Method for generating enhanced image of bright field microscope

Abstract

The invention discloses a method for generating an enhanced image of a bright-field microscope, and the method comprises the steps: simulating an optical imaging model of optical Zernike phase contrast microscope imaging through a computer system based on the optical imaging principle of a conventional optical Zernike phase contrast microscope; extra hardware such as an annular diaphragm and an annular phase plate does not need to be added in an illumination light path and an imaging light path of the bright-field microscope, enhancement of the phase contrast of a bright-field digital microscopic image is achieved, energy loss of an illumination optical system is avoided, and the design and manufacturing cost of the microscopic optical system is greatly reduced; the optical Zernike phase contrast microscope can be universally used for different bright field microscopes, halo artifacts caused by interference effects in a traditional optical Zernike phase contrast microscope do not exist, finer structural features in biological cells can be distinguished, the resolution of the optical Zernike phase contrast microscope can be close to the resolution limit of objective lens design, and the technical requirements for users are greatly reduced.

Description

Technical Field

[0001] The present invention relates to a method for generating an enhanced image, in particular to a method for generating an enhanced image of a bright field microscope. Background Art

[0002] In basic research in biology, medicine, materials, etc., in order to achieve high contrast observation of transparent samples such as biological cells and scratches on glass surfaces, it is usually necessary to enhance the image of the bright field microscope. Currently, the commonly used methods for observing samples are optical Zernike phase contrast microscope and optical differential interference microscope. The structure of the traditional optical Zernike phase contrast microscope is as follows: Figure 1 As shown, a white light source 1 (such as an LED or a 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 adjusted at its exit angle by a reflector 3 so that the beam is parallel to the optical axis. The Gaussian parallel beam passes through an annular aperture 11 and becomes an annular beam. It is then focused by a converging lens 4 to illuminate a sample on a sample stage 5, thereby stimulating diffracted light from the sample. The annular transmitted light and the diffracted light from the sample are collected by an objective lens 6. After passing through an annular phase plate 12 in the objective lens 6, a relative phase delay of π / 2 or 3π / 2 is generated between the annular transmitted light and the diffracted light from the sample. The annular transmitted light and the diffracted light from the sample with relative phase delay pass through a tube lens 7 and an imaging lens 8, whereupon interference occurs at an imaging plane where a digital image sensor 9 is located to produce an enhanced sample microscopic image signal. The digital image sensor 9 sends the recorded digital signal to a computer system 10 for displaying and storing the contrast-enhanced sample microscopic image.

[0003] Traditional methods incorporate an annular aperture and annular phase plate into brightfield microscopy to alter the phase difference between diffracted light and transmitted light passing through the sample, converting minute phase changes in weakly absorbing transparent objects into variations in light intensity, thereby improving the visibility of structures. However, when observing samples with an optical Zernike phase contrast microscope, halo artifacts are present at the sample edges, making it only suitable for observing thinner samples. Furthermore, to avoid degradation in image quality, precise alignment of the conjugate positions between the annular aperture and the annular phase plate is required, placing high demands on the microscope user. Furthermore, the introduction of an annular aperture blocks most of the illumination beam, resulting in significant energy loss in the illumination system. 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 bright-field microscopes suitable for thick samples, which can enhance the imaging contrast of weakly absorbing objects such as biological cells without the need for additional hardware such as annular apertures and annular phase plates, avoid energy loss of the illumination optical system, and is suitable for use with thick samples.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a method for generating an enhanced image of a bright field microscope, comprising the following specific steps: Step 1: Obtain the physical parameters of the bright field microscope when taking bright field digital microscopic images; Step 2: using the bright field microscope to obtain the light intensity distribution of the bright field digital microscopic image of the sample to be tested; 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: Map the light intensity distribution of the de-noised bright-field digital microscope image into a phase distribution to obtain an approximate phase distribution of the sample to be tested; Step 5: According to the optical imaging model of positive optical Zernike phase contrast microscope and the optical imaging model of negative optical Zernike phase contrast microscope, the approximate phase distribution of the sample to be measured is converted into an enhanced image of bright field microscope.

[0006] Compared with the existing technology, the advantage of the present invention is that it is based on the optical imaging principle of the traditional optical Zernike phase contrast microscope, and simulates the optical imaging model of the optical Zernike phase contrast microscope through a computer system. There is no need to add additional hardware such as annular apertures and annular phase plates in the illumination and imaging light paths of the bright field microscope, thereby achieving phase contrast enhancement of bright field digital microscopic images, avoiding energy loss of the illumination optical system, and greatly reducing the design and manufacturing costs of the microscopic optical system. The present invention does not rely on additional optical elements and is therefore applicable to different bright field microscopes. The present invention achieves phase contrast enhancement microscopic effects through digital phase shifting in the frequency domain, so there is no halo artifact caused by interference effects in traditional optical Zernike phase contrast microscopes, and finer structural features in biological cells can be distinguished, and its resolution can be close to the resolution limit of the objective lens design. The technical solution of the present invention does not require complex debugging of the illumination optical system, which greatly reduces the technical requirements for users.

