CN116755313A - High-resolution imaging method based on digital holographic microscopy - Google Patents
High-resolution imaging method based on digital holographic microscopy Download PDFInfo
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- CN116755313A CN116755313A CN202310296621.8A CN202310296621A CN116755313A CN 116755313 A CN116755313 A CN 116755313A CN 202310296621 A CN202310296621 A CN 202310296621A CN 116755313 A CN116755313 A CN 116755313A
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- 238000003384 imaging method Methods 0.000 title claims abstract description 38
- 238000009647 digital holographic microscopy Methods 0.000 title claims abstract description 15
- 238000000034 method Methods 0.000 claims abstract description 21
- 238000001228 spectrum Methods 0.000 claims abstract description 11
- 230000003287 optical effect Effects 0.000 claims description 18
- 238000005259 measurement Methods 0.000 claims description 11
- 238000001914 filtration Methods 0.000 claims description 6
- 230000021615 conjugation Effects 0.000 claims description 2
- 238000012937 correction Methods 0.000 claims description 2
- 230000004075 alteration Effects 0.000 abstract description 16
- 238000011160 research Methods 0.000 abstract description 3
- 238000010586 diagram Methods 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 8
- 238000012634 optical imaging Methods 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 210000001747 pupil Anatomy 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 239000004973 liquid crystal related substance Substances 0.000 description 2
- 238000001000 micrograph Methods 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000001093 holography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000009022 nonlinear effect Effects 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 238000012876 topography Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/0005—Adaptation of holography to specific applications
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/04—Processes or apparatus for producing holograms
- G03H1/0443—Digital holography, i.e. recording holograms with digital recording means
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/0005—Adaptation of holography to specific applications
- G03H2001/005—Adaptation of holography to specific applications in microscopy, e.g. digital holographic microscope [DHM]
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- G—PHYSICS
- G03—PHOTOGRAPHY; CINEMATOGRAPHY; ANALOGOUS TECHNIQUES USING WAVES OTHER THAN OPTICAL WAVES; ELECTROGRAPHY; HOLOGRAPHY
- G03H—HOLOGRAPHIC PROCESSES OR APPARATUS
- G03H1/00—Holographic processes or apparatus using light, infrared or ultraviolet waves for obtaining holograms or for obtaining an image from them; Details peculiar thereto
- G03H1/04—Processes or apparatus for producing holograms
- G03H1/0443—Digital holography, i.e. recording holograms with digital recording means
- G03H2001/045—Fourier or lensless Fourier arrangement
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Abstract
The invention relates to the research field of digital holographic microscopic imaging, in particular to a high-resolution imaging method based on digital holographic microscopy. The invention uses a digital holographic microscopic system with a small hole structure to obtain a hologram and a reference image of a sample, and uses an angular spectrum method and a digital refocusing algorithm to obtain an intensity image of sample focusing. The method is characterized in that the two methods are unified in the light path from the two angles of light collection efficiency and aberration, so that the light path has two functions of optimizing resolution, and the imaging resolution is further improved.
Description
Technical Field
The invention relates to the research field of digital holographic microscopic imaging, in particular to a high-resolution imaging method based on digital holographic microscopy.
Background
Far field imaging resolution is an important technical parameter for digital holographic microscopy applied to measuring micro-nano geometry. High resolution imaging is of great importance for improving holographic performance.
In recent years, far-field imaging technology breaking through diffraction limit has become a research hot spot, and various far-field imaging technologies for improving resolution are observed, and although imaging utilizes far-field transmission light waves, from principle, near-field evanescent waves are collected for conversion into far-field light waves, so that the collection efficiency of evanescent waves is improved, aberration is corrected, and the light field quality is optimized. The collecting method mainly comprises a method of utilizing nonlinear effect, special light illumination, coupling element and microsphere; and the optimization method is mainly a digital optimization method.
The aberration may cause a decrease in resolution of the image and a decrease in positioning accuracy. The purpose of optimizing the aberration is to regulate the light wave collection quality of the light field and optimize the point spread function PSF.
