CN108362643B - Double-height illumination Fourier laminated microscopic imaging method - Google Patents

Double-height illumination Fourier laminated microscopic imaging method Download PDF

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CN108362643B
CN108362643B CN201810137985.0A CN201810137985A CN108362643B CN 108362643 B CN108362643 B CN 108362643B CN 201810137985 A CN201810137985 A CN 201810137985A CN 108362643 B CN108362643 B CN 108362643B
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戎路
王大勇
唐超
赵洁
王云新
王红红
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Abstract

The invention discloses a double-height illumination Fourier laminated microscopic imaging method, and belongs to the technical field of optics. The optical path of the imaging system for realizing the method comprises a spiral height adjusting frame, an LED array, a tested sample, a microscope objective, an imaging lens, a camera and a microscope. According to the invention, by changing the distance between the sample and the LED plane and jointly carrying out reconstruction iteration on the low-resolution intensity images recorded at two heights according to the sequence of increasing the illumination incidence angle in sequence, the sampling rate of the low-frequency part can be increased, so that the overlapping rate and the data redundancy of the low-frequency region in the frequency spectrum are effectively increased under the condition of keeping other imaging system parameters unchanged, and the imaging precision and the convergence speed of the FPM are further improved.

Description

Double-height illumination Fourier laminated microscopic imaging method
Technical Field
The invention relates to a double-height illumination Fourier laminated microscopic imaging method, in particular to a method for improving imaging precision and convergence rate of a double-plane illumination Fourier laminated, and belongs to the technical field of optics.
Background
Optical microscopes have found great application in biomedical testing, but conventional optical microscopes suffer from two drawbacks: first, a phase contrast image of the sample cannot be obtained; second, as resolution increases, the field of view decreases substantially. The FPM imaging system is formed by modifying a traditional optical microscope, a two-dimensional thin sample is illuminated by plane waves from different angles, and the frequency spectrum of the sample on the focal plane behind an objective lens is translated to different corresponding positions, so that frequency components which originally exceed the numerical aperture of the objective lens are translated into the numerical aperture of the objective lens, and can be transmitted to an imaging plane for imaging. The frequency components are synthesized in a frequency domain through a reconstruction program to obtain a spread spectrum of the sample, and the intensity and phase distribution of the sample can be recovered by performing inverse Fourier transform. According to the method, the low-frequency region of the sample needs a higher frequency spectrum overlapping rate to meet the requirement of image reconstruction, but the frequency spectrum overlapping rate formed by LED illumination in the frequency domain at a single height has the condition that the overlapping rate of the low-frequency region is lower and the overlapping rate of the high-frequency region is higher, so that the distance between the sample and an LED plane can be changed, the low-resolution intensity images recorded at two heights are subjected to reconstruction iteration together according to the sequence of increasing the illumination incidence angles in sequence, the sampling rate of the low-frequency part can be increased, the overlapping rate and the data redundancy of the low-frequency region in the frequency spectrum can be effectively increased under the condition that other imaging system parameters are kept unchanged, and the imaging precision and the convergence speed of the FPM are further improved.
Disclosure of Invention
The invention aims to change the distance between a sample and an LED plane, and jointly perform reconstruction iteration on low-resolution intensity images recorded at two heights according to the sequence of increasing the illumination incidence angle in sequence, so that the sampling rate of a low-frequency part can be increased, the overlapping rate and the data redundancy of a low-frequency region in a frequency spectrum are effectively increased under the condition of keeping other imaging system parameters unchanged, and the imaging precision and the convergence speed of the FPM are further improved.
In order to achieve the purpose, the technical scheme adopted by the invention is a double-height illumination Fourier laminated microimaging method, and an optical path of an imaging system for achieving the method comprises a spiral height adjusting frame, an LED array, a measured sample, a microobjective, an imaging lens, a camera and a microscope. The spiral height adjusting frame is used for fixing the LED array, and the lighting height of the LED array is adjusted through the spiral height adjusting frame; the LED array sequentially lights the light-emitting units to provide illumination light at different angles for the sample to be measured; the sample to be measured is arranged between the LED array and the microscope objective, and the sample to be measured is positioned on the focal plane of the microscope objective; the microscope objective and the imaging lens are combinedForming a 4f system, wherein the illumination light beams are emitted from the LED array, are scattered by a tested sample, then are imaged on the back focal plane of the imaging lens through the 4f system, and are recorded by a camera; the camera is used for recording low-resolution intensity images I of a tested sample under different heights of illuminationmi(x, y) and Imi' (x, y), i equals 1,2,3 … N, N indicates the total number of LEDs; the microscope objective and the imaging lens are both arranged on a microscope, and the objective table of the microscope is used for fixing a sample.
