CN112651884B - Method and device for acquiring chromatography super-resolution image and electronic equipment - Google Patents
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Abstract
The invention discloses a method, a device and electronic equipment for acquiring a chromatography super-resolution image, wherein the method comprises the following steps: acquiring a plurality of multi-focus scanning original wide field images; performing power operation on the brightness of each multi-focus original wide field image for k times respectively to obtain a plurality of multi-focus images after power operation, wherein k is equal to 2 or 3; carrying out variance calculation on the multi-focus images subjected to power calculation to obtain a modulation degree image; setting a point spread function, and performing deconvolution processing on the modulation degree image according to the point spread function to obtain a deconvolved modulation degree image; and performing k-th power root operation on the deconvolved modulation degree image to obtain a final chromatography super-resolution image. Not only the capability of inhibiting background defocused signals is maintained, but also the spatial resolution is further improved.
Description
Technical Field
The invention relates to the technical field of computational imaging, in particular to a method and a device for acquiring a chromatography super-resolution image and electronic equipment.
Background
When thick tissue samples or cell fluorescence imaging is carried out, imaging results are often influenced by defocused signals, so that the image contrast is greatly reduced, and even structural information of the samples can not be identified. In the field of imaging research such as biology and medicine, a device or a method capable of effectively inhibiting background defocusing signals and obtaining a structure image with a clear focal plane is very popular, and the capability is also called tomography capability. Confocal microscopy enables very sharp focal plane images to be obtained by using a single point scanning mode and a pinhole. However, the single-point scanning mode has low imaging efficiency and is not suitable for tomography of fast-moving dynamic processes.
In contrast, the multi-focus scanning imaging technology proposed in recent years can greatly increase the imaging speed, which is helpful for realizing the fast tomography. The conventional multifocal scanning technology mainly depends on a repositioning algorithm to realize resolution improvement, and a digital pinhole is used for inhibiting background defocusing signals. The methods not only need to accurately calibrate the central position of each projection laser focus in advance, but also the digital pinhole cannot eliminate deep defocusing signals, and the tomography capability of the technology is limited. The variance tomography method proposed in the present year can more effectively inhibit defocused signals (particularly deep defocused signals) and obtain tomographic images with higher contrast. In addition, the method does not need to calibrate the central position of each projection laser focus, and is very convenient to use. The variance tomography method has the defects that the resolution improvement effect is limited, and tomography super-resolution imaging cannot be realized.
Disclosure of Invention
The invention provides a method, a device and an electronic device for acquiring a tomography super-resolution image, which are used for solving one or more technical problems in the prior art and at least providing a beneficial selection or creation condition.
In a first aspect, an embodiment of the present invention provides a method for acquiring a tomographic super-resolution image, including:
acquiring a plurality of multi-focus scanning original wide field images;
performing power operation on the brightness of each multi-focus original wide field image for k times respectively to obtain a plurality of multi-focus images after power operation, wherein k is equal to 2 or 3;
carrying out variance calculation on the multi-focus images subjected to power calculation to obtain a modulation degree image;
setting a point spread function, and carrying out deconvolution processing on the modulation degree image according to the point spread function to obtain a deconvolved modulation degree image;
and performing k-th power root operation on the deconvolved modulation degree image to obtain a final chromatography super-resolution image.
Further, the obtaining of the multiple multifocal images after performing the power operation k times on the luminance of each multifocal original wide field image specifically includes:
the ith multifocal original wide field image in the multiple multifocal original wide field images is I i ，I i The brightness at position (x, y) is I i (x, y), the k power operations are as follows:
I′ i (x，y)＝I i (x，y) k ；
wherein, I' i (x, y) is a multifocal image I 'after the ith power calculation' i The luminance value at position (x, y), x and y representing the coordinate position of the image row and column, k being an index of the power.
