CN111062889B - Light intensity correction method for Fourier stacked microscopic imaging technology - Google Patents
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Abstract
The light intensity correction method for the Fourier laminated microscopic imaging technology can realize the effect of correcting the brightness of an image, thereby achieving the purpose of correcting the inconsistent light intensity error. Comprising the following steps: (1) acquiring an original image to form an image dataset; (2) Setting an image intensity multiple change interval [ A, B ], setting an initial value of an intensity correction coefficient corresponding to an acquired original image as 1, converting the value of the intensity correction coefficient, and adjusting the intensity of the image; sequentially changing the initial image intensity correction coefficient value according to the values in the intervals [ A, B ] according to t, multiplying different intensity correction coefficients by each measured image, and calculating an evaluation function after each change; after a plurality of iterations, finding out the most suitable brightness multiple value; (3) Adjusting each low-resolution image according to the most appropriate brightness multiple value, and completing brightness correction of the image; (4) And carrying out high-resolution reconstruction on the corrected image to obtain a reconstructed image.
Description
Technical Field
The invention relates to the technical field of microscopic imaging, in particular to a light intensity correction method for a Fourier laminated microscopic imaging technology.
Background
The microscopic imaging technology is a technology for observing the morphological structure and the characteristics of a tiny object by utilizing an optical system or electronic equipment, and the Fourier laminated microscopic imaging technology (FPM) can break through the limitation of the numerical aperture of an objective lens of an imaging system in a calculation reconstruction mode, and simultaneously realize large-view-field and super-resolution imaging, and has wide application in the aspects of cytology, biology, medicine and the like.
As shown in the prior study, the FPM adopts illumination light with consistent illumination brightness to provide plane waves with different angles and respectively illuminate the sample, and under ideal conditions, the intensity of the illumination light with different angles reaching the sample is consistent, and the restored sub-frequency components keep the original frequency proportion relation of each part of the sample.
However, there are a variety of illumination non-uniformity types in current systems, and error sources include: the current flowing through the LED lamp is unstable, the resistances of different LED lamp beads are different, and the heat dissipation function of the LED lamp is reduced, the brightness is attenuated and the like along with the longer service time of the LED lamp. These all result in different intensities of incident light reaching the sample surface at different angles, resulting in poor quality reconstructed high resolution images.
The existing method for solving the problems is mainly to compensate from the algorithm, the purpose of error correction is generally achieved by reducing the amplitude difference between the measured image and the target complex amplitude image or the amplitude difference between the measured image and the target complex amplitude image, but when the acquired original image has a large degree of uneven illumination error or a nonlinear error exists in the sub-spectrum during reconstruction, many correction methods cannot achieve a good correction effect.
Disclosure of Invention
In order to overcome the defects of the prior art, the technical problem to be solved by the invention is to provide a light intensity correction method for a Fourier laminated microscopic imaging technology, which can realize the effect of correcting the brightness of an image and further achieve the purpose of correcting the inconsistent light intensity error.
The technical scheme of the invention is as follows: the light intensity correction method for the Fourier stacked microscopy imaging technology comprises the following steps:
(1) Collecting an original image to form an image data set;
(2) Setting an image intensity multiple change interval [ A, B ], changing the intensity correction coefficient value based on the initial value of the intensity correction coefficient corresponding to the collected original image to be 1, and adjusting the intensity of the image; the method for adjusting is that the initial image intensity correction coefficient value is changed in sequence according to the value in the interval [ A, B ] according to a certain step length t, each time the image is measured, different intensity correction coefficients are multiplied, and each time the value is changed, an evaluation function is calculated; finding the most suitable brightness multiple value through a process of finding the optimal evaluation function and the optimal brightness multiple through a plurality of iterations;
(3) Adjusting each low-resolution image according to the most appropriate brightness multiple value, and completing brightness correction of the image;
(4) And carrying out high-resolution reconstruction on the corrected image to obtain a reconstructed image.
