmetry is calculated in advance, so that the index is interpolated conveniently and quickly. Normal vector to a mirror surface
And two points of symmetry
The following inequalities are present:
where k is a non-zero value, n T Is the transpose of the normal vector n, based on which equation D can be solved:
then, the position of each discrete point in the whole volume after the mirror symmetry is further calculated, and the mapping matrix W is saved P In order to calculate the mirror estimates and the volume reconstruction next:
wherein V ∈ R 3×N Is a voxelization process of a three-dimensional vector of each point position in the reconstructed volume, N being the number of voxels. It should be noted that each time the position of the mirror surface changesThe corresponding mapping matrix needs to be recalculated. In performing mirror estimation, the position of the mirror is affected by three variables: the vertical distance Δ z between the center of the reconstruction volume and its point projected on the mirror along the z-axis, the angle α of the mirror to the horizontal, and the angle θ of rotation of the mirror about the z-axis. However, since the holder with the mirror is prefabricated, its angle is determined when the objective lens is selected. The angle α between the mirror plane and the plane is usually considered to be a fixed value and is measured in advance. In this case, the problem to be solved is reduced from the conventional three-variable optimization problem to a two-variable problem. Normal vector based on mirror surface and horizontal plane
And
comprises the following steps:
thereby obtaining
In practice, a negative sign is usually used:
thus, the plane equation is further rewritten as:
P(b 1 ,b 2 )→z＝b 1 +b 2 x+F(b 2 )y (2.12)
the corresponding can be obtained:
in the optimization process, in order to ensure the optimization effect, b 2 Is limited to a small range close to zero. Since the rotation angle theta is usually a very small value. Finally, the preliminary reconstructed volume V from the light field deconvolution and the mirror-based position P (b) are used 1 ,b 2 ) Is mapped to P To optimize the plane
The parameters of (2):
where |, represents the voxelized dot product process. In a specific implementation, the fminsearch function in MATLAB is used to search for the optimal point. In short, the only optimal mirror position is determined by optimizing the maximum similarity between the reconstructed body and the symmetrical reconstructed body.
And finally, obtaining a mirror-surface-based symmetric body of the reconstructed body through the mirror surface position obtained after optimization, and performing combined secondary reconstruction on the sample by using the symmetric body, the primary reconstructed body and the light field original image to obtain a final optimization result. Specifically, once the mirror position P is determined * (b 1 ,b 2 ) The final mapping matrix of the data can be calculated
And deconvoluted using two-step deconvolution in each iteration. In light field microscopy imaging, each phase space measurement is independently input into the reconstruction algorithm to optimize the entire volume, which can be described as:
furthermore, for each input angle, a symmetric map deconvolution process may be added. Since the measurement values are from the sum of two symmetric volumes, the symmetric volumes should also satisfy the light field imaging model. Based on this, a second deconvolution is performed as follows:
here first by symmetrical mapping
Mapping volumes
A similar deconvolution reconstruction process is then performed, and the volume is flipped back to the original view. Finally, to make the result more pleasing, the visual effect is often enhanced by also cutting away portions of the symmetric body (under the mirror) after the final result is achieved.
In this way, the entire acquisition and reconstruction process is completed. Fig. 5 shows the general light field reconstruction result and the mirror-enhanced isotropic light field reconstruction result of a group of chlamydomonas, and fig. 6 shows the general light field reconstruction result and the mirror-enhanced isotropic light field reconstruction result of a cell, and it can be seen that the result of the mirror-enhanced isotropic light field reconstruction is clearer in the z-axis direction, has higher resolution, and can achieve the isotropic effect.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A scanning light field imaging system of isotropic resolution, characterized by: the device comprises a sample inclined module and a scanning light field acquisition module, wherein the sample inclined module comprises an object stage and a plane mirror, an inclined surface is arranged on the object stage, the plane mirror is arranged on the inclined surface, the mirror surface of the plane mirror is parallel to the inclined surface, and a sample to be sampled is placed on the plane mirror; the scanning light field acquisition module comprises a microscope, a relay lens pair with a phase space scanning function, a micro lens array and an imaging sensor, wherein the objective lens of the microscope is positioned above a sample to be sampled, and the image of the sample to be sampled is sequentially composed of the microscope, the relay lens pair with the phase space scanning function, the micro lens array and the imaging sensor.
2. A scanning light-field imaging system of isotropic resolution as claimed in claim 1, wherein: an installation groove is formed in the inclined surface, and the plane mirror is installed in the installation groove.
3. A scanning light-field imaging system of isotropic resolution as claimed in claim 1, wherein: the inclination angle of the inclined plane is less than or equal to 45 degrees.
4. A scanning light-field imaging system of isotropic resolution as claimed in claim 3, wherein: the sum of the observation angle a3 corresponding to the objective lens NA of the microscope and the objective lens chamfer a1 of the microscope is less than or equal to 90 degrees, and the inclination angle a2 of the inclined surface is less than or equal to the objective lens chamfer a1 of the microscope.
5. A scanning light-field imaging system of isotropic resolution as claimed in claim 1, wherein: the objective table is a front-back closed structure, a culture solution is added into the objective table, and the sample to be sampled is located in the culture solution.
6. A scanning light field imaging method with isotropic resolution is characterized by comprising the following steps:
s1, obtaining a sample light field original image, and performing standard scanning light field reconstruction to obtain a primary reconstructed body;
s2, carrying out mirror surface estimation on the primary reconstructed body, and determining the only optimal mirror surface position;
s3, determining the only optimal mirror surface position to obtain a mirror surface-based symmetric body of the primary reconstructed body;
s4, performing combined secondary reconstruction on the sample by using the symmetric body of the S3, the primary reconstruction body of the S1 and the original image of the sample light field; and obtaining a final reconstruction result.
7. The method of claim 6, wherein: the step S1 specifically comprises the following substeps:
s11, acquiring a sample light field original image under phase space scanning through a scanning light field imaging system with isotropic resolution;
and S12, performing standard scanning light field reconstruction through a light field deconvolution algorithm to obtain a primary three-dimensional reconstruction result.
8. The method of claim 7, wherein: the step S2 specifically includes the following substeps:
s21, carrying out parametric modeling on the mirror surface position to obtain a parametric model of the mirror surface position;
s22, obtaining the parametric modeling of the mirror symmetric body of the preliminary three-dimensional reconstruction result based on the parametric model of the mirror position and the preliminary three-dimensional reconstruction result to obtain the parametric model of the mirror symmetric body;
and S23, taking the coincidence degree of the parameterized model of the mirror symmetry body and the preliminary three-dimensional reconstruction result as an optimization function, and obtaining parameters corresponding to the optimal mirror position through an optimization method.
9. A method of isotropic resolution scanned-light-field imaging as claimed in claim 6, wherein: the step S4 specifically includes the following substeps:
s41, constructing an initialized three-dimensional reconstruction body combined with secondary reconstruction based on the weighted average value of the symmetric body of S3 and the primary reconstruction body of S1;
and S42, combining the initialized three-dimensional reconstruction body of the secondary reconstruction, the original image of the sample light field and the fixed constraint of the optimal mirror surface position, and carrying out scanning light field reconstruction containing the fixed constraint to obtain a final three-dimensional reconstruction result with isotropic resolution.
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