Further, the calculating the modulation degree image from the plurality of multi-focus images includes:
acquiring a multifocal image I 'for each of the respective occupant calculations' i (x, y) the luminance value at position (x, y) constitutes a vector M xy Wherein M is xy ∈R 1×N ，R 1×N Representing a set of real numbers of dimension 1*N, M xy Is a real number with dimension 1*N, N is the number of multifocal images, I' i I =1, …, N for the multifocal image after the ith power operation;
according to M xy Obtaining a luminance value V (x, y) at a position (x, y) of the modulation degree image V, where V (x, y) is:
wherein
M xy (i) Is a multifocal image I 'after the ith power calculation' i Luminance value I 'at position (x, y)' i (x，y)。
Further, the deconvolving the modulation degree image according to the point spread function, and obtaining the deconvolved modulation degree image includes:
respectively carrying out Fourier transformation on the modulation degree image and the point spread function to obtain a modulation degree image and an optical transfer function after the Fourier transformation;
and carrying out frequency domain deconvolution on the Fourier transformed modulation degree image according to the optical transfer function to obtain a deconvolved image:
and carrying out inverse Fourier transform on the deconvolved image to obtain a deconvolved modulation degree image.
Further, frequency domain deconvolution is performed on the fourier-transformed modulation degree image according to the optical transfer function, and the deconvolved image is obtained specifically as follows:
wherein, delta is a preset coefficient, V f OTF is an optical transfer function, V' f Is the deconvolved image.
Further, the k-th power root operation is performed on the deconvolved modulation degree image to obtain a final tomographic super-resolution image specifically as follows:
the modulation degree image after deconvolution is V ', and the luminance of V' at the position (x, y) is V '(x, y), and the k-th power root operation is performed on V' (x, y) as follows:
where V 'is the final tomographic super-resolution image, V' (x, y) is the intensity value of the final tomographic super-resolution image at position (x, y), and x and y represent the coordinate positions of the image rows and columns.
In a second aspect, an embodiment of the present invention further provides an apparatus for acquiring a tomographic super-resolution image, including:
the acquisition module is used for acquiring a plurality of multi-focus scanning original wide field images;
the power operation module is used for respectively carrying out power operation on the brightness of each multi-focus original wide field image for k times to obtain a plurality of multi-focus images after power operation, wherein k is equal to 2 or 3;
the calculation module is used for carrying out variance calculation on the multi-focus images subjected to the power calculation to obtain modulation degree images;
the deconvolution module is used for setting a point spread function and deconvoluting the modulation degree image according to the point spread function to obtain a deconvoluted modulation degree image;
and the square root operation module is used for performing k-th square root operation on the deconvolved modulation degree image to obtain a final chromatographic super-resolution image.
In a third aspect, an embodiment of the present invention further provides an apparatus for acquiring a tomographic super-resolution image, including:
a processor;
a memory for storing a computer readable program;
the computer readable program, when executed by the processor, causes the processor to implement the method of the first aspect.
In a fourth aspect, an embodiment of the present invention further provides an electronic device, including:
a processor;
a memory for storing a computer readable program;
the computer readable program, when executed by the processor, causes the processor to implement the method of the first aspect.
The embodiment of the invention at least has the following beneficial effects: obtaining a plurality of multifocal scanning original wide field images of a sample by scanning the sample, and respectively performing k times of power calculation on the brightness of each multifocal scanning original wide field image so as to improve
The resolution is multiplied, the variance calculation is carried out on the multifocal image after the power calculation to obtain a modulation degree image, the effect of restraining defocusing signals is achieved, and in addition, the defocusing signals are not only in the prior art
Multiple spaceOn the basis of improving the resolution, the spatial resolution improving effect close to 2 times is further realized, the point spread function is set, the deconvolution processing is carried out on the modulation degree image, the central position of each projection laser focus is not required to be calibrated (the position can change along with the slippage deformation of the system structure, and the calibration is frequently carried out in the existing method), the resolution is further improved, the k-th power operation is carried out on the modulation degree image after deconvolution, the final chromatography super-resolution image is obtained, the capability of inhibiting background defocusing signals is maintained, and the spatial resolution is further improved.
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The accompanying drawings are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the example serve to explain the principles of the invention and not to limit the invention.