The invention continuously adjusts the intensity value of each image by setting a series of intensity correction coefficients, achieves the aim of correcting the light intensity, and reconstructs the corrected image to weaken the influence of uneven illumination intensity on the reconstruction result, thereby realizing the effect of correcting the brightness of the image and further achieving the aim of correcting the inconsistent light intensity error.
Drawings
Fig. 1 is an acquired original image.
Fig. 2 is a reconstructed image without illumination non-uniformity error correction.
Fig. 3 is a reconstructed image with illumination non-uniformity error correction added.
Fig. 4 is a flow chart of a light intensity correction method for fourier stack microscopy imaging technique according to the invention.
Detailed Description
In the FPM system, the reconstruction quality is affected by the illumination intensity, and when the illumination intensity reaching the sample surface is inconsistent, an illumination non-uniformity error is generated, and the intensity of each original image needs to be corrected, so that the image intensity is closer to the real intensity value. The invention continuously adjusts the intensity value of each image by setting a series of intensity correction coefficients, achieves the aim of light intensity correction, and can weaken the influence of uneven illumination intensity on a reconstruction result by reconstructing the corrected image.
The conventional FPM reconstruction method directly performs GS reconstruction on uncorrected images multiple times. Firstly, constructing a high-resolution initial estimation image, carrying out Fourier transform on the initial estimation image to obtain an initial estimation spectrum, searching a sub-region spectrogram on the initial estimation spectrum, carrying out Fourier inverse transform on the sub-region spectrogram to obtain a target complex amplitude image, then directly replacing an amplitude part of the target complex amplitude image with an acquired original image, keeping a phase part unchanged, carrying out Fourier transform on the obtained new target complex amplitude image, and updating the spectrogram at a corresponding position on the initial estimation spectrogram. And then replacing the next sub-spectrum until the replacement of all the sub-spectrums is completed, and thus, one iteration is completed.
As shown in fig. 4, the light intensity correction method for fourier stack microscopy according to the present invention comprises the steps of:
(1) Collecting an original image to form an image data set;
(2) Setting an image intensity multiple change interval [ A, B ], changing the intensity correction coefficient value based on the initial value of the intensity correction coefficient corresponding to the collected original image to be 1, and adjusting the intensity of the image; the method for adjusting is that the initial image intensity correction coefficient value is changed in sequence according to the value in the interval [ A, B ] according to a certain step length t, each time the image is measured, different intensity correction coefficients are multiplied, and each time the value is changed, an evaluation function is calculated; finding the most suitable brightness multiple value through a process of finding the optimal evaluation function and the optimal brightness multiple through a plurality of iterations;
(3) Adjusting each low-resolution image according to the most appropriate brightness multiple value, and completing brightness correction of the image;
(4) And carrying out high-resolution reconstruction on the corrected image, for example, carrying out GS iterative reconstruction to obtain a reconstructed image.
The invention continuously adjusts the intensity value of each image by setting a series of intensity correction coefficients, achieves the aim of correcting the light intensity, and reconstructs the corrected image to weaken the influence of uneven illumination intensity on the reconstruction result, thereby realizing the effect of correcting the brightness of the image and further achieving the aim of correcting the inconsistent light intensity error.
Preferably, the evaluation function in the step (2) is the square of the difference between the target complex amplitude image and the measured image intensity value, and the calculation method is as follows: firstly, a traditional GS reconstruction method is utilized to reconstruct once to obtain a high-resolution image as an initial estimated value, and the high-resolution image is processedFourier transform is carried out to obtain an initial estimated frequency spectrum, a sub-region spectrogram is searched on the initial estimated frequency spectrum, and inverse Fourier transform is carried out to obtain a target complex amplitude image I 1 The intensity value is recorded as Er 1 Its corresponding measurement image is denoted as I 2 The intensity value is recorded as Er 2 The initial evaluation function value was denoted as er= (Er 1 -Er 2 ) 2 And recording Er as an optimal evaluation function Er best The method comprises the steps of carrying out a first treatment on the surface of the After the image is adjusted by the brightness correction coefficient, a new measurement image I is obtained 2new The intensity value is recorded as Er 2new Updating the target complex amplitude image by using the new measurement image to obtain a new target complex amplitude image I 1new The intensity value is recorded as Er 1new Calculate a new evaluation function value Er new ＝(Er 1new -Er 2new ) 2 When Er new ＜Er best At the time, er new Is marked as Er best The corresponding intensity correction coefficient is the optimal correction coefficient, when Er new ＞Er best At the time of Er best Is unchanged.
