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KR-20260062657-A - HIGH-RESOLUTION OBLIQUE BACK ILLUMINATION MICROSCOPY AND IMAGE RESTORATION METHOD THEREOF

KR20260062657AKR 20260062657 AKR20260062657 AKR 20260062657AKR-20260062657-A

Abstract

A high-resolution backward-slanted microscope and a method for reconstructing images thereof are disclosed. The high-resolution backward-slanted microscope minimizes image distortion caused by optical aberrations and can obtain high-resolution images even in various scattering media.

Inventors

  • 주철민
  • 문태균
  • 윤경로

Assignees

  • 연세대학교 산학협력단

Dates

Publication Date
20260507
Application Date
20241029

Claims (13)

  1. A specimen forming part in which a specimen complex comprising a specimen to be observed and a scatterer formed around said specimen can be disposed; A light source capable of irradiating light onto the above-mentioned sample portion; A light processing unit comprising at least one beam splitter and configured such that light emitted by scattering from the specimen complex from light irradiated from the light source proceeds; A specimen area image sensor that senses light emitted in a first direction from the beam splitter above; A fill area image sensor that senses light emitted from the beam splitter and emitted in a second direction different from the first direction; and A processing unit comprising imaging the observation target specimen from an image obtained from the specimen area image sensor and the perforation area image sensor, High-resolution back-angle microscope.
  2. In paragraph 1, The above operation unit performs a first operation to derive a light source pattern from an image obtained from the above-mentioned fill area image sensor using the following formula (A). High-resolution backward-slanted microscope: (A) Here, u is a 2D spatial frequency coordinate, and I_pupil() is an image obtained from the above-mentioned pupil area image sensor, and S() is a light source pattern, and const is a constant, and |P()| is an ideal pupil function expressed by the following formula (B), and (B) Here, NA is the numerical aperture of the objective lens, and λ is the wavelength of the light source.
  3. In paragraph 2, The above operation unit performs a second operation to derive the transmission function of the specimen from the above-derived light source pattern through the following equation (C). High-resolution backward-slanted microscope: (C) Here, t() is the transfer function of the specimen, and u' is a 2D spatial frequency coordinate, and r is a 2D spatial coordinate, and F[] is the Fourier transform, and j represents the imaginary part, P() is a pupil function modeled through the following equation (D): (D) Here, z(m) is the weight (Zernike coefficient) of the m-th Zernike function, and Zm() is the m-th Zernike function.
  4. In paragraph 3, The above-described operation unit performs a third operation to derive the light absorption and light phase of the specimen from the above-described specimen transfer function through the following equation (E). High-resolution backward-slanted microscope: (E) Here, μ() is the light absorption of the specimen, and Φ() is the optical phase of the specimen.
  5. In paragraph 4, The above-mentioned operation unit performs a fourth operation to derive a predicted acquisition image through the following equation (F) from the light absorption and light phase of the specimen derived above. High-resolution backward-slanted microscope: (F) Here, ^I_l() is a predicted acquired image predicted under specific light irradiation condition l, and H_l^μ() and H_l^Φ() are the light absorption transfer function and the light phase transfer function, respectively, under specific light irradiation condition l.
  6. In paragraph 5, The above operation unit performs a fifth operation to derive the weight (Zernike coefficient) of the Zernike function of the pupil function through an optimization technique expressed by the following equation (G) from the above derived prediction acquisition image and the image obtained from the specimen area image sensor. High-resolution backward-slanted microscope: (G) Here, I_l() is the above-mentioned specimen area image sensor image obtained under specific light irradiation conditions l, and argmin is the argument of the minimum.
  7. In paragraph 6, The above optimization technique uses Gradient descent or the Nelder-Mead simplex method, High-resolution back-angle microscope.
  8. In Paragraph 7, The above operation unit updates the above-described function using the weights of the Zernike function obtained through the above-described fifth operation, and repeats the above-described fourth operation and the above-described fifth operation at least once. High-resolution back-angle microscope.
  9. In paragraph 8, The above specimen complex, the beam splitter, and the specimen area image sensor are formed on a first axis, High-resolution back-angle microscope.
  10. In Paragraph 9, The light processing unit comprises one or more lenses formed on the first axis, High-resolution back-angle microscope.
  11. In Paragraph 9, At least some of the constituent members of the above specimen complex, the above specimen area image sensor, or the above light processing unit are movable forward and backward on the first axis, High-resolution back-angle microscope.
  12. In Paragraph 11, The above-described operation unit performs operations on two or more different phases obtained by moving at least some of the components of the above-described specimen complex, the above-described specimen area image sensor, or the above-described light processing unit forward and backward along the first axis to image the specimen to be observed. High-resolution back-angle microscope.
  13. In Paragraph 12, The above-mentioned operation unit visualizes three-dimensional physical quantity information regarding the specimen, High-resolution back-angle microscope.

Description

High-resolution back-oblique microscope and image restoration method thereof The present invention relates to a high-resolution backward-slanted microscope and a method for reconstructing images thereof. Oblique back illumination microscopy (OBM) is a representative technique capable of imaging specimens in two or three dimensions using a reflective method, such as thick or reflective specimens that are difficult to access with transmission light microscopes. Other reflective imaging techniques, such as optical coherence tomography (OCT) and reflective confocal microscopy (RCM), can also image such specimens, but this is only possible if the material has a significant difference in refractive index along the depth direction. However, OBM is a technique that can image materials with relatively weak scattering characteristics, such as biological samples, without such limitations. For this reason, OBM technology has been utilized for the analysis of various biological specimens within scattering media. However, OBM technology has disadvantages, such as degradation of resolution and image quality due to optical aberrations and the need for additional simulations to predict light source patterns, which need to be overcome. In OBM imaging, problems arise where resolution is degraded and the image of the specimen is distorted due to optical aberrations inherent in the optical system itself and sample-induced aberrations caused by scattering within the biological specimen. A representative method to address this is hardware-based optical aberration correction (Hardware adaptive optics), which measures wavefront distortion using a wavefront sensor and directly corrects it using equipment such as a deformable mirror. However, this method has the disadvantage of high system complexity and the need for expensive equipment. On the other hand, computational optical aberration correction (Computational adaptive optics) expresses the relationship between the specimen and the image as an optical aberration function and corrects optical aberrations through an arithmetic algorithm; compared to hardware-based methods, this approach has the advantage of allowing for a simpler and more affordable optical system configuration. Conventional OBM technology requires performing simulations such as Monte-Carlo to predict light irradiation patterns, but the present invention does not require additional simulations because it directly predicts the light source pattern using an image sensor in the pupil area. Furthermore, conventional light source pattern prediction simulations require optical information regarding the scattering medium, making it difficult to apply OBM technology when such information is unavailable; however, the light source pattern image acquisition method of the present invention overcomes this limitation, enabling imaging of specimens located within any scattering medium. This invention proposes two directions of advancement from existing OBM technology for high-resolution imaging using a reflective back-tilt microscope. First, high-resolution images are acquired by combining an arithmetic optical aberration restoration method. Second, specimens within any scattering medium can be imaged by directly acquiring the light source pattern in the pupil area. This enables the application of OBM imaging technology in a wider range of environments and allows for the acquisition of high-resolution images of improved quality through optical aberration correction. Figure 1 shows (a) a schematic diagram of an optical system and a structure composed of a light source and a pupil modulation imaging system. (b1-b2) illustrates a method in which the light source irradiates light onto a specimen, allowing for a detailed explanation of how light is irradiated onto the specimen. Figure 2 illustrates the process of light irradiation into a scattering medium and visually explains the process in which multiple scattered light within the scattering medium is scattered backward to illuminate the specimen in a transmissive manner. Figure 3 illustrates axial scanning in a 3D OBM image and shows that multiple components can move in the manner of ①-④ to obtain data from various focal planes of the specimen. Figure 4 shows a schematic diagram of the optical aberration restoration algorithm to visually explain the process of aberration restoration. Figure 5 shows the results of optical aberration restoration and correction in OBM images to which the present technology is applied. (a) shows the results of aberration correction in a 2D OBM image of a mouse retinal specimen, and (b) shows the results of aberration correction in a 3D OBM image of a Siemens phase target specimen, visually demonstrating that this technology can help improve image quality by correcting optical aberrations. Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. Since the present invention is susceptible to v