FIG. 1 is a flowchart of a method for acquiring a tomographic super-resolution image according to an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of an apparatus for acquiring a tomographic super-resolution image according to an embodiment of the present invention;
FIG. 3 is a schematic structural diagram of an apparatus for acquiring a tomographic super-resolution image according to another embodiment of the present invention;
fig. 4 is a schematic structural diagram of an electronic device according to an embodiment of the present invention;
FIG. 5 (a) is a wide-field image with an out-of-focus background signal obtained by a conventional microscope provided by an embodiment of the present invention;
FIG. 5 (b) is an image processed using a prior art variance tomography method;
fig. 5 (c) is an image processed by the method provided in the embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
It should be noted that although functional block divisions are provided in the system drawings and logical orders are shown in the flowcharts, in some cases, the steps shown and described may be performed in different orders than the block divisions in the systems or in the flowcharts. The terms first, second and the like in the description and in the claims, as well as in the drawings described above, are used for distinguishing between similar elements and not necessarily for describing a particular sequential or chronological order.
FIG. 1 is a method for acquiring a tomographic super-resolution image according to an embodiment of the present invention, including the following steps:
s101, acquiring a plurality of multifocal scanning original wide field images;
specifically, a multipoint laser scanning device is used for scanning a sample, and a plurality of sparsely distributed multifocal templates are used for scanning different positions of the sample respectively during scanning; the multi-focus template refers to a template distributed with a plurality of points and is used for inputting the template into projection equipment, so that uniform illumination projected by laser becomes a multi-focus light source, and a sample is scanned;
and simultaneously, synchronously acquiring the multifocal scanning original wide field image corresponding to each multifocal template by using a camera to obtain a plurality of multifocal scanning original wide field images, wherein the camera is a CCD (charge coupled device) camera or a CMOS (complementary metal oxide semiconductor) camera.
S102, performing power operation on the brightness of each multi-focus original wide field image for k times respectively to obtain a plurality of multi-focus images after power operation, wherein k is equal to 2 or 3;
specifically, the ith multifocal original wide field image of the multifocal original wide field images is I i ，I i The brightness at position (x, y) is I i (x, y), the k power operations are as follows:
I′ i (x,y)＝I i (x,y) k ；
where x and y represent the image row and column coordinate positions and k is an index of the power.
It should be noted that directly squaring a wide-field image does not improve the spatial resolution of the image. The principle of the invention for improving the spatial resolution lies in that the fluorescence of the sampleThe wide field imaging result can be regarded as an imaging result set of a large number of fluorescence molecules. According to the diffraction limit theory, when each extremely minute fluorescence molecule is imaged, the imaging result is a diffraction spot (corresponding to a point spread function). Therefore, the fluorescence wide-field imaging result of the sample can be regarded as a collection of a plurality of diffraction spots. When these diffraction spots are gathered together to form a wide field image, the image resolution cannot be changed by performing a power operation. However, when the diffraction spots are dispersed in different images and there is a certain distance between the diffraction spots in each image, we can change the distribution of the diffraction spots by performing power calculation on each of the images, and reduce the diffraction spots. We can use a two-dimensional gaussian function model to describe the point spread function and the diffraction spots, i.e. the distribution of the single diffraction spot can be expressed as:
when we perform a k-th power operation on a single diffraction spot, the distribution of the diffraction spot becomes:
wherein the content of the first and second substances,
σ is the standard deviation parameter of the Gaussian function, and x0 and y0 represent the diffraction spot row and column coordinate positions. It can be seen that the standard deviation of a single diffraction spot is reduced after k power operations
And (4) doubling. This is equivalent to making the point spread function of the system smaller, and when these diffraction spots of varying distribution are re-superimposed, an improvement in spatial resolution is obtained
Doubled super-resolution image.
It should be noted that, in the conventional fluorescence scintillation image power operation method, the power operation is directly followed by the superposition processing, and although the image resolution can be improved, the tomography capability of inhibiting background defocused signals is not provided, and the method is often only applicable to ultra-high resolution imaging of thin samples.
Since a plurality of multi-focus images with sparse distribution are acquired during multi-focus scanning imaging, the light intensity distribution for each focus area can be expressed as:
wherein S is the sample structure distribution, E is the excitation light intensity distribution, and PSF is the system point spread function. Because the focused light beam is adopted to excite the sample during the multi-focus scanning imaging, the excitation light intensity distribution E is approximately the same as the point spread function PSF of the system, at the moment, the focused light beam sequentially selects a very small area on the sample to carry out fluorescence excitation and imaging, so that the original fluorescence molecules which should be subjected to luminescence imaging together are dispersed on different images to carry out luminescence imaging and form light spots. Therefore, the multi-focus image can be processed by using a power operation method, and the image resolution is improved.
In the invention, a plurality of multifocal images after power processing are not directly superposed, the multifocal images are not real fluorescent molecule sparse distribution images, and the spatial resolution improvement effect realized by only using power calculation and superposition is limited, so that the value of k is 2 or 3 (k =2 or 3). After the power operation is carried out, the calculation of the step S103 is carried out on the multifocal image after the power operation, so that the spatial resolution is further improved, and the good defocusing signal inhibition capability (namely tomography capability) can be realized.
S103, calculating the multiple multifocal images subjected to power calculation to obtain a modulation degree image;
specifically, step S103 includes:
s1031, obtaining the multi-focus image I 'after each power calculation' i (x, y) at position (x, y)The luminance value of (a) constitutes a vector M xy Wherein M is xy ∈R 1×N ，R 1×N Representing a set of real numbers, M, of dimension 1*N xy Is a real number with dimension 1*N, N is the number of multifocal images, I' i I =1, …, N for the multifocal image after the ith power operation;
s1032, according to M xy Obtaining a luminance value V (x, y) at a position (x, y) of the modulation degree image V, where V (x, y) is:
wherein
M xy (i) Is a multifocal image I 'after the ith power calculation' i Luminance value I 'at position (x, y)' i (x，y)。
After the power calculation, the calculation of step S103 is performed at this time, which not only has the effect of suppressing the defocus signal, but also has an effect of suppressing the defocus signal
On the basis of improving the spatial resolution, the spatial resolution which is close to 2 times is further improved, and the chromatography super-resolution imaging effect is preliminarily realized.
S104, setting a point spread function, and performing deconvolution processing on the modulation degree image according to the point spread function to obtain a deconvolved modulation degree image;
the point spread function is described by using a two-dimensional Gaussian distribution model, and the size of the point spread function is determined by a standard deviation parameter sigma of the Gaussian function. The resolution is further improved by deconvoluting the modulated image with a point spread function.
Further, in step S104, deconvolving the modulation degree image according to the point spread function, and obtaining a deconvolved modulation degree image includes:
s1041, respectively carrying out Fourier transform on the modulation degree image and the point spread function to obtain a modulation degree image and an optical transfer function after Fourier transform;
s1042, performing frequency domain deconvolution on the Fourier transformed modulation degree image according to the optical transfer function to obtain a deconvolved image;
step S1042 specifically is:
wherein, delta is a preset coefficient and ranges from 0.001 to 1,V f OTF is an optical transfer function, V' f Is the deconvolved image.
And S1043, performing inverse Fourier transform on the deconvolved image to obtain a deconvolved modulation degree image.
Step S1043 specifically includes:
to V' f And performing inverse Fourier transform to obtain a modulation degree image V' after deconvolution.
And S105, performing k-th power root operation on the deconvolved modulation degree image to obtain a final chromatography super-resolution image.
Specifically, the deconvolved modulation degree image is V ', the luminance of V' at the position (x, y) is V '(x, y), and the k-th power root operation is performed on V' (x, y) as follows:
where V 'is the final tomographic super-resolution image, V' (x, y) is the intensity value of the final tomographic super-resolution image at position (x, y), and x and y represent the coordinate positions of the image rows and columns.
During fluorescence imaging, the concentration of the fluorescent dye at different positions of the sample is not uniform, so that the light intensity of the sample is inconsistent, the uneven light intensity of the sample is amplified during the power operation of the step S102, and the uneven light intensity of the sample can be prevented from being amplified by performing the square root operation in the step S105, and the loss of the spatial resolution can be avoided.
FIG. 2 is a device for acquiring a tomographic super-resolution image according to an embodiment of the present invention, including:
the acquisition module is used for acquiring a plurality of multi-focus scanning original wide field images;
the power operation module is used for respectively carrying out power operation on the brightness of each multi-focus original wide field image for k times to obtain a plurality of multi-focus images after power operation, wherein k is equal to 2 or 3;
the calculation module is used for carrying out variance calculation on the multi-focus images after the power calculation to obtain modulation degree images;
the deconvolution module is used for setting a point spread function and carrying out deconvolution processing on the modulation degree image according to the point spread function to obtain a deconvolved modulation degree image;
and the square root operation module is used for performing k-th square root operation on the deconvolved modulation degree image to obtain a final chromatographic super-resolution image.
The processing process of the obtaining module, the power operation module, the calculating module, the deconvolution module and the square root operation module is similar to that of the method in fig. 1, and is not described herein again.
FIG. 3 is a schematic diagram of an apparatus for acquiring tomographic super-resolution images according to another embodiment of the present invention, including:
a processor;
a memory for storing a computer readable program;
the computer readable program, when executed by a processor, causes the processor to implement the method as described in fig. 1.
The processor and memory may be connected by a bus or other means, such as by a bus in FIG. 3.
The memory, as a non-transitory computer-readable storage medium, may be used to store non-transitory software programs as well as non-transitory computer-executable programs. Further, the memory may include high speed random access memory, and may also include non-transitory memory, such as at least one disk storage device, flash memory device, or other non-transitory solid state storage device.
The image processing apparatus in fig. 2 and 3 may be a terminal, which includes but is not limited to a mobile terminal such as a mobile phone and a fixed terminal such as a computer, and may also be an application installed on these terminals.
In an embodiment, the present invention further provides an electronic device, as shown in fig. 4, including:
a processor;
a memory for storing a computer readable program;
the computer readable program, when executed by a processor, causes the processor to implement the method as described in fig. 1.
In one embodiment, the present application also provides a computer-readable storage medium storing computer-executable instructions that are executable by one or more processors and when executed implement the steps of the above embodiments.
Fig. 5 (a) is a wide-field image with a defocused background signal obtained by a normal microscope, fig. 5 (b) is an image processed by a conventional variance tomography method, and fig. 5 (c) is an image processed by the method of the present invention.
Compared with wide-field images, the conventional variance tomography method can effectively inhibit background defocusing signals and improve the image contrast, but the resolution improvement effect is limited, and tomography super-resolution imaging cannot be realized. The invention not only keeps the capability of inhibiting background defocusing signals, but also can further improve the spatial resolution.
One of ordinary skill in the art will appreciate that all or some of the steps, systems, and methods disclosed above may be implemented as software, firmware, hardware, and suitable combinations thereof. Some or all of the physical components may be implemented as software executed by a processor, such as a central processing unit, digital signal processor, or microprocessor, or as hardware, or as an integrated circuit, such as an application specific integrated circuit. Such software may be distributed on computer readable media, which may include computer storage media (or non-transitory media) and communication media (or transitory media). The term computer storage media includes volatile and nonvolatile, removable and non-removable media implemented in any method or technology for storage of information such as computer readable instructions, data structures, program modules or other data, as is well known to those of ordinary skill in the art. Computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or other memory technology, CD-ROM, digital Versatile Disks (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used to store the desired information and which can accessed by a computer. In addition, communication media typically embodies computer readable instructions, data structures, program modules or other data in a modulated data signal such as a carrier wave or other transport mechanism and includes any information delivery media as known to those skilled in the art.
While the preferred embodiments of the present invention have been described, the present invention is not limited to the above embodiments, and those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and such equivalent modifications or substitutions are to be included within the scope of the present invention defined by the appended claims.