According to the invention, the small holes are added in the optical path, so that the two methods of improving the collection efficiency of evanescent waves and optimizing the aberration model method are unified in the optical path, the optical path has two functions of optimizing the resolution, and the imaging resolution is further improved.
Disclosure of Invention
The invention aims to improve the resolution of a digital holographic microscopic measurement system, proves the effect of improving the resolution of the digital holographic microscopic measurement system by a small hole structure, and provides a technical method for realizing high-resolution measurement of digital holography.
The technical scheme adopted by the invention is as follows:
the high-resolution imaging method based on digital holographic microscopy is characterized by comprising the following steps of: the method comprises the following steps:
step 1: designing a digital holographic off-axis microscopic light path system, and recording an off-axis hologram of a sample and a reference hologram without characteristic sample information;
step 2: carrying out real image filtering on the spectrums of the hologram and the reference image, carrying out two-dimensional inverse Fourier transform on the spectrums of the hologram and the reference image after filtering, and multiplying the complex conjugate of the hologram real image and the reference image real image to obtain out-of-focus object wavefront;
step 3: reconstructing and reproducing the object light wave by using an angular spectrum method to obtain focused object light wave information;
Step 4: adding a small hole structure into the optical path system, debugging the optical path, and repeating the steps 1-3 by using a new optical path system to obtain new object light wave information；
Step 5: and comparing and analyzing the information of the two object light waves, and finding that the resolution of the holographic optical path system added with the small hole structure is better than that of the holographic system not added with the small hole structure.
Further, in the step 3Expressed as:
wherein, the liquid crystal display device comprises a liquid crystal display device,factor representing distortion correction +_>For reference wavefront, < >>For its conjugation, ->Is the object wavefront.
Furthermore, the small hole structure in the step 4 is processed into an array type small hole structure, and the array imaging view fields are spliced to realize full view field measurement.
The invention has the advantages and positive effects that:
the high resolution imaging technology mainly comprises a near field optical imaging technology and a far field optical imaging technology. Near field optical imaging technology equipment is expensive and complex, and the real-time measurement is more limited by the scanning characteristic. The far-field optical imaging technology has the characteristics of no damage, long working distance and strong operability. In order to better test the manufacturing process and performance level of the micro-nano device and system, the micro-nano scale geometric quantity and mechanical quantity test should have real-time performance and dynamic performance, and the far-field optical imaging technology is more applicable.
Researchers at home and abroad improve the collection efficiency of evanescent waves from a path of regulating and controlling PSF or optical transfer function OTF and optimize aberration by an aberration model method to realize far-field high-resolution imaging, and both methods have certain achievements. Therefore, from the two angles of light collection efficiency and aberration, a small hole structure is added in a light path, and the imaging characteristics of the small hole structure are utilized to improve the light collection efficiency, optimize the aberration and realize high-resolution far-field imaging.
Drawings
FIG. 1 is a light path diagram of a transmissive high resolution digital holographic system of the present invention;
FIG. 2 is a schematic diagram of a microscopic magnification system without the addition of a pinhole structure;
FIG. 3 is a schematic diagram of a microscopic magnification system incorporating a pinhole structure;
FIG. 4 is an aberration diagram without the addition of a pinhole structure;
FIG. 5 is an aberration diagram incorporating a pinhole structure;
FIG. 6 is a defocused microscopic image of a resolution plate without the addition of a pinhole structure;
FIG. 7 is a microscopic image of the position of FIG. 6 of a resolution plate after addition of a pinhole structure;
FIG. 8 is a focusing microscopy image of a resolution plate without the addition of a pinhole structure;
FIG. 9 is a microscopic image of the position of FIG. 8 of a resolution plate after addition of a pinhole structure;
FIG. 10 is a diagram of the physical light path of the multi-angle digital holographic system;
FIG. 11 is an atomic force microscope test resolution plate structure;
FIG. 12 is an intensity plot without the high resolution test method;
FIG. 13 is an intensity map using high resolution holographic measurements;
Detailed Description
The invention will now be further illustrated by the following examples, which are intended to be illustrative, not limiting, and are not intended to limit the scope of the invention.