Preferably, in the step (2), the process of searching the optimal evaluation function and the optimal brightness multiple through multiple iterations is performed, so as to find the most suitable brightness multiple value of each image.
Preferably, in the step (4), the image is reconstructed, and the FPM algorithm is performed on the acquired multiple low-resolution images.
Preferably, in the step (4), the image reconstruction is performed by super-resolution reconstruction by a GS phase recovery algorithm in the phase recovery algorithm.
Taking the GS phase recovery algorithm as an example (but not limited to this way), the step (4) includes the following sub-steps:
(4.1) carrying out interpolation processing on an image shot by a central LED lamp on the LED array to serve as an initial airspace estimated value;
(4.2) carrying out Fourier transform on the interpolated image to obtain a frequency domain initial estimated value;
(4.3) selecting a sub-region from the obtained spectrogram to carry out inverse Fourier transform to obtain a target complex amplitude image, wherein the target complex amplitude image comprises amplitude information and phase information;
(4.4) keeping the phase information of the target complex amplitude image unchanged, and replacing the amplitude information of the target complex amplitude image by using an actual image shot by the LED lamp at the corresponding position on the LED array to obtain an updated target complex amplitude image;
(4.5) carrying out Fourier transform on the updated target complex amplitude image to obtain an updated spectrogram, and replacing a corresponding sub-spectrum region of the initial spectrogram with the updated spectrogram;
(4.6) repeating the steps (4.3) - (4.5) to finish all sub-spectrum updating;
and (4.7) repeating the steps (4.3) - (4.6) to enable the result to be converged, obtaining a high-resolution frequency spectrum image with enhanced image high-frequency information, and then carrying out inverse Fourier transform to obtain a high-resolution image in a space domain.
Preferably, the method further comprises the step (5) of checking that, after comparing the original low resolution image, the reconstructed image without the illumination non-uniformity error correction and the reconstructed image with the illumination non-uniformity error correction, the quality of the reconstructed image is poor due to the illumination non-uniformity error, after correction, the resolution is improved, and meanwhile, the image background is more uniform.
For a better description of the objects and advantages of the present invention, the following detailed description of the invention refers to the accompanying drawings and examples.
Example 1:
because the system has the errors of unstable current flowing through the LED lamp, different resistances of different LED lamp beads, reduced heat dissipation function of part of the LED lamp, reduced brightness, and the like along with the longer service time, the errors of inconsistent light intensity reaching the surface of the sample can be caused, and the errors can be effectively corrected by adopting the light intensity correction method which can be used for the Fourier laminated microscopic imaging technology. The light source used in this embodiment is an LED array. The LED array participating in illumination is 31×31, the distance between the LED lamps is 2.5mm, the distance between the LED array and a sample is 96mm, the wavelength of illumination light is 630nm, the numerical aperture of an objective lens is 0.09, the image acquisition device is a CCD camera, and the imaging pixel size is 2.45 μm. The LED lamps at different positions on the LED array collect a sub-aperture image, a certain brightness error exists between each sub-aperture image, and when the collected original image is directly used for reconstruction, the quality of the reconstructed image is reduced. An error correction method is required to correct this error.
The image enhancement method for the Fourier stacked microscopic imaging technology disclosed by the embodiment comprises the following specific steps:
step one: collecting image dataset, total 961 original low resolution images
Step two: performing primary GS iterative reconstruction on 961 original images to obtain an initial estimated image, and performing Fourier transform to obtain a high-resolution spectrogram which is used as the initial estimated spectrum for correcting the illumination non-uniformity errors.
Step three: sorting original images, correcting brightness from the first image, selecting corresponding sub-spectrum image from high-resolution spectrogram, performing inverse Fourier transform to obtain a target complex amplitude image I 1 The intensity value is recorded as Er 1 The first image is denoted as I 2 The intensity value is recorded as Er 2 The initial evaluation function value was denoted as er= (Er 1 -Er 2 ) 2 And recording Er as an optimal evaluation function Er best 。
Step four: setting brightness correction coefficient variation interval [0.4,1.2 ]]The step length is 0.01, the brightness correction coefficient C is changed from 0.4, each time the brightness correction coefficient C is increased by 0.01 until 1.2 is finished, the original low-resolution image is changed according to the brightness correction coefficient, and a new measurement image I is obtained after each change 2new The intensity value is recorded as Er 2new Updating the target complex amplitude image by using the new measurement image to obtain a new target complex amplitude image I 1new The intensity value is recorded as Er 1new Calculate a new evaluation function value Er new ＝(Er 1new -Er 2new ) 2 When Er new ＜Er best At the time, er new Is marked as Er best The corresponding intensity correction coefficient is the optimal correction coefficient, when Er new ＞Er best At the time of Er best Is unchanged.
Step five: and finding the most suitable brightness multiple value of each image through the process of finding the optimal evaluation function and the optimal brightness multiple through five iterations. And adjusting each low-resolution image according to the most appropriate brightness multiple value to finish brightness correction of the image.
Step six: image reconstruction, performing FPM algorithm reconstruction on a plurality of acquired low-resolution images, and performing super-resolution reconstruction by selecting a classical GS phase recovery algorithm, wherein the method comprises the following steps of:
[1] interpolation processing is carried out on the image shot by the central LED lamp on the LED array, and the image is used as an initial airspace estimated value;
[2] carrying out Fourier transformation on the interpolated image to obtain a frequency domain initial estimated value;
[3] selecting a sub-region from the obtained spectrogram to carry out inverse Fourier transform to obtain a target complex amplitude image, wherein the target complex amplitude image comprises amplitude information and phase information;
[4] keeping the phase information of the target complex amplitude image unchanged, and replacing the amplitude information of the target complex amplitude image by using an actual image shot by the LED lamps at the corresponding positions on the LED array to obtain an updated target complex amplitude image;
[5] performing Fourier transform on the updated target complex amplitude image to obtain an updated spectrogram, and replacing a corresponding sub-spectrum region of the initial spectrogram with the updated spectrogram;
[6] repeating the steps [3] to [5] to finish updating all the sub-spectrums;
[7] repeating the steps [3] - [6] to converge the result, obtaining a high-resolution frequency spectrum image with enhanced image high-frequency information, and then carrying out inverse Fourier transform to obtain a high-resolution image in the space domain.
Step seven: the result is checked, the original low resolution image is shown in fig. 1, the reconstructed image without illumination non-uniformity error correction is shown in fig. 2, and the reconstructed image with illumination non-uniformity error correction is shown in fig. 3; the contrast shows that the uneven illumination error can cause the quality of the reconstructed image to be poor, the resolution is improved after correction, and the image background is more uniform.
The present invention is not limited to the preferred embodiments, but can be modified in any way according to the technical principles of the present invention, and all such modifications, equivalent variations and modifications are included in the scope of the present invention.
Claims (5)
1. A light intensity correction method for a Fourier laminated microscopic imaging technology is characterized by comprising the following steps of: which comprises the following steps:
(1) Collecting an original image to form an image data set;
(2) Setting an image intensity multiple change interval [ A, B ], changing the intensity correction coefficient value based on the initial value of the intensity correction coefficient corresponding to the collected original image to be 1, and adjusting the intensity of the image; the method for adjusting is that the initial image intensity correction coefficient value is changed in sequence according to the value in the interval [ A, B ] and according to the step length t, each time the image is measured, the image is multiplied by different intensity correction coefficients, and after each change, an evaluation function is calculated once; finding out the optimal brightness multiple value through a process of finding out the optimal evaluation function and the optimal brightness multiple through a plurality of iterations;
(3) Adjusting each low-resolution image according to the optimal brightness multiple value, and completing brightness correction of the image;
(4) Carrying out high-resolution reconstruction on the corrected image to obtain a reconstructed image;
the evaluation function in the step (2) is the square of the difference value between the target complex amplitude image and the measured image intensity value, and the calculation method is as follows: firstly, performing primary reconstruction by using a traditional GS reconstruction method to obtain a high-resolution image as an initial estimated value, performing Fourier transform on the high-resolution image to obtain an initial estimated spectrum, searching a sub-region spectrogram on the initial estimated spectrum, and performing inverse Fourier transform on the sub-region spectrogram to obtain a target complex amplitude image I 1 The intensity value is recorded as Er 1 Its corresponding measurement image is denoted as I 2 The intensity value is recorded as Er 2 The initial evaluation function value was denoted as er= (Er 1 -Er 2 ) 2 And recording Er as an optimal evaluation function Er best The method comprises the steps of carrying out a first treatment on the surface of the When the image goes brightAfter the adjustment of the degree correction coefficient, a new measurement image I is obtained 2new The intensity value is recorded as Er 2new Updating the target complex amplitude image by using the new measurement image to obtain a new target complex amplitude image I 1new The intensity value is recorded as Er 1new Calculate a new evaluation function value Er new ＝(Er 1new -Er 2new ) 2 When Er new ＜Er best At the time, er new Is marked as Er best The corresponding intensity correction coefficient is the optimal correction coefficient, when Er new ＞Er best At the time of Er best Is unchanged.
2. The method for intensity correction for fourier ptychographic techniques of claim 1, wherein: in the step (4), the image is reconstructed, and the FPM algorithm is performed on the acquired multiple low-resolution images.
3. The method for intensity correction for fourier ptychographic techniques of claim 2, wherein: in the step (4), the image reconstruction is performed with super-resolution reconstruction by a GS phase recovery algorithm in the phase recovery algorithm.
4. A light intensity correction method for fourier ptychographic techniques as claimed in claim 3, wherein: the step (4) is to reconstruct super resolution by GS phase recovery algorithm, and comprises the following sub-steps:
(4.1) carrying out interpolation processing on an image shot by a central LED lamp on the LED array to serve as an initial airspace estimated value;
(4.2) carrying out Fourier transform on the interpolated image to obtain a frequency domain initial estimated value;
(4.3) selecting a sub-region from the obtained spectrogram to carry out inverse Fourier transform to obtain a target complex amplitude image, wherein the target complex amplitude image comprises amplitude information and phase information;
(4.4) keeping the phase information of the target complex amplitude image unchanged, and replacing the amplitude information of the target complex amplitude image by using an actual image shot by the LED lamp at the corresponding position on the LED array to obtain an updated target complex amplitude image;
(4.5) carrying out Fourier transform on the updated target complex amplitude image to obtain an updated spectrogram, and replacing a corresponding sub-spectrum region of the initial spectrogram with the updated spectrogram;
(4.6) repeating the steps (4.3) - (4.5) to finish all sub-spectrum updating;
(4.7) repeating the steps (4.3) - (4.6) to converge the result, thereby obtaining the image high-frequency information
And (3) carrying out inverse Fourier transform on the high-resolution frequency spectrum image with enhanced information to obtain a high-resolution image in the space domain.
5. The method for intensity correction for fourier ptychographic techniques of claim 4, wherein: and (5) checking the result, and comparing the original low-resolution image, the reconstructed image without illumination non-uniformity error correction and the reconstructed image with illumination non-uniformity error correction to find that the quality of the reconstructed image is poor due to the illumination non-uniformity error, and improving the resolution after correction, and meanwhile, the image background is more uniform.
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| CN113671682B (en) * | 2021-08-23 | 2023-05-23 | 北京理工大学重庆创新中心 | Frequency domain light source position accurate correction method based on Fourier laminated microscopic imaging |
| CN114998161A (en) * | 2022-06-02 | 2022-09-02 | 中国科学院西安光学精密机械研究所 | Fourier stack microscopy high-precision image reconstruction method based on perfect Fourier transform |
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