Claims (8)
1. A method for acquiring a tomographic super-resolution image, comprising:
acquiring a plurality of multi-focus scanning original wide field images;
performing power operation on the brightness of each multifocal original wide field image for k times respectively to obtain a plurality of multifocal images after power operation, wherein k is equal to 2 or 3;
carrying out variance calculation on the multi-focus images subjected to power calculation to obtain a modulation degree image;
setting a point spread function, and performing deconvolution processing on the modulation degree image according to the point spread function to obtain a deconvolved modulation degree image;
and performing k-th power root operation on the deconvolved modulation degree image to obtain a final chromatography super-resolution image.
2. The method for acquiring the tomographic super-resolution image according to claim 1, wherein the obtaining of the plurality of multifocal images by performing the k-times power operation on the luminance of each multifocal raw wide field image is specifically:
the ith multifocal original wide field image in the multiple multifocal original wide field images is I i ，I i The brightness at position (x, y) is I i (x, y), the k power operations are as follows:
I′ i (x,y)＝I i (x,y) k ；
wherein, I' i (x, y) is the multifocal image I 'after the ith power calculation' i The luminance value at position (x, y), x and y representing the coordinate position of the image row and column, k being an index of the power.
3. The method for acquiring tomographic super-resolution images according to claim 2, wherein the calculating a modulation degree image for the plurality of power-operated multifocal images comprises:
acquiring a multifocal image I 'for each of the respective occupant calculations' i (x, y) the luminance value at position (x, y) constitutes a vector M xy Wherein M is xy ∈R x×N ，R 1×N Representing a set of real numbers of dimension 1*N, M xy Is a real number with dimension 1*N, N is the number of multifocal images, I' i I =1, …, N for the multifocal image after the ith power operation;
according to M xy Obtaining a luminance value V (x, y) of the modulation degree image V at a position (x, y), V (x, y) being:
wherein
M xy (i) For the multifocal image I after the ith power operation i ' luminance value I at position (x, y) i ′(x，y)。
4. The method for acquiring a tomographic super-resolution image according to claim 1, wherein the deconvoluting the modulation level image according to the point spread function to obtain a deconvolved modulation level image comprises:
respectively carrying out Fourier transformation on the modulation degree image and the point spread function to obtain a modulation degree image and an optical transfer function after the Fourier transformation;
carrying out frequency domain deconvolution on the modulation degree image after Fourier transformation according to the optical transfer function to obtain a deconvolved image;
and carrying out inverse Fourier transform on the deconvolved image to obtain a deconvolved modulation degree image.
5. The method for acquiring a tomographic super-resolution image according to claim 4, wherein the frequency domain deconvolution is performed on the Fourier transformed modulation degree image according to the optical transfer function, and the obtaining of the deconvolved image is specifically:
wherein, delta is a preset coefficient, V f OTF is an optical transfer function, V' f Is the deconvolved image.
6. The method for acquiring a tomographic super-resolution image according to claim 5, wherein the k-th power operation is performed on the deconvolved modulation degree image to obtain a final tomographic super-resolution image, specifically:
the deconvolved modulation degree image is V ', and the luminance of V' at the position (x, y) is V '(x, y), and the k-th power root operation is performed on V' (x, y) as follows:
where V 'is the final tomographic super-resolution image, V' (x, y) is the intensity value of the final tomographic super-resolution image at position (x, y), and x and y represent the coordinate positions of the image rows and columns.
7. An apparatus for acquiring a tomographic super-resolution image, comprising:
the acquisition module is used for acquiring a plurality of multi-focus scanning original wide field images;
the power operation module is used for respectively carrying out power operation on the brightness of each multi-focus original wide field image for k times to obtain a plurality of multi-focus images after power operation, wherein k is equal to 2 or 3;
the calculation module is used for carrying out variance calculation on the multi-focus images subjected to the power calculation to obtain modulation degree images;
the deconvolution module is used for setting a point spread function and deconvoluting the modulation degree image according to the point spread function to obtain a deconvolved modulation degree image;
and the square root operation module is used for performing k-th power root operation on the deconvolved modulation degree image to obtain a final chromatographic super-resolution image.
8. An apparatus for acquiring a tomographic super-resolution image, comprising:
a processor;
a memory for storing a computer readable program;
the computer readable program, when executed by the processor, causes the processor to implement the method of any of claims 1-6.
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