The invention discloses a high-resolution imaging method based on digital holographic microscopy, which is characterized by comprising the following steps of:
step 1: designing a digital holographic off-axis microscopic light path system, and recording an off-axis hologram of a sample and a reference hologram without characteristic sample information;
step 2: carrying out real image filtering on the spectrums of the hologram and the reference image, carrying out two-dimensional inverse Fourier transform on the spectrums of the hologram and the reference image after filtering, and multiplying the complex conjugate of the hologram real image and the reference image real image to obtain out-of-focus object wavefront;
step 3: reconstructing and reproducing the object light wave by using an angular spectrum method to obtain focused object light wave information;
Step 4: adding a small hole structure into the optical path system, debugging the optical path, and repeating the steps 1-3 by using a new optical path system to obtain new object light wave information；
Step 5: and comparing and analyzing the information of the two object light waves, and finding that the resolution of the holographic optical path system added with the small hole structure is better than that of the holographic system not added with the small hole structure.
Before experiments are carried out, the invention carries out optical path analysis on the small hole structure in the optical path.
The principle of the aperture structure to improve imaging resolution is shown in fig. 2 and 3. In FIG. 2, the maximum aperture angle of the edge point of the sample to be measured is as shown in the figure due to the limited entrance pupil aperture of the microscopic magnification systemThe angle is shown, whereas in FIG. 3, where an array of small holes is added, greater than +.>Angle of incidence of angle, e.g.)>The incident light rays at the angle can be projected to the aperture array according to the aperture imaging principle, and after passing through the aperture, the incident light rays reach the entrance pupil boundary of the microscopic amplifying system through diffraction, enter the microscopic amplifying system and are collected by the imaging system to participate in imaging. Compared with an imaging system without a small hole array structure, the small holes have the functions of turning and collecting high-frequency light, the spatial bandwidth product of a frequency spectrum is increased, the OTF of the system is enlarged, and the super diffraction limit resolution is realized.
Imaging aberration analysis of a microscopic magnification system as shown in fig. 4 and 5, taking spherical aberration as an example, a point on the object plane is imaged as a diffuse spot on the image plane. The size of the diffuse spots is related to the entrance pupil aperture of the magnifying system, and after the small hole structure is added, the size of the entrance pupil aperture is changed, the size of the diffuse spots is reduced, and the influence of aberration on imaging resolution is reduced, as shown in fig. 5. Although the aberration is optimized, the light flux is greatly reduced relative to a system without the small hole structure, and the imaging intensity and the field of view are affected, so that the small hole structure is processed into an array form, and the array imaging field of view is spliced, so that full field of view measurement is realized.
Then, a physical light path without a small hole structure is built according to a light path diagram of the digital holographic system shown in fig. 1, in the light path diagram, a Laser is a Laser, the wavelength is 670nm, NF is a middle gray mirror for adjusting the intensity of incident light, BS1 and BS2 are light splitting prisms, M1 and M2 are reflectors, a CCD is a photoelectric coupling recording device, MO1 and MO2 are micro objective lenses (NA=0.42, the magnification is 50), and SH is a small hole structure. The incident light is divided into two beams of coherent light through the beam splitting prism, one beam of light transmits the measured object, the object light passes through the small hole structure because of carrying object information, the light collecting efficiency is improved, aberration is corrected, and high-resolution measurement is realized; the other beam of light is reference light, and interferes with the object light. The interference information is recorded on the CCD to form a digital hologram.
The light path before addition of the pinhole structure was tested by the transmission digital holographic microscopy system according to steps 1-4 described in this example, as shown in fig. 6 and 8, which are microscopic imaging images of the resolution plate (USAF standard resolution plate) without the addition of the pinhole structure. Fig. 6 is a micrograph of a resolution plate in an out-of-focus state, with the sample imaging blurred. By adding the pinhole structure, the axial position of the pinhole structure is adjusted, the blurred image becomes clear, and as shown in fig. 7, the pinhole structure is proved to have the effect of reducing the diameter of the diffuse spots. In fig. 8, for a resolution plate image in a focus state, a sample in a red circle in the figure is in a blurred state due to diffraction limit, and by adding a pinhole structure and adjusting an appropriate axial position, a resolution plate imaging map as in fig. 9 can be obtained. By comparing the partial samples in circles of fig. 8 and 9, fig. 9 can distinguish the blurred bar sample in fig. 8. According to the comparison result, the diffraction limit can be broken through by adding the small hole structure, super-resolution measurement is realized, and the method is a method for improving the measurement resolution of the digital holographic microscopy system. According to the characteristic structure information of the resolution plate, when no small hole is added, the resolution of the holographic system is 1.1 mu m, and after the small hole is added, the resolution is 800nm.
In addition, the practicality of the present invention was again verified by establishing a multi-angle illumination digital holographic microscopy system using a plurality of mirrors as shown in fig. 10, and the topography of the reflective test piece was measured, and the result of testing the resolution plate using an atomic force microscope is shown in fig. 11. Fig. 12 shows the intensity image obtained by synthesizing the multi-angle sub-aperture hologram without using the small pore structure, and fig. 13 shows the lateral resolution of the synthesized multi-angle sub-aperture hologram using the method of the present invention, which can reach 800nm. The above is the principle and example results of a high resolution imaging method based on digital holographic microscopy.
The application process of the invention comprises the following steps:
in the invention, as shown in fig. 1, a system light path diagram of a high-resolution imaging method based on digital holographic microscopy is shown, a middle gray mirror NF and a beam splitting prism BS1 are arranged at the emergent end of a laser, and after the emergent laser beam attenuates the intensity, object light and reference light are formed by the beam splitting prism BS1 in a beam splitting way. The object beam is transmitted and emitted from the tested sample through the reflector M1, carries sample information and is amplified by the microscope objective MO1 after passing through the small hole structure, so as to form an amplified object image; the reference beam passes through the microscope objective MO2 via the mirror M2, and the object light and the reference light are converged by the beam splitter prism BS2, and interfere on the CCD plane to form a hologram of the sample to be measured.
In order to ensure that only sample differences exist in object reference light, the microscope objective MO1 and the microscope objective MO2 are arranged as identical devices. By adjusting the angle of the beam splitting prism, the off-axis included angle between the object light and the reference light can be changed. For the obtained sample hologram and reference image, the sample hologram is digitally processed as described in example steps 1-3 to obtain a high resolution measured sample intensity image.
Claims (3)
1. The high-resolution imaging method based on digital holographic microscopy is characterized by comprising the following steps of: the method comprises the following steps:
step 1: designing a digital holographic off-axis microscopic light path system, and recording an off-axis hologram of a sample and a reference hologram without characteristic sample information;
step 2: carrying out real image filtering on the spectrums of the hologram and the reference image, carrying out two-dimensional inverse Fourier transform on the spectrums of the hologram and the reference image after filtering, and multiplying the complex conjugate of the hologram real image and the reference image real image to obtain out-of-focus object wavefront;
step 3: reconstructing and reproducing the object light wave by using an angular spectrum method to obtain focused object light wave information;
Step 4: adding a small hole structure into the optical path system, debugging the optical path, and repeating the steps 1-3 by using a new optical path system to obtain new object light wave information；
Step 5: and comparing and analyzing the information of the two object light waves, and finding that the resolution of the holographic optical path system added with the small hole structure is better than that of the holographic system not added with the small hole structure.
2. The digital holographic microscopy high resolution imaging method of claim 1, wherein: said step 3Expressed as:
wherein->Factor representing distortion correction +_>For reference wavefront, < >>For its conjugation, ->Is the object wavefront.
3. The digital holographic microscopy high resolution imaging method of claim 1, wherein: and (3) processing the small hole structure in the step (4) into an array type small hole structure, and splicing the array imaging view fields to realize full view field measurement.
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| CN202310296621.8A CN116755313A (en) | 2023-03-24 | 2023-03-24 | High-resolution imaging method based on digital holographic microscopy |
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