[0007] Compared with the existing optical Zernike phase contrast microscope, the method of the present invention has the following beneficial effects: (1) It can be used in bright field illumination digital microscopy imaging systems without the need for additional complex optical components and debugging, reducing costs while making the operation of label-free microscopy more convenient; (2) Due to diffraction and halo effects, the resolution of a conventional optical Zernike phase contrast microscope is usually not as good as that of a bright field microscope of the same specification. However, the method of the present invention is a digitally enhanced microscopic imaging method based on a bright field microscope, so its resolution is consistent with that of a bright field image, and fine structures can be better distinguished. (3) Traditional optical Zernike phase contrast microscopes are only suitable for imaging thin samples. When imaging thick samples, the imaging contrast will decrease due to the halo problem. However, the method of the present invention can still achieve phase contrast enhancement for thick samples through phase mapping.

[0008] The physical parameters of the bright field microscope are the central wavelength and numerical aperture of the illumination light of the bright field microscope, and the optical transfer function of the bright field microscope is obtained for the preprocessing of step three.

[0009] Preferably, the specific method of step three is to perform Fourier transform on the light intensity distribution of the bright field digital microscopic image of the sample to be tested obtained by the bright field microscope, then perform 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 microscopic image to obtain the light intensity distribution of the bright field digital microscopic image after noise reduction, as follows: The light intensity distribution of the bright field digital microscopic image of the sample to be tested obtained by the bright field microscope is calculated according to the following formula: Perform preprocessing: ,in, represents the light intensity distribution of the bright field digital microscope image after noise reduction, Represents the average noise floor of bright field digital microscope images, and represent Fourier transform and inverse Fourier transform respectively, is the optical transfer function of the brightfield microscope, , where represents the central wavelength of the illumination light, NA represents the numerical aperture, represents the spatial frequency, represents the horizontal spatial frequency, Indicates the longitudinal spatial frequency.

[0010] Preferably, the specific method of step 4 is: The light intensity distribution of the bright field digital microscope image after noise reduction is calculated according to the following formula Mapped to 0- π Phase area, obtain the approximate phase distribution of the sample to be tested : ,in nor represents the normalization operation, mean Indicates finding the average value.

[0011] The brightfield microscope is composed 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 diffracted light of the sample to be tested. The numerical aperture and optical transfer function are the numerical aperture and optical transfer function of the imaging optical system. After the transmitted illumination light passes through the sample to be tested and the diffracted light of the sample to be tested passes through the imaging optical system together, the digital image sensor obtains the light intensity distribution of the brightfield digital microscopic image of the sample to be tested and sends it to the computer system.

[0012] Preferably, the actual physical size of a unit pixel of the bright field digital microscopic image acquired by the digital image sensor is less than half of the imaging resolution of the imaging optical system.

[0013] The illumination optical system is composed of a white light source, a collimating lens, a reflector and a converging lens. The imaging optical system is composed of an objective lens, a tube lens and an imaging lens. The white light source emits illumination light, which is collimated into Gaussian parallel illumination light after passing through the collimating lens. The collimated Gaussian parallel illumination light is adjusted to an emission angle by the reflector so that the Gaussian parallel illumination light is parallel to the optical axis. The Gaussian parallel illumination light is directly converged to illuminate the sample to be tested placed on the sample stage after passing through the converging lens to stimulate diffraction light of the sample to be tested. The transmitted illumination light after passing through the sample to be tested and the diffraction light of the sample to be tested pass through the objective lens, the tube lens and the imaging lens together, and are received by the digital image sensor for imaging.

[0014] Preferably, the white light source is an LED or a halogen lamp. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram of the structure of a traditional optical Zernike phase contrast microscope; Figure 2 A schematic diagram of the structure of a bright field microscope used in the method of the present invention; Figure 3 A flow chart of a method for generating an enhanced image of a bright field microscope of the present invention; Figure 4 A comparison diagram of the imaging of thin samples by the prior art and the method of the present invention; Figure 5 A comparison diagram of the imaging of thick samples by the prior art and the method of the present invention; Figure 6 This is an image showing the imaging effect of the method of the present invention on a hyaline cartilage slice sample; Figure 7 This is an image showing the imaging effect of the method of the present invention on a human chromosome section sample; Figure 8 This is an imaging effect diagram of a scratched sample on the surface of a metallographic sample glass using the method of the present invention.

[0016] Description of reference numerals: 1. White light source; 2. Collimating lens; 3. Reflector; 4. Converging lens; 5. Sample stage; 6. Objective lens; 7. Tubular lens; 8. Imaging lens; 9. Digital image sensor; 10. Computer system; 11. Annular aperture; 12. Annular phase plate. DETAILED DESCRIPTION

[0017] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0019] The inventive concept of the present invention is to combine the physical mechanism of optical label-free phase contrast imaging with modern digital imaging technology, and simulate the phase contrast optical imaging process of a bright field microscope through a computer system, thereby achieving an enhancement effect on the digital image of a label-free transparent sample without the need for an optical phase contrast element.

[0020] Figure 2 The figure shows a schematic diagram of the structure of the bright field microscope used in the method of the present invention. A white light source 1 emits illumination light, which can usually be an LED or a halogen lamp. The white light source 1 is collimated into Gaussian parallel illumination light by a collimating lens 2. The collimated Gaussian parallel illumination light is adjusted to its emission angle by a reflector 3 so that the Gaussian parallel illumination light is parallel to the optical axis. The Gaussian parallel illumination light is directly converged by a converging lens 4 to illuminate the sample to be tested placed on the sample stage 5, thereby stimulating diffraction light of the sample to be tested. The transmitted illumination light after passing through the sample to be tested and the diffraction light of the sample to be tested pass through the objective lens 6, the tube lens 7 and the imaging lens 8, and are then received by the digital image sensor 9 for imaging. The digital image sensor 9 obtains the light intensity distribution of the bright field digital microscope image of the sample to be tested and sends it to the computer system 10. The computer system 10 calculates the intensity of the image according to the image. Figure 3 The process shown maps the light intensity distribution of a brightfield digital microscopy image of the sample to be measured into a phase distribution, obtaining an approximate phase distribution of the sample. Phase contrast imaging is then further simulated to convert this approximate phase distribution into an enhanced image for brightfield microscopy. This method eliminates the need for precise alignment between the annular aperture and the annular phase plate, reducing user requirements and improving the utilization of illumination light energy.

[0021] The specific method is: Step 1: Obtain the physical parameters of the bright field microscope when taking bright field digital microscopic images and transmit 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 thus the optical transfer function of the imaging optical system is obtained ; Step 2: Obtain the light intensity distribution of the bright field digital microscope image of the sample to be tested by the digital image sensor 9 and transmit it to the computer system 10; Step 3: The computer system 10 calculates the light intensity distribution of the bright field digital microscopic image of the sample to be tested obtained by the bright field microscope according to the following formula: Perform preprocessing: ,in, represents the light intensity distribution of the bright field digital microscope image after noise reduction, Represents the average noise floor of bright field digital microscope images, and represent Fourier transform and inverse Fourier transform respectively, is the optical transfer function of the imaging optical system, , where represents the central wavelength of the illumination light, NA represents the numerical aperture of the imaging optical system, represents the spatial frequency, represents the horizontal spatial frequency, Indicates the longitudinal spatial frequency; Step 4: The computer system 10 converts the light intensity distribution of the bright field digital microscope image after noise reduction into Mapped to 0- π Phase area, obtain the approximate phase distribution of the sample to be tested , ,in nor represents the normalization operation, mean Indicates finding the average value; Step 5: The computer system 10 Figure 1 The optical imaging model of the traditional optical Zernike phase contrast microscope shown in the figure approximates the phase distribution of the sample to be measured. Converting to enhanced images for brightfield microscopy , and its conversion relationship is: ,in, Re Indicates taking the real part information, e represents a natural constant, j represents the imaginary unit, , and Parameters that affect the imaging effect of the enhanced image of the bright field microscope, and the imaging effect of the optical imaging model of the negative optical Zernike phase contrast microscope, 0.1~0.5, for ; For the imaging effect of the optical imaging model of the positive optical Zernike phase contrast microscope, 2~10, for .

[0022] In this embodiment, the applicant conducted imaging experiments on a bright field microscope model NIB 950. Figure 4 and Figure 5 The imaging effects of the traditional optical Zernike phase contrast microscope and the imaging effects of the method of the present invention were compared for thin samples and thick samples respectively.

[0023] Figure 4 This is an imaging comparison of thin samples of oral epithelial cells. Figure 4 In the figure, (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 the present invention. As can be seen from the figures, the method of the present invention significantly improves contrast, further avoids the effects of haloing, and enhances the ability to resolve cell edges.

[0024] Figure 5 This is a comparison of imaging of thick sample mouse lung sections. Figure 5 In the figure, (a) is the original image, (b) is the image obtained using a conventional Zernike phase contrast microscope, and (c) is the image obtained using the method of the present invention. As can be seen from the figures, the presence of halo in conventional Zernike phase contrast microscope images completely obscures detailed information about thick samples. However, the method of the present invention, through digital phase normalization, achieves enhanced imaging of the sample's structural information.

