阅读说明：本技术 一种高对比度编码显微成像系统及方法 (High-contrast coding microscopic imaging system and method ) 是由 陈硕 路交 王大珩 于 2020-04-27 设计创作，主要内容包括：本发明属于光学领域,公开了一种高对比度编码显微成像系统及方法,系统包括编码光源模块和显微成像模块。其中,编码光源模块包含白光光源、光纤、扩束镜、光栅、消色差透镜、数字微镜器件、透镜组、锥型光纤及计算机,用于实现任意输出光源光谱的编码光源；显微成像模块包含载物台、物镜、透镜、相机以及计算机,用于采集编码显微图像。该方法利用光谱数据后处理方法计算可实现高对比度显微成像所需的波长位置以及该波长对于提高对比度的权重,随后通过控制编码光源模块实现对应的光源输出光谱,最后通过在该编码光源照明下成像获得高对比度的编码显微图像。(The invention belongs to the field of optics and discloses a high-contrast coding microscopic imaging system and a high-contrast coding microscopic imaging method. The encoding light source module comprises a white light source, an optical fiber, a beam expander, a grating, an achromatic lens, a digital micromirror device, a lens group, a tapered optical fiber and a computer, and is used for realizing an encoding light source of any output light source spectrum; the microscopic imaging module comprises an object stage, an objective lens, a camera and a computer and is used for acquiring a coded microscopic image. The method utilizes a spectrum data post-processing method to calculate the wavelength position required by realizing high-contrast microscopic imaging and the weight of the wavelength on improving the contrast, then realizes the corresponding light source output spectrum by controlling a coding light source module, and finally obtains a high-contrast coding microscopic image by imaging under the illumination of the coding light source.)

1. A high-contrast coding microscopic imaging system is characterized by comprising a coding light source module, a microscopic imaging module and a computer (13); the encoding light source module comprises a white light source (1), an optical fiber (2), a beam expander (3), a grating (4), an achromatic lens (5), a digital micromirror device (6), a lens group (7) and a tapered optical fiber (8) and is used for outputting an encoding light source; the microscopic imaging module comprises an object stage (9), an objective lens (10), a lens (11) and a camera (12) and is used for acquiring a coding microscopic image; white light emitted by a white light source (1) in the coding light source module forms parallel beams after passing through an optical fiber (2) and a beam expander (3), is split by a grating (4), then is converged on the same micromirror unit of a digital micromirror device (6) by an achromatic lens (5), and is reflected by the digital micromirror device (6) and then is coupled into a single beam of light by a lens group (7) and a tapered optical fiber (8); the angle of each micro-mirror unit on the digital micro-mirror device (6) is controlled by a computer (13) and is used for outputting light with specified wavelength to form coded light and irradiating the coded light on a sample; a sample is placed on the objective table (9) in the microscopic imaging module and imaged by the objective lens (10), and after the magnification of the sample is adjusted by the lens (11), a coding microscopic image is collected by the camera (12) and displayed on the computer (13).

2. A high contrast coded microscopic imaging system according to claim 1, characterized in that in the coded light source module, the light converged by the lens group (7) is further condensed and collimated by the tapered optical fiber (8) for improving the intensity and uniformity of the illumination on the sample; one end of the tapered optical fiber is formed by a plurality of optical fibers into an optical fiber bundle with a thicker diameter, and is used for collecting coded light as much as possible; the other end is a single optical fiber with relatively thin diameter and is used for realizing high-intensity and uniform illumination; the optical fiber bundle is connected with the single optical fiber through an optical cone and used for shrinking the light beam.

3. The method for high-contrast coded microscopic imaging by adopting the system of claim 1 or 2 is characterized in that the high-spectrum data with label information and the spectral data post-processing method are utilized, the spectral data post-processing method is converted into a one-dimensional vector through a series of linear operations, the wavelength position required by the high-contrast microscopic imaging and the weight of the wavelength for improving the contrast are further obtained, a coding matrix for controlling the digital micromirror device (6) is generated, the coding light source module is controlled to output a coding light source capable of realizing the high-contrast coded microscopic imaging based on the coding matrix, and the microscopic imaging module is utilized to acquire microscopic images under the illumination of the coding light source, namely the high-contrast coded microscopic images are obtained.