The method comprises the steps that the LED light-emitting units at two lighting heights illuminate point light sources at the same height, and low-resolution intensity images I are shot respectivelymi(x, y) and Imi' (x, y) sequentially increasing the intensity images obtained at the two heights according to the incident angle and sequencing and numbering clockwise, and then performing iterative reconstruction according to the sequence of sequentially increasing the illumination incident angle by a reconstruction algorithm to obtain the recovered high-resolution intensity and phase image.
A biplane illumination Fourier laminated microscopic imaging method is characterized in that the process of improving imaging precision and convergence speed is divided into three steps:
s1 adjusting the screw height adjusting frame to make the distance between the LED array and the tested sample be h1Sequentially lightening each light-emitting unit on the LED array; corresponding to each light-emitting unit, the illuminating beams with different angles are emitted from the LED light-emitting units, and after being scattered by a tested sample, the illuminating beams form a low-resolution intensity image I on the back focal plane of the imaging lens through a 4f system consisting of a microscope objective and the imaging lensmi(x, y) and recorded by the camera.
S2 adjusting the height of the screw to make the distance between the LED array and the tested sample h2Repeating the step S1 to record the low-resolution intensity image recorded on the back focal plane of the imaging lens as Imi′(x,y)。
S3 Using a reproduction Algorithm on the recorded Low resolution intensity image Imi(x, y) and Imi' (x, y) for reconstruction. The reconstruction process is divided into the following four steps:
s3.1 at height h1Next, the coordinates of the light emitting unit at the center of the LED array are noted as (ξ)0,η0) Selecting the low-resolution intensity image I recorded under illumination of the light-emitting unitm1(x, y) and interpolated to generate a guessed high-resolution initial intensity distribution Ih(x, y), multiplied by the guessed random phase
Figure BDA0001576828440000031
Generating a guessed sample high-resolution complex amplitude distribution:
Figure BDA0001576828440000032
fourier transform of the high resolution complex amplitude distribution yields a guessed spread spectrum distribution Uh(kx,ky)：
Figure BDA0001576828440000033
S3.2 at height h2Lower LED array center lighting unit (ξ)0′,η0') Low resolution intensity image recorded by illumination Im1' (x, y) wherein h2Greater than h1And all low-resolution intensity images I taken at two illumination levelsmi(x, y) and Imi' (x, y) are ordered chronologically from small to large in the angle of incidence to give Imk(x, y), k is 1,2,3, … … 2i-1, corresponding to an angle of incidence of αk。
S3.3 selecting the incident angle as αkWhen k is 1, the corresponding spectral region (-f)c,fc) Inverse Fourier transform to obtain the guessed low resolution complex amplitude Ul(x,y):
Figure BDA0001576828440000041
Wherein f iscNA/λ is the cut-off frequency of the imaging system, NA is the Numerical Aperture (NA) of the microscope objective, and λ is the center wavelength of the LED light-emitting unit. Maintaining phase
Figure BDA0001576828440000042
Unchanged, the guessed low resolution complex amplitude UlAmplitude I of (x, y)lk(x, y) intensity image I recorded experimentallymk(x, y) substitution to obtain a substituted complex amplitude Ulm(x,y):
Figure BDA0001576828440000043
Then Fourier transform is carried out on the spectrum region to fill back to the corresponding frequency spectrum region (-f)c,fc) The center region of the spread spectrum is updated.
S3.4 selecting the incident angle as αkWhen k is 2,3, … … 2i-1, corresponding to the spectral region (-f)c+fx,fc+fy) Wherein f isx、fyRepresenting an angle of incidence of αkIn the frequency domain kx,kyDirection forming frequency shift fx、fy：
Figure BDA0001576828440000044
Wherein, αkxAnd αkyAngle of incidence α representing stereokAnd the included angle is formed between the X coordinate axis and the Y coordinate axis. Repeating step S3.3 updates the other regions of the spread spectrum.
And repeating the step S3.3 and the step S3.4 until the overlapping parts of adjacent regions of the frequency spectrum are continuous, so as to converge and obtain an updated spread spectrum. And performing inverse Fourier transform on the spread spectrum to obtain the high-resolution complex amplitude distribution of the measured sample.
The test result of the typical embodiment of the invention shows that by changing the height between the sample and the LED array, and when the size and the density of the LED array are fixed, the low-resolution intensity images recorded at the two heights are reconstructed and iteratively reconstructed together, so that the overlapping rate and the data redundancy of the low-frequency region in the frequency spectrum are effectively increased, and further, the improvement of the imaging precision and the convergence speed of the FPM are further improved under the condition of keeping the parameters of other imaging systems unchanged.
Compared with the prior art, the method for improving the imaging precision and the convergence speed by the bi-plane illumination LED array multi-angle illumination provided by the invention has the advantages that the reconstruction iteration is carried out on the low-resolution intensity images recorded at two heights together, the range of the incidence angle is ensured by the closer high illumination, and the recoverable frequency domain size and the final resolution are ensured; and through illumination of a far height, the overlapping rate and the data redundancy of low-frequency regions in a frequency spectrum can be effectively increased on the premise of not changing the array arrangement of the LED light-emitting units, and the imaging precision and the convergence speed of the FPM are further improved under the condition of keeping other imaging system parameters unchanged.
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FIG. 1 is a system optical path based on a biplane illumination Fourier stack microscopy imaging method.
In the figure: 1. the device comprises a spiral height adjusting frame, a 2, 32X32 LED light emitting array, 3, a sample to be tested, 4, a microscope objective, 5, an imaging lens, 6, a scientific grade sCMOS camera, 7 and an optical microscope.
Detailed Description
As shown in fig. 1, a dual-height illumination fourier stack microscopy imaging method is characterized in that: a double-height illumination Fourier laminated microscopic imaging method is characterized in that an optical path of an imaging system of the double-height illumination Fourier laminated microscopic imaging method comprises a spiral height adjusting frame 1, a 32x32 LED array 2 of a light emitting unit, a sample 3 to be measured, a microscope objective (4 x, 0.1NA)4, an imaging lens 5, a scientific-grade sCMOS camera 6 and a microscope 7. The screw height adjusting rack 1 is used for fixing the LED array 2 of the 32x32 luminous unit and adjusting the lighting height h of the LED array 21，h2(ii) a The LED array 2 of the 32x32 light-emitting units sequentially lights the light-emitting units to provide illumination light with different angles for the tested sample 3, the distance between the light-emitting units is 5mm, and the wavelength is 625 nm; the tested sample 3 is arranged between the LED array 2 and the microscope objective 4 and is positioned on the focal plane of the microscope objective 4; the microscope objective 4 and the imaging lens 5 jointly form a 4f system, the illuminating light beam is emitted from the LED array, is scattered by the tested sample 3, then is imaged on the back focal plane of the imaging lens 5 through the 4f system, and is recorded by a scientific-grade sCMOS camera 6; the scientific grade sCMOS camera 6 has resolution of 2560 x 2160 and pixel sizeSetting the particle size at 6.5 mu m; used for recording the measured sample at h1，h2Low resolution intensity image under high illumination Imi(x, y) and Imi' (x, y) (i ═ 1,2,3 … N, N indicates the total number of LEDs); the resolution was 2560 x 2160, and the pixel size was set to 6.5 μm; the entire experimental system is built on the basis of a microscope 7, the stage of which is used to fix the sample 2.
The method comprises the steps that the LED light-emitting units at two lighting heights illuminate point light sources at the same height, and low-resolution intensity images I are shot respectivelymi(x, y) and Imi' (x, y) sequentially increasing the intensity images obtained at the two heights according to the incident angle and sequencing and numbering clockwise, and then performing iterative reconstruction according to the sequence of sequentially increasing the illumination incident angle by a reconstruction algorithm to obtain the recovered high-resolution intensity and phase image.
A biplane illumination Fourier laminated microscopic imaging method is characterized in that the process of improving imaging precision and convergence speed is divided into three steps:
s1 adjusting the screw height adjusting frame 1 to make the distance between the LED array 2 and the tested sample 3 be h1Sequentially lighting each light-emitting unit on the LED array 2; corresponding to each light-emitting unit, the illumination beams with different angles are emitted from the LED light-emitting units, scattered by the tested sample 3 and then form a low-resolution intensity image I on the back focal plane of the imaging lens 5 through a 4f system consisting of a microscope objective 4 and the imaging lens 5mi(x, y) and recorded by the scientific grade sCMOS camera 6.
