SHG confocal endoscopic imaging system based on structured light

Abstract

The invention discloses an SHG confocal endoscopic imaging system based on structured light. The SHG confocal endoscopic imaging system comprises a laser light source used for generating exciting light; the half-wave plate is placed on a light path of exciting light emitted by the exciting light source; the electro-optical modulator is placed on a light path after the exciting light passes through the half-wave plate; the excitation light filter is placed on a light path of the excitation light modulated by the electro-optical modulator; the dichroic mirror is placed on a light path after the exciting light passes through the exciting light filter; the first objective lens is placed on a light path after the exciting light passes through the dichroic mirror; and the confocal pinhole is placed on a light path formed after the exciting light is converged by the first objective lens. The SHG confocal endoscopic imaging system based on the structured light can make up for the high imaging resolution and the optical layer cutting capability to thicker samples which are not possessed by the existing fluorescent confocal microscopic imaging system, and solves the technical problem that imaging cannot be performed when some smaller biological cells and thicker samples are encountered.

Description

Technical Field

[0001] The present invention relates to the technical field of confocal endoscopic imaging, and particularly to a SHG confocal endoscopic imaging system based on structured light. Background Art

[0002] The second harmonic generation process (SHG) is a second-order nonlinear process. The SHG imaging technology is a label-free imaging technology for non-centrosymmetric biological tissues, with good optical sectioning ability and the ability to reveal the nonlinearity of materials, and has become one of the important research means in life science. During the SHG imaging process, the second harmonic intensity is proportional to the square of the laser intensity, has an excitation threshold, and almost all the photon energies excited are near the focus, so the detectable signals are all effective signals. Due to the existence of the diffraction limit, the SHG microscopy imaging technology cannot image fine structures below the diffraction limit.

[0003] In order to break through the limitation of the diffraction limit on imaging resolution, scientists have proposed a series of super-resolution imaging methods: stimulated emission depletion technology (STED), reversible saturable fluorescence transition microscopy (RSOLFT), stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), and wide-field structured light super-resolution microscopy (SIM). Among them, STED and RSOLFT consume the excited-state fluorescent molecules in the edge region of the excitation spot through stimulated loss or ground-state depletion and other spontaneous emission transition methods to narrow the fluorescence emission range, thereby improving the imaging resolution; STORM and PALM achieve super-resolution imaging by controlling the random emission of fluorescent molecules and combining the centroid localization algorithm; wide-field SIM generates a sinusoidal fringe pattern through grating diffraction or grid projection, and the sinusoidal fringe pattern is superimposed on the sample pattern to produce a Moiré fringe effect. The Moiré fringe effect can move the high-frequency information in the sample into the OTF passband, and then the high-frequency information of the sample can be detected, and a super-resolution image can be obtained through a specific algorithm. Compared with other technologies, SIM has the characteristics of specific fluorescent dyes and fast imaging speed, and thus has a broad application scenario.

[0004] A fluorescence confocal microscopy imaging system is a confocal microscopy imaging system that observes a sample by irradiating the sample with a laser to excite fluorescence and then receiving the fluorescence by a detector. Although the fluorescence confocal microscopy imaging system has high contrast and resolution, due to the absorption and scattering of visible light by biological tissues, the penetration depth of the laser in biological tissues is limited, so it is only used for in vitro cell imaging or tissue superficial imaging. Summary of the Invention

[0005] Aiming at the above technical problems, the present invention proposes a SHG confocal endoscopic imaging system based on structured light.

[0006] To achieve the above object, the present invention adopts the following technical solutions: The present invention provides a SHG confocal endoscopy imaging system based on structured light, and the system includes: A laser light source for generating excitation light; A half-wave plate placed on the optical path of the excitation light emitted by the excitation light source; An electro-optic modulator placed on the optical path of the excitation light after passing through the half-wave plate; An excitation filter placed on the optical path of the excitation light after being modulated by the electro-optic modulator; A dichroic mirror placed on the optical path of the excitation light after passing through the excitation filter; A first objective lens placed on the optical path of the excitation light transmitted through the dichroic mirror; A confocal pinhole placed on the optical path of the excitation light converged by the first objective lens; A second objective lens placed on the optical path of the excitation light after passing through the confocal pinhole; A two-dimensional scanner placed on the optical path of the excitation light collimated by the second objective lens; A scanning lens placed on the optical path of the excitation light two-dimensionally scanned by the two-dimensional scanner, and the two-dimensional scanner is placed on the focal plane of the scanning lens; A tube lens placed on the optical path of the excitation light after passing through the scanning lens, and the excitation light is collimated by the tube lens and then passes through a third objective lens; An optical fiber bundle placed on the optical path of the excitation light converged by the third objective lens; A microscopic lens placed on the optical path of the excitation light after exciting the optical fiber bundle; An emission filter placed on the optical path of the fluorescence obtained by exciting the sample with the excitation light after being reflected by the dichroic mirror; A focusing lens placed on the optical path of the fluorescence filtered by the emission filter; A detector placed on the optical path of the fluorescence focused by the focusing lens, and the detector is placed on the rear focal plane of the focusing lens.

[0007] Further, the electro-optic modulator is placed between the half-wave plate and the excitation filter.

[0008] Further, the two sides of the dichroic mirror are respectively the first objective lens and the emission filter.

[0009] Further, the confocal pinhole is placed between the first objective lens and the second objective lens, and the tube lens is placed between the scanning lens and the third objective lens.

[0010] Further, the optical fiber bundle connects the microscopic lens and the third objective lens.

[0011] Further, it is placed between the emission filter and the detector.

[0012] Further, the two-dimensional scanner is a two-dimensional galvanometer or an acousto-optic device.

[0013] Further, the detector is a photomultiplier tube.

[0014] Further, the electro-optic modulator, the two-dimensional scanner, and the detector are all controlled, displayed, and stored by a mobile communication terminal.

[0015] The SHG confocal endoscopy imaging system based on structured light of the present invention combines SHG and SIM, and can make up for the high imaging resolution and optical sectioning ability for thicker samples that the existing fluorescence confocal microscopy imaging system does not have, solves the technical problem of inability to image when encountering some smaller biological cells and thicker samples, and is conducive to the industrial application of the system. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a structural light SHG confocal fluorescence imaging system structure diagram of the SHG confocal endoscopy imaging system based on structured light of the present invention; Figure 2 It is Figure 1 A schematic diagram of the ordinary wide-field spectrum of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 3 It is Figure 1 A schematic diagram of the structured illumination stripes of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 4 It is Figure 1 A schematic diagram of the non-linear SIM structured light spectrum of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 5 It is Figure 1 A schematic diagram of the extended spectrum of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 6 It is Figure 1 A schematic diagram of obtaining an isotropic extended spectrum from multi-directional stripes of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 7 It is Figure 1 A schematic diagram of the fluorescence saturation phenomenon of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 8 It is Figure 1 A schematic diagram of the peak flattening of the sine stripe of the SHG confocal endoscopy imaging system based on structured light shown in; Figure 9 For Figure 1 Schematic diagram of the resolution and excitation light power of the SHG confocal endoscopy imaging system based on structured light shown in the figure; In the figure: excitation light source 1, half-wave plate 2, electro-optic modulator 3, excitation filter 4, dichroic mirror 5, first objective lens 6, confocal pinhole 7, second objective lens 8, two-dimensional scanner 9, scanning lens 10, tube lens 11, third objective lens 12, fiber optic bundle 13, microscopic lens 14, sample 15, emission filter 16, focusing lens 17, detector 18. Specific implementation mode

[0017] Please refer to Figures 1-9 , the present invention provides a SHG confocal endoscopy imaging system based on structured light, and the system includes: A laser light source 1 for generating excitation light; A half-wave plate 2 placed on the optical path of the excitation light emitted by the excitation light source; An electro-optic modulator 3 placed on the optical path of the excitation light after passing through the half-wave plate; An excitation filter 4 placed on the optical path of the excitation light after being modulated by the electro-optic modulator; A dichroic mirror 5 placed on the optical path of the excitation light after passing through the excitation filter; A first objective lens 6 placed on the optical path of the excitation light after passing through the dichroic mirror; A confocal pinhole 7 placed on the optical path of the excitation light converged by the first objective lens; A second objective lens 8 placed on the optical path of the excitation light after passing through the confocal pinhole; A two-dimensional scanner 9 placed on the optical path of the excitation light after being collimated by the second objective lens; A scanning lens 10 placed on the optical path of the excitation light after being two-dimensionally scanned by the two-dimensional scanner, and the two-dimensional scanner is placed on the focal plane of the scanning lens; A tube lens 11 placed on the optical path of the excitation light after passing through the scanning lens, and the excitation light passes through the third objective lens 12 after being collimated by the tube lens; A fiber optic bundle 13 placed on the optical path of the excitation light converged by the third objective lens; A microscopic lens 14 placed on the optical path of the excitation light after exciting the fiber optic bundle; An emission filter 16 placed on the optical path of the fluorescence obtained by exciting the sample 15 by the excitation light after being reflected by the dichroic mirror; A focusing lens 17 placed on the optical path of the fluorescence after being filtered by the emission filter; Detector 18 placed on the optical path after the fluorescence is focused by the focusing lens, and the detector is placed on the rear focal plane of the focusing lens.

[0018] In one embodiment, the electro-optic modulator is placed between the half-wave plate and the excitation filter.

[0019] In one embodiment, the dichroic mirror has the first objective lens and the emission filter on its opposite sides respectively.

[0020] In one embodiment, the confocal pinhole is placed between the first objective lens and the second objective lens, and the tube lens is placed between the scanning lens and the third objective lens.

[0021] In one embodiment, the fiber bundle connects the microscopic lens and the third objective lens.

[0022] In one embodiment, it is placed between the emission filter and the detector.

[0023] The SHG confocal endoscopy imaging system based on structured light provided by the present invention, on the basis of the existing confocal imaging optical path, additionally adds an electro-optic modulator, generates a point-scanning structured illumination pattern by sinusoidally modulating the excitation light, the point-scanning structured illumination pattern interacts with the sample structure to produce a Moiré fringe effect, moves the high-frequency information of the sample that was originally undetectable into the OTF passband, and uses a photomultiplier tube (PMT) to detect. Finally, a super-resolution image is reconstructed using a specific algorithm.

[0024] The working principle of the SHG confocal endoscopy imaging system based on structured light provided by the present invention is: Generally, at a low excitation light intensity level, the fluorescence intensity is linearly related to the excitation light intensity, and the fluorescence intensity distribution is consistent with the excitation light intensity distribution. However, when the excitation light is enhanced and the SHG phenomenon occurs, the fluorescence intensity becomes the square of the excitation light intensity. At this time, in the frequency domain space, there is a second harmonic term in the SHG signal, corresponding to a spatial frequency that is twice the frequency of the illumination structured light. The information of these second harmonic spatial frequencies is modulated into the range of the optical transfer function (OTF) supported by the objective lens, and then a spectral space image larger than linear SIM is obtained after algorithmic separation and addition, thereby further improving the resolution. Theoretically, as long as the excitation light intensity is continuously increased, higher-order harmonic signals will be generated, which can support the down-conversion of higher spatial frequency information to be recorded by the detector through the objective lens. However, the problems of photobleaching and photo-damage effects caused by strong light are difficult to solve. At the same time, the high-frequency information signal has a weak intensity and a low signal-to-noise ratio, and it is easy to generate reconstruction artifacts.

[0025] For the acquisition of three-dimensional tomographic images, first, an excitation light is emitted by a laser light source 1. The excitation light can be continuous light or pulsed light, and its wavelength is located in the absorption region of fluorescent dyes or autofluorescent substances. Then, after passing through a half-wave plate 2, the excitation light is modulated by an electro-optic modulator 3, and the intensity of the modulated laser light changes rapidly with time in a cosine manner. Then, the excitation light filtered by an excitation filter 4 is transmitted through a dichroic mirror 5 to a first objective lens 6 and converged by the first objective lens 6 to a confocal pinhole 7. The confocