A method for generating an enhanced image of a brightfield microscope

Abstract

The application discloses a kind of generation methods of enhanced image of bright field microscope, based on the optical imaging principle of traditional optical Zernike phase contrast microscope, the optical imaging model of optical Zernike phase contrast microscope imaging is simulated by computer system, without adding annular diaphragm and annular phase plate and other additional hardware in the illumination light path and imaging light path of bright field microscope, the enhancement of phase contrast of bright field digital microscopic image is realized, avoid the energy loss of illumination optical system, greatly reduce the design and manufacturing cost of microscopic optical system;The present application can be used for different bright field microscopes, there is no halo artifact caused by interference effect in traditional optical Zernike phase contrast microscope, can distinguish more fine structure characteristics in biological cells, its resolution can be close to the resolution limit of objective design, and greatly reduce the technical requirements of user.

Description

Technical Field

[0001] This invention relates to a method for generating enhanced images, and more particularly to a method for generating enhanced images using a bright-field microscope. Background Technology

[0002] In basic research in biology, medicine, and materials science, high-contrast observation of transparent samples such as biological cells and glass surface scratches often requires image enhancement from bright-field microscopy. Currently, optical Zernike phase-contrast microscopy and optical differential interferometry are commonly used for sample observation. The structure of a traditional optical Zernike phase-contrast microscope is as follows: Figure 1 As shown, a white light source 1 (such as an LED or halogen lamp) emits an illumination beam, which is collimated into a Gaussian parallel beam by a collimating lens 2. The collimated Gaussian parallel beam is then adjusted at its exit angle by a reflecting mirror 3 to make the beam parallel to the optical axis. The Gaussian parallel beam becomes a ring beam after passing through an annular aperture 11, and is then focused by a converging lens 4 to illuminate the sample on the sample stage 5, exciting the sample's diffracted light. The annular transmitted light and the sample's diffracted light are collected together by the objective lens 6. After passing through the annular phase plate 12 in the objective lens 6, the annular transmitted light and the sample's diffracted light have a relative phase delay of π / 2 or 3π / 2. The annular transmitted light and the sample's diffracted light with the relative phase delay pass together through the tube lens 7 and the imaging lens 8, and interfere at the imaging plane where the digital image sensor 9 is located to generate an enhanced sample microscopic image signal. The digital image sensor 9 sends the recorded digital signal to the computer system 10 to display and store the contrast-enhanced sample microscopic image.

[0003] Traditional methods improve the visibility of weakly absorbing transparent objects by altering the phase difference between diffracted and transmitted light in bright-field microscopes by adding an annular aperture and an annular phase plate. This converts the minute phase changes of the weakly absorbing transparent object into changes in light intensity. However, when observing samples with an optical Zernike phase-contrast microscope, halo artifacts appear at the sample edges, making it suitable only for observing thinner samples. Furthermore, to avoid image quality degradation, precise alignment of the conjugate positions of the annular aperture and the annular phase plate is crucial, placing high demands on the microscope user. In addition, the introduction of the annular aperture blocks most of the illumination beam, resulting in significant energy loss in the illumination system; and the presence of the phase plate also affects the aberrations of the microscope's optical system, indirectly increasing the design and manufacturing costs of the optical system. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for generating enhanced images of weakly absorbing objects such as biological cells without the need for additional hardware such as annular apertures and annular phase plates, avoiding energy loss of the illumination optical system, and applicable to bright-field microscopy of thick samples.

[0005] The technical scheme adopted by the present application to solve the above technical problems is: a method for generating an enhanced image of a bright field microscope, comprising the following specific steps:

[0006] Step one: obtaining the physical parameters of the bright field microscope when shooting a bright field digital microscope image;

[0007] Step two: using the bright field microscope to obtain the light intensity distribution of the bright field digital microscope image of the sample to be measured;

[0008] Step three: pre-processing the light intensity distribution of the bright field digital microscope image of the sample to be measured according to the physical parameters of the bright field microscope, to obtain the light intensity distribution of the denoised bright field digital microscope image;

[0009] Step four: mapping the light intensity distribution of the denoised bright field digital microscope image to a phase distribution to obtain the approximate phase distribution of the sample to be measured;

[0010] Step five: converting the approximate phase distribution of the sample to be measured into an enhanced image of the bright field microscope according to the optical imaging model of the positive optical Zernike phase contrast microscope and the optical imaging model of the negative optical Zernike phase contrast microscope.

[0011] Compared with the prior art, the present application has the advantages that based on the optical imaging principle of the traditional optical Zernike phase contrast microscope, the optical imaging model of the optical Zernike phase contrast microscope imaging is simulated by a computer system, without adding additional hardware such as a ring-shaped diaphragm and a ring-shaped phase plate in the illumination and imaging light path of the bright field microscope, the contrast of the bright field digital microscope image is enhanced by phase contrast, the energy loss of the illumination optical system is avoided, and the design and manufacturing cost of the microscopic optical system is greatly reduced. The present application does not depend on additional optical elements, and can be used for different bright field microscopes. The present application realizes the phase contrast enhancement microscopic effect by digital phase shift in the frequency domain, and therefore does not have the halo artifact caused by the interference effect in the traditional optical Zernike phase contrast microscope, can distinguish more fine structural features in biological cells, and the resolution can approach the resolution limit of the objective lens design. The technical scheme of the present application does not need complex debugging of the illumination optical system, greatly reducing the technical requirements for the user.

[0012] Compared with the existing optical Zernike phase contrast microscope, the method of the present application has the following beneficial effects:

[0013] (1) It can be used for bright field illumination digital microscopic imaging system without additional complex optical elements and debugging, reducing cost and making the operation of the label-free microscope more convenient;

[0014] (2) Due to diffraction and halo effects, the resolving power of traditional optical Zernike phase contrast microscopes is usually not as good as that of bright field microscopes of the same specifications; however, the method of the present invention is a digital intensified microscopy imaging method based on bright field microscopes, so its resolving power is consistent with that of bright field images, and it can better distinguish fine structures.

[0015] (3) Traditional optical Zernike phase contrast microscopes are only suitable for imaging thin samples. In imaging thick samples, the imaging contrast will decrease due to halo problems. However, the method of the present invention can still achieve phase contrast enhancement for thick samples through phase mapping.

[0016] The physical parameters of the bright-field microscope are the center wavelength and numerical aperture of the illumination light of the bright-field microscope, and then the optical transfer function of the bright-field microscope is obtained for the preprocessing in step three.

[0017] Preferably, the specific method of step three is to perform a Fourier transform on the light intensity distribution of the bright-field digital micrograph of the sample to be tested obtained by the bright-field microscope, then perform an inverse Fourier transform on the product of the Fourier transform result and the optical transfer function of the bright-field microscope, and then subtract the average noise floor of the bright-field digital micrograph to obtain the light intensity distribution of the noise-reduced bright-field digital micrograph, as follows:

[0018] The light intensity distribution of the bright-field digital micrograph of the sample acquired by the bright-field microscope is described by the following formula. Preprocessing: ,in, This represents the light intensity distribution of the denoised bright-field digital micrograph. This represents the average noise floor of a bright-field digital micrograph. and These represent the Fourier transform and the inverse Fourier transform, respectively. It is the optical transfer function of the bright-field microscope. In the formula, Indicates the center wavelength of the illuminating light. NA Indicates numerical aperture. Indicates spatial frequency, Indicates the spatial frequency in the lateral direction. It represents the spatial frequency in the longitudinal direction.

[0019] Preferably, the specific method of step four is as follows:

[0020] The light intensity distribution of the denoised brightfield digital micrograph is calculated using the following formula. Mapping to 0- π The phase region is used to obtain the approximate phase distribution of the sample under test. : ,innor represents a normalization operation, mean represents an averaging operation.

[0021] The bright field microscope consists of an illumination optical system, a sample stage, an imaging optical system, a digital image sensor and a computer system, the illumination optical system emits illumination light and converges the illumination light on the sample to be tested on the sample stage to excite the diffraction light of the sample to be tested, the numerical aperture and the optical transfer function are the numerical aperture and the optical transfer function of the imaging optical system, the transmitted illumination light after the sample to be tested and the diffraction light of the sample to be tested together pass through the imaging optical system, and the digital image sensor acquires the light intensity distribution of the bright field digital microscope image of the sample to be tested and sends it to the computer system.

[0022] Preferably, the actual physical size of a unit pixel of the bright field digital microscope image acquired by the digital image sensor is less than half of the imaging resolution of the imaging optical system.

[0023] The illumination optical system consists of a white light source, a collimating lens, a mirror and a converging lens, the imaging optical system consists of an objective lens, a tube lens and an imaging lens, the white light source emits illumination light, which is collimated into Gaussian type parallel illumination light after passing through the collimating lens, the Gaussian type parallel illumination light is adjusted by the mirror to have an exit angle, so that the Gaussian type parallel illumination light is parallel to the optical axis, and the Gaussian type parallel illumination light passes through the converging lens to directly converge the illumination on the sample to be tested on the sample stage to excite the diffraction light of the sample to be tested, the transmitted illumination light after the sample to be tested and the diffraction light of the sample to be tested together pass through the objective lens, the tube lens and the imaging lens, and are received by the digital image sensor for imaging.

[0024] Preferably, the white light source is an LED or a halogen lamp. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 It is a structural schematic diagram of a traditional optical Zernike phase contrast microscope;

[0026] Figure 2 It is a structural schematic diagram of a bright field microscope used in the method of the present application;

[0027] Figure 3 It is a flow chart of the method for generating an enhanced image of the bright field microscope of the present application;

[0028] Figure 4 It is a comparison diagram of the imaging of a thin sample by the prior art and the method of the present application;

[0029] Figure 5 It is a comparison diagram of the imaging of a thick sample by the prior art and the method of the present application;

[0030] Figure 6 Imaging effect diagram of the method of the present application for transparent cartilage section sample;

[0031] Figure 7 Imaging effect diagram of the method of the present application for human chromosome section sample;

[0032] Figure 8 Imaging effect diagram of the method of the present application for glass surface scratch sample of metallographic sample.

[0033] Explanation of reference signs:

[0034] 1, white light source; 2, collimating lens; 3, reflecting mirror; 4, converging lens; 5, sample stage; 6, objective lens; 7, tube lens; 8, imaging lens; 9, digital image sensor; 10, computer system; 11, annular diaphragm; 12, annular phase plate. DETAILED DESCRIPTION

[0035] The present application will be further described below in conjunction with the accompanying drawings.

[0036] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0037] The inventive concept of the present application is that the physical mechanism of optical label-free phase contrast imaging is combined with modern digital image technology, and the phase contrast optical imaging process of bright field microscope is simulated by computer system, so that the enhancement effect of digital image of label-free transparent sample can be realized without optical phase contrast element.

[0038] Figure 2The structure diagram of bright field microscope used in the method of the present application is shown. The white light source 1 emits illumination light, which can be selected as LED or halogen lamp, and the parallel illumination light of Gaussian type is collimated by the collimating lens 2. The exit angle of the parallel illumination light of Gaussian type is adjusted by the reflecting mirror 3, so that the parallel illumination light of Gaussian type is parallel to the optical axis. The parallel illumination light of Gaussian type is directly converged by the converging lens 4 to illuminate the sample to be measured placed on the sample stage 5, so as to excite the diffraction light of the sample to be measured. The transmission light after the sample to be measured and the diffraction light of the sample to be measured are jointly passed through the objective lens 6, the tube lens 7 and the imaging lens 8, and then the imaging is received by the digital image sensor 9. The digital image sensor 9 obtains the light intensity distribution of the bright field digital microscope image of the sample to be measured and sends it to the computer system 10. The computer system 10 maps the light intensity distribution of the bright field digital microscope image of the sample to be measured into phase distribution according to the following formula, obtains the approximate phase distribution of the sample to be measured, and further simulates the phase contrast imaging process to convert the approximate phase distribution of the sample to be measured into the enhanced image of the bright field microscope. Figure 3 The flow chart is shown. The light intensity distribution of the bright field digital microscope image of the sample to be measured is mapped into phase distribution, the approximate phase distribution of the sample to be measured is obtained, and the approximate phase distribution of the sample to be measured is further converted into the enhanced image of the bright field microscope. The method of the present application does not need the accurate registration between the annular diaphragm and the annular phase plate, reduces the requirement for the user, and improves the utilization rate of the illumination light energy.