S2, adjusting the screw height adjusting frame 1 to make the distance between the LED array 2 and the tested sample 3 be h2Repeating the above step S1, recording the low resolution intensity image recorded on the back focal plane of the imaging lens 5 by the scientific sCMOS camera 6 as Imi′(x,y)。
S3 Using a reproduction Algorithm on the recorded Low resolution intensity image Imi(x, y) and Imi' (x, y) for reconstruction. The reconstruction process is divided into the following four steps:
s3.1 at h1The coordinates of the light emitting unit at the center of the LED array 1 are noted as (ξ) in height0,η0) Under the selected illumination of the light-emitting unitRecorded low resolution intensity image Im1(x, y) and interpolated to generate a guessed high-resolution initial intensity distribution Ih(x, y), multiplied by the guessed random phase
Figure BDA0001576828440000071
Generating a guessed sample high-resolution complex amplitude distribution:
Figure BDA0001576828440000072
fourier transforming the high resolution complex amplitude to obtain a guessed spread spectrum distribution Uh(kx,ky)：
Figure BDA0001576828440000073
S3.2 leaving out a greater height h2 Lower LED array 1 central lighting unit (ξ)0′,η0') Low resolution intensity image recorded by illumination Im1' (x, y) and all low resolution intensity images I taken at two illumination heightsmi(x, y) and Imi' (x, y) are ordered chronologically from small to large in the angle of incidence to give Imk(x, y) ( k 1,2,3, … … 2i-1) corresponding to an angle of incidence of αk。
S3.3 selecting the incident angle as αk(k-1) corresponding to the spectral region (-f)c,fc) Inverse Fourier transform to obtain the guessed low resolution complex amplitude Ul(x,y):
Figure BDA0001576828440000081
Wherein f iscNA/λ is the cut-off frequency of the imaging system, NA is the Numerical Aperture (NA) of the microscope objective 4, and λ is the center wavelength of the LED lighting unit 2. Maintaining phase
Figure BDA0001576828440000082
Unchanged, the guessed low resolution complex amplitude UlAmplitude I of (x, y)lk(x, y) intensity image I recorded experimentallymk(x, y) substitution to obtain a substituted complex amplitude Ulm(x,y):
Figure BDA0001576828440000083
Then Fourier transform is carried out on the spectrum region to fill back to the corresponding frequency spectrum region (-f)c,fc) The center region of the spread spectrum is updated.
S3.4 selecting the incident angle as αk(k-2, 3, … … 2i-1) corresponding to the spectral region (-f)c+fx,fc+fy) Wherein f isx、fyRepresenting an angle of incidence of αkIn the frequency domain kx,kyDirection forming frequency shift fx、fy：
Figure BDA0001576828440000084
Wherein, αkxAnd αkyAngle of incidence α representing stereokAnd the included angle is formed between the X coordinate axis and the Y coordinate axis. Repeating step S3.3 updates the other regions of the spread spectrum.
And repeating the step S3.3 and the step S3.4 until the overlapping parts of adjacent regions of the frequency spectrum are continuous, so as to converge and obtain an updated spread spectrum. The spread spectrum is inverse fourier transformed to obtain a high-resolution complex amplitude distribution of the sample 3 to be measured.
The test result of the typical embodiment of the invention shows that by changing the height between the sample 2 and the LED array 1, when the size and the density of the LED array are fixed, the low-resolution intensity images recorded at the two heights are reconstructed and iteratively reconstructed together, so that the overlapping rate and the data redundancy of low-frequency regions in a frequency spectrum are effectively increased, and further, the improvement of the imaging precision and the convergence speed of the FPM are further improved under the condition of keeping other imaging system parameters unchanged.