4. The method of claim 3, wherein the step of acquiring the high contrast encoded microscope image under illumination by the encoded light source comprises:

step one, obtaining a transformation vector U by utilizing principal component analysis₁(ii) a Calculating a covariance matrix D of the hyperspectral data H of the sample, arranging the covariance matrix D according to the eigenvalues from large to small, and taking the first eigenvectors of a specific number as transformation vectors U₁And calculating a corresponding characteristic value s;

step two, obtaining a transformation vector U by utilizing a linear discrimination method₂(ii) a Calculating an intra-class divergence matrix M based on the eigenvalue s and the label matrix Y obtained in the step one_inAnd the interspecies divergence matrix M_outThen transform the vector U₂Is M_inInverse matrix of and M_outThe product of (a);

step three, calculating the wavelength position required for realizing high-contrast microscopic imaging and the weight of the wavelength for improving the contrast, and generating a coding matrix T-U for controlling the digital micro-mirror device (6)₁U₂；

If the coding matrix T is not negative, directly generating an imaging coding light source by using a coding light source module, and acquiring a microscopic image under the illumination of the coding light source by using a microscopic imaging module to obtain a high-contrast coding microscopic image; if negative values exist in the coding matrix T, decomposing the coding matrix T into non-negative imaging coding matrix T₁And a non-negative compensation coding matrix T₂(ii) a Wherein the coding matrix T is compensated₂The value of each element in the coding matrix T is equal to the absolute value of the minimum value in the coding matrix T, and the coding matrix T and the imaging coding matrix T₁And compensating the coding matrix T₂Satisfy T ═ T₁-T₂Continuing to execute the step five;

step five, according to the imaging coding matrix T in the step four₁And compensating the coding matrix T₂Controlling the coding light source module to respectively generate an imaging coding light source and a compensation coding light source;

sixthly, respectively obtaining microscopic images I under the illumination of the imaging coding light source by using the microscopic imaging module₁And compensating the microscopic image under the illumination of the coded light source I₂；

Seventhly, the microscopic image I of the sample collected under the illumination of the imaging coding light source₁And compensating the microscopic image I of the sample acquired under the illumination of the coding light source₂Subtraction can be equivalent to a high contrast sample encoded microscopic image acquired under illumination by an encoding light source.

5. The method of claim 3, wherein acquiring the high contrast encoded microscope image under illumination by the encoded light source comprises the steps of:

step one, calculating wavelength position required by high-contrast microscopic imaging by utilizing a pseudo-inverse method based on sample hyperspectral data H and a label matrix YAnd setting the weight of the wavelength to improve the contrast, and generating an encoding matrix T for controlling the digital micromirror device (6), wherein the encoding matrix T is YH^T(HH^T)^-1；

If the coding matrix T is not negative, directly generating an imaging coding light source by using a coding light source module, and acquiring a microscopic image under the illumination of the coding light source by using a microscopic imaging module to obtain a high-contrast coding microscopic image; if negative values exist in the coding matrix T, decomposing the coding matrix T into non-negative imaging coding matrix T₁And a non-negative compensation coding matrix T₂(ii) a Wherein the coding matrix T is compensated₂The value of each element in the coding matrix T is equal to the absolute value of the minimum value in the coding matrix T, and the coding matrix T and the imaging coding matrix T₁And compensating the coding matrix T₂Satisfy T ═ T₁-T₂Continuing to execute the third step;

step three, encoding the matrix T according to the imaging in the step four₁And compensating the coding matrix T₂Controlling the coding light source module to respectively generate an imaging coding light source and a compensation coding light source;

step four, respectively obtaining a microscopic image I under the illumination of an imaging coding light source by using a microscopic imaging module₁And compensating the microscopic image under the illumination of the coded light source I₂；

Fifthly, the microscopic image I of the sample collected under the illumination of the imaging coding light source₁And compensating the microscopic image I of the sample acquired under the illumination of the coding light source₂Subtraction can be equivalent to a high contrast sample encoded microscopic image acquired under illumination by an encoding light source.

Technical Field

The invention belongs to the field of optics, and relates to a high-contrast coding microscopic imaging system and method.

Background

Optical microscopy imaging can provide information about the microstructure of a sample and has been widely used in many fields such as biology, medicine, and material science. The contrast is one of the key technical indexes for measuring the imaging quality of the optical microscopic imaging system, and is always a hot problem for the research in the field of microscopic imaging. Due to the selective absorption of light by the sample to be measured, that is, the absorption of light at different wavelengths by the composition components at different positions in the sample to be measured is different, the contrast of the microscopic images acquired at different wavelengths generally has significant difference. However, due to the complexity of the components of biological samples, it is often difficult to achieve high imaging contrast using only microscopic imaging at a single wavelength, and complex color light sources are often spectrally fixed and also difficult to use as illumination sources for high contrast microscopic imaging. Therefore, what kind of light source is used to output the spectrum of the complex color light source can realize high contrast imaging, and how to really realize the spectrum of the light source output the complex color light source is a critical technical bottleneck to be solved urgently for realizing high contrast optical microscopic imaging.

Disclosure of Invention

The invention provides a high-contrast coding microscopic imaging system and a high-contrast coding microscopic imaging method for realizing high-contrast optical microscopic imaging.

The specific scheme of the invention is as follows: a high-contrast coding microscopic imaging system comprises a coding light source module, a microscopic imaging module and a computer; the encoding light source module comprises a white light source, an optical fiber, a beam expander, a grating, an achromatic lens, a digital micromirror device, a lens group and a tapered optical fiber and is used for outputting an encoding light source; the microscopic imaging module comprises an object stage, an objective lens, a lens and a camera and is used for collecting a coded microscopic image; white light emitted by the white light source in the coding light source module forms parallel beams after passing through the optical fiber and the beam expander, light with the same wavelength is converged on the same micromirror unit of the digital micromirror device by the achromatic lens after being split by the grating, and is coupled into single-beam light by the lens group and the tapered optical fiber after being reflected by the digital micromirror device; controlling the angle of each micro-mirror unit on the digital micro-mirror device through a computer, outputting light with specified wavelength to form coded light, and irradiating the coded light onto a sample; and a sample is placed on the objective table in the microscopic imaging module and imaged by the objective lens, and after the magnification of the sample is adjusted by the lens, a coded microscopic image is collected by the camera and displayed on a computer.

In the coding light source module, the light converged by the lens group is further condensed and collimated by the conical optical fiber, so that the illumination intensity and uniformity irradiated on a sample are improved; one end of the tapered optical fiber is formed by a plurality of optical fibers into an optical fiber bundle with a thicker diameter, and is used for collecting coded light as much as possible; the other end is a single optical fiber with relatively thin diameter and is used for realizing high-intensity and uniform illumination; the optical fiber bundle is connected with the single optical fiber through an optical cone and used for shrinking the light beam.

The method for high-contrast coded microscopic imaging by adopting the system comprises the steps of converting a spectral data post-processing method into a one-dimensional vector through a series of linear operations by utilizing a hyperspectral data with label information and a spectral data post-processing method, further obtaining a wavelength position required by the high-contrast microscopic imaging and the weight of the wavelength on improving the contrast, generating a coding matrix for controlling a digital micromirror device, controlling the digital micromirror device to output a coding light source capable of realizing the high-contrast coded microscopic imaging based on the coding matrix, and acquiring a microscopic image under the illumination of the coding light source by utilizing a microscopic imaging module, namely obtaining the high-contrast coded microscopic image.

The invention has the beneficial effects that: the invention provides a high-contrast coding microscopic imaging system and a high-contrast coding microscopic imaging method.

Drawings

FIG. 1 is an optical path diagram of a high contrast encoded microscopic imaging system of the present invention;

in the figure: 1 a white light source; 2, an optical fiber; 3 a beam expander; 4, grating; 5 an achromatic lens; 6 digital micromirror device; 7 lens groups; 8 a tapered optical fiber; 9 an object stage; 10 objective lens; 11 a lens; 12 a camera; 13 computer.

FIG. 2 shows the result of coded microscopy imaging on osteoblast samples using a high contrast coded microscopy imaging system according to example 1; (a) is an osteoblast microscopic image collected by white light illumination; (b) is a spectrogram of an imaging coding light source and a compensation coding light source for obtaining a high-contrast microscopic image; (c) is equivalent to a high-contrast osteoblast coding microscopic image acquired under the illumination of a coding light source; (d) the average cross-sectional diagram is normalized by the cell boundaries in the area circled by the dotted lines under white light illumination and under coded light source illumination.

Detailed Description

The following detailed description of embodiments of the invention refers to the accompanying drawings.