[0039] The specific method is as follows:

[0040] Step 1: Obtain the physical parameters of the bright field microscope when the bright field digital microscope image is taken and send them to the computer system 10. The physical parameters are the central wavelength of the illumination light emitted by the white light source 1 and the numerical aperture of the imaging optical system NA , and the optical transfer function of the imaging optical system is obtained ;

[0041] Step 2: Obtain the light intensity distribution of the bright field digital microscope image of the sample to be measured by the digital image sensor 9 and send it to the computer system 10;

[0042] Step 3: The computer system 10 pre-processes the light intensity distribution of the bright field digital microscope image of the sample to be measured obtained by the bright field microscope according to the following formula: , wherein represents the light intensity distribution of the bright field digital microscope image after noise reduction, represents the average value of the noise of the bright field digital microscope image, and respectively represent the Fourier transform and the inverse Fourier transform, is the optical transfer function of the imaging optical system, , wherein represents the central wavelength of the illumination light, NA represents the numerical aperture of the imaging optical system,​ Indicates spatial frequency, Indicates the spatial frequency in the lateral direction. Indicates the spatial frequency in the longitudinal direction;

[0043] Step 4: The computer system 10 calculates the light intensity distribution of the denoised brightfield digital micrograph according to the following formula. Mapping to 0- π The phase region is used to obtain the approximate phase distribution of the sample under test. , ,in nor This indicates a normalization operation. mean This indicates calculating the average value;

[0044] Step 5: Computer system 10 according to Figure 1 The optical imaging model shown is based on a conventional optical Zernike phase-contrast microscope, which approximates the phase distribution of the sample under test. Enhanced images converted to bright-field microscopy The conversion relationship is as follows: ,in, Re This indicates that the real part information is taken. e Represents the natural constant. j Represents the imaginary unit. , and The parameters that affect the imaging effect of enhanced images from a bright-field microscope, and the imaging effect of the optical imaging model of a negative-optics Zernike phase-contrast microscope. The value is 0.1~0.5. for Regarding the imaging effect of the optical imaging model of the Zernike phase-contrast microscope with positive optics, The range is 2 to 10. for .

[0045] In this embodiment, the applicant conducted imaging experiments on a bright-field microscope of model NIB 950. Figure 4 and Figure 5 The imaging effects of the conventional optical Zernike phase-contrast microscope and the imaging effect of the method of this invention were compared for thin samples and thick samples, respectively.

[0046] Figure 4 It is an imaging comparison of thin samples of oral epithelial cells. Figure 4 In the figures, (a) is the original image, (b) is the image obtained using a conventional optical Zernike phase-contrast microscope, and (c) is the image obtained using the method of this invention. As can be seen from the figures, the method of this invention can significantly improve contrast, further avoid the influence of halos, and improve the resolution of cell edges.

[0047] Figure 5 is the imaging contrast of a thick sample mouse lung section. Figure 5 In the figure, (a) is the original image, (b) is the imaging image of the traditional optical Zernike phase contrast microscope, and (c) is the imaging image of the method of the present application. As can be seen from the figure, the imaging of the traditional optical Zernike phase contrast microscope completely covers the detailed information of the thick sample due to the existence of the halo, while the method of the present application realizes the enhanced imaging of the structural information of the sample to be measured through the normalization processing of the digital phase.

[0048] As shown in Figure 6~Figure 8 , the method of the present application can be used for the enhanced imaging of biological samples and metallographic samples, and the positive contrast enhanced microscopic imaging and the negative contrast enhanced microscopic imaging can be realized in the same system without changing the hardware.

[0049] Figure 6 is the imaging effect diagram of the method of the present application on a transparent cartilage section sample. Figure 6 In the figure, (a) is the original image, (b) is the positive contrast enhanced image obtained by the method of the present application, and (c) is the negative contrast enhanced image obtained by the method of the present application. As can be seen from the figure, Figure 6 , the contrast of the original image is relatively low, and the details are difficult to distinguish. However, the method of the present application can obtain the positive contrast enhanced image and the negative contrast enhanced image of the low-contrast transparent sample without changing the hardware, which is helpful for the user to quickly identify the boundary information and internal structure and other detailed information of the transparent cartilage section sample.

[0050] Figure 7 is the imaging effect diagram of the method of the present application on a human chromosome section sample. Figure 7 In the figure, (a) is the original image, (b) is the positive contrast enhanced image obtained by the method of the present application, and (c) is the negative contrast enhanced image obtained by the method of the present application. As can be seen from the figure, Figure 7 , the imaging effect of the method of the present application is also very good.

[0051] Figure 8 is the imaging effect diagram of the method of the present application on a scratch sample of a glass surface of a metallographic sample. Figure 8 In the figure, (a) is the original image, (b) is the positive contrast enhanced image obtained by the method of the present application, and (c) is the negative contrast enhanced image obtained by the method of the present application. As can be seen from the figure, Figure 8 , the imaging effect of the method of the present application on the metallographic sample is also very good.

[0052] Finally, it should be noted that the above examples are only used to illustrate the technical solutions and advantages of the present application, and are not intended to limit the same; although the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art will understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method of generating an enhanced image of a brightfield microscope, characterized by The method comprises the following specific steps: Step 1: obtaining the physical parameters of the bright field microscope when shooting the bright field digital microscopic image; Step 2: obtaining the light intensity distribution of the bright field digital microscopic image of the sample to be measured by using the bright field microscope, wherein the bright field microscope comprises an illumination optical system, a sample stage, an imaging optical system, a digital image sensor and a computer system, the illumination optical system emits illumination light and converges the illumination light on the sample to be measured on the sample stage to excite the diffraction light of the sample to be measured, the transmitted light after passing through the sample to be measured and the diffraction light of the sample to be measured pass through the imaging optical system together, then the light intensity distribution of the bright field digital microscopic image of the sample to be measured is obtained by the digital image sensor and sent to the computer system; Step 3: pre-processing the light intensity distribution of the bright field digital microscopic image of the sample to be measured according to the physical parameters of the bright field microscope to obtain the light intensity distribution of the bright field digital microscopic image after noise reduction; Step 4: mapping the light intensity distribution of the bright field digital microscopic image after noise reduction to the phase distribution to obtain the approximate phase distribution of the sample to be measured; Step 5: converting the approximate phase distribution of the sample to be measured into the enhanced image of the bright field microscope according to the optical imaging model of the positive optical Zernike phase contrast microscope and the optical imaging model of the negative optical Zernike phase contrast microscope.

2. A method of generating an enhanced image of a bright field microscope according to claim 1, characterized in that The physical parameters of the bright field microscope are the central wavelength and the numerical aperture of the illumination light of the bright field microscope, and further the optical transfer function of the bright field microscope is obtained for the pre-processing of step 3.

3. A method of generating an enhanced image of a brightfield microscope according to claim 2, wherein The specific method of step 3 is that the light intensity distribution of the bright field digital microscopic image of the sample to be measured obtained by the bright field microscope is subjected to Fourier transform, then the product of the Fourier transform result and the optical transfer function of the bright field microscope is subjected to inverse Fourier transform, and finally the average value of the noise floor of the bright field digital microscopic image is subtracted to obtain the light intensity distribution of the bright field digital microscopic image after noise reduction.

4. A method of generating an enhanced image of a brightfield microscope according to claim 3, wherein The specific method of the fourth step is to map the light intensity distribution of the noise-reduced bright field digital microscopic image to the phase region of 0- to obtain the approximate phase distribution of the sample to be measured.

5. A method of generating an enhanced image of a brightfield microscope according to claim 4, characterized in that The numerical aperture and the optical transfer function are the numerical aperture and the optical transfer function of the imaging optical system.

6. A method of generating an enhanced image of a brightfield microscope according to claim 5, wherein The actual physical size of a unit pixel of the bright field digital microscopic image obtained by the digital image sensor is less than half of the imaging resolution of the imaging optical system.

7. A method of generating an enhanced image of a brightfield microscope as claimed in claim 5, characterized in that The illumination optical system comprises a white light source, a collimating lens, a reflecting mirror and a converging lens, and the imaging optical system comprises an objective lens, a tube lens and an imaging lens, the white light source emits illumination light, the collimating lens collimates the illumination light into Gaussian-type parallel illumination light, the reflecting mirror adjusts the exit angle of the Gaussian-type parallel illumination light so that the Gaussian-type parallel illumination light is parallel to the optical axis, the converging lens converges the Gaussian-type parallel illumination light to directly illuminate the sample to be measured on the sample stage to excite the diffraction light of the sample to be measured, the transmitted light after passing through the sample to be measured and the diffraction light of the sample to be measured pass through the objective lens, the tube lens and the imaging lens together, and then the digital image sensor receives the image.

8. A method of generating an enhanced image of a brightfield microscope according to claim 7, characterized in that The white light source is an LED or a halogen lamp.

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