Claims (2)

1. A double-height illumination Fourier laminated microscopic imaging method is characterized in that an optical path of an imaging system for realizing the method comprises a spiral height adjusting frame, an LED array, a sample to be detected, a microscope objective lens in a microscope, an imaging lens and a camera; the spiral height adjusting frame is used for fixing the LED array, and the lighting height of the LED array is adjusted through the spiral height adjusting frame; the LED array sequentially lights the light-emitting units to provide illumination light at different angles for the sample to be measured; the sample to be measured is arranged between the LED array and the microscope objective, and the sample to be measured is positioned on the focal plane of the microscope objective; the microscope objective and the imaging lens jointly form a 4f system, the illumination light beam is emitted from the LED array, is scattered by a tested sample, then is imaged on the back focal plane of the imaging lens through the 4f system, and is recorded by a camera; the camera is used for recording low-resolution intensity images Imi (x, y) and Imi' (x, y) of the tested sample under different heights of illumination, wherein i is 1,2,3 … N, and N represents the total number of LEDs; the microscope objective and the imaging lens are both arranged on a microscope, and an objective table of the microscope is used for fixing a sample; the method is characterized in that:
the method comprises the steps that LED array light-emitting units at two illumination heights are used as point light sources, and low-resolution intensity images I are shot respectivelymi(x, y) and Imi(x, y), sequentially increasing the intensity images obtained at the two heights according to the incident angle and sequencing and numbering clockwise, and then performing iterative reconstruction according to the sequentially increasing order of the illumination incident angle by a reproduction algorithm to obtain the recovered high-resolution intensity and phase image;
the process of improving the imaging precision and the convergence rate is divided into three steps:
s1 adjusting the screw height adjusting frame to make the distance between the LED array and the tested sample be h1Sequentially lightening each light-emitting unit on the LED array; corresponding to each light-emitting unit, the illuminating beams with different angles are emitted from the LED light-emitting units, and after being scattered by a tested sample, the illuminating beams form a low-resolution intensity image I on the back focal plane of the imaging lens through a 4f system consisting of a microscope objective and the imaging lensmi(x, y) and recorded by a camera;
s2 adjusting the height of the screw to make the distance between the LED array and the tested sample h2Repeating the step S1 to let the camera in the imaging processThe low-resolution intensity image recorded on the focal plane behind the mirror is marked as Imi′(x,y)；
S3 Using a reproduction Algorithm on the recorded Low resolution intensity image Imi(x, y) and Imi' (x, y) for reconstruction.
2. The method of dual-height illumination fourier stack microscopy imaging of claim 1, wherein: the reconstruction process is divided into the following four steps,
s3.1 at height h1Next, the coordinates of the light emitting unit at the center of the LED array are noted as (ξ)0,η0) Selecting the low-resolution intensity image I recorded under illumination of the light-emitting unitm1(x, y) and interpolated to generate a guessed high-resolution initial intensity distribution Ih(x, y), multiplied by the guessed random phase
Figure FDA0002456991700000021
Generating a guessed sample high-resolution complex amplitude distribution:
Figure FDA0002456991700000022
fourier transform of the above equation yields a guessed spread spectrum distribution Uh(kx,ky)：
Figure FDA0002456991700000023
S3.2 at height h2Lower LED array center lighting unit (ξ)0′,η0') Low resolution intensity image recorded by illumination Im1' (x, y) wherein h2Greater than h1And all low-resolution intensity images I taken at two illumination levelsmi(x, y) and Imi' (x, y) are ordered chronologically from small to large in the angle of incidence to give Imk(x, y), k is 1,2,3, … … 2i-1, corresponding to an angle of incidence of αk；
S3.3 selecting the incident angle as αkWhen k is equal to1, corresponding spectral region (-f)c,fc) Inverse Fourier transform to obtain the guessed low resolution complex amplitude Ul(x,y):
Figure FDA0002456991700000024
Wherein f iscThe NA/lambda is the cut-off frequency of the imaging system, the NA is the numerical aperture of the microscope objective, and the lambda is the central wavelength of the LED light-emitting unit; maintaining phase
Figure FDA0002456991700000031
Unchanged, the guessed low resolution complex amplitude UlAmplitude I of (x, y)lk(x, y) intensity image I recorded experimentallymk(x, y) substitution to obtain a substituted complex amplitude Ulm(x,y):
Figure FDA0002456991700000032
Then Fourier transform is carried out on the spectrum region to fill back to the corresponding frequency spectrum region (-f)c,fc) Updating a center region of the spread spectrum;
s3.4 selecting the incident angle as αkWhen k is 2,3, … … 2i-1, corresponding to the spectral region (-f)c+fx,fc+fy) Wherein f isx、fyRepresenting an angle of incidence of αkIn the frequency domain kx,kyDirection forming frequency shift fx、fy：
Figure FDA0002456991700000033
Wherein, αkxAnd αkyAngle of incidence α representing stereokIncluded angles formed with the x and y coordinate axes; repeating the step S3.4, and updating other areas of the spread spectrum;
repeating the step S3.3 and the step S3.4 until the overlapping parts of adjacent regions of the frequency spectrum are continuous to achieve convergence so as to obtain an updated spread spectrum; and performing inverse Fourier transform on the spread spectrum to obtain the high-resolution complex amplitude distribution of the measured sample.
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