[0025] like Figures 6 to 8 As shown, the method of the present invention can be generally applied to enhanced imaging of biological samples and metallographic samples, and positive phase contrast enhanced microscopy imaging and negative phase contrast enhanced microscopy imaging can be realized in the same system without changing the hardware.

[0026] Figure 6 This is an image showing the imaging effect of the method of the present invention on a hyaline cartilage slice sample. Figure 6 In the figure, (a) is the original image, (b) is the enhanced image of positive phase contrast obtained by the method of the present invention, and (c) is the enhanced image of negative phase contrast obtained by the method of the present invention. Figure 6As can be seen in the original image, the contrast is relatively low, making details difficult to discern. However, the method of the present invention can generate positive and negative phase contrast enhanced images of low-contrast transparent samples without changing hardware, helping users quickly identify details such as the boundaries and internal structure of hyaline cartilage slices.

[0027] Figure 7 This is an image showing the imaging effect of the method of the present invention on a human chromosome section sample. Figure 7 In the figure, (a) is the original image, (b) is the enhanced image of positive phase contrast obtained by the method of the present invention, and (c) is the enhanced image of negative phase contrast obtained by the method of the present invention. Figure 7 It can be seen from the figure that the imaging effect of the method of the present invention is also very good.

[0028] Figure 8 This is an imaging effect diagram of a scratch sample on the surface of a metallographic sample glass using the method of the present invention. Figure 8 In the figure, (a) is the original image, (b) is the enhanced image of positive phase contrast obtained by the method of the present invention, and (c) is the enhanced image of negative phase contrast obtained by the method of the present invention. Figure 8 It can be seen from the figure that the method of the present invention also has a good effect on the enhanced imaging of metallographic samples.

[0029] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions and beneficial effects of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for generating an enhanced image of a bright field microscope, characterized in that The specific steps include: Step 1: Obtain the physical parameters of the bright field microscope when taking bright field digital microscopic images; Step 2: using the bright field microscope to obtain the light intensity distribution of the bright field digital microscopic image of the sample to be tested; 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: Map the light intensity distribution of the de-noised bright-field digital microscope image into a phase distribution to obtain an approximate phase distribution of the sample to be tested; Step 5: According to the optical imaging model of positive optical Zernike phase contrast microscope and the optical imaging model of negative optical Zernike phase contrast microscope, the approximate phase distribution of the sample to be measured is converted into an enhanced image of bright field microscope.

2. The method for generating an enhanced image of a bright field microscope according to claim 1, wherein The physical parameters of the bright field microscope are the central wavelength and numerical aperture of the illumination light of the bright field microscope, and the optical transfer function of the bright field microscope is obtained for the preprocessing of step three.

3. The method for generating an enhanced image of a bright field microscope according to claim 2, wherein The specific method of step three is to perform Fourier transform on the light intensity distribution of the bright field digital microscopic image of the sample to be tested obtained by the bright field microscope, and then perform inverse Fourier transform on the product of the above Fourier transform result and the optical transfer function of the bright field microscope, and then subtract the background noise average value of the bright field digital microscopic image to obtain the light intensity distribution of the bright field digital microscopic image after noise reduction.

4. The method for generating an enhanced image of a bright field microscope according to claim 3, wherein The specific method of step 4 is to map the light intensity distribution of the bright field digital microscope image after noise reduction to 0- π The phase region of the sample to be tested is obtained by calculating the approximate phase distribution of the sample to be tested.

5. The method for generating an enhanced image of a bright field microscope according to claim 4, characterized in that The brightfield microscope is composed 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 diffracted light of the sample to be tested. The numerical aperture and optical transfer function are the numerical aperture and optical transfer function of the imaging optical system. After the transmitted illumination light passes through the sample to be tested and the diffracted light of the sample to be tested passes through the imaging optical system together, the digital image sensor obtains the light intensity distribution of the brightfield digital microscopic image of the sample to be tested and sends it to the computer system.

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

7. The method for generating an enhanced image of a bright field microscope according to claim 5, wherein The illumination optical system is composed of a white light source, a collimating lens, a reflector and a converging lens. The imaging optical system is composed of an objective lens, a tube lens and an imaging lens. The white light source emits illumination light, which is collimated into Gaussian parallel illumination light after passing through the collimating lens. The collimated Gaussian parallel illumination light is adjusted to an emission angle by the reflector so that the Gaussian parallel illumination light is parallel to the optical axis. The Gaussian parallel illumination light is directly converged to illuminate the sample to be tested placed on the sample stage after passing through the converging lens to stimulate diffraction light of the sample to be tested. The transmitted illumination light after passing through the sample to be tested and the diffraction light of the sample to be tested pass through the objective lens, the tube lens and the imaging lens together, and are received by the digital image sensor for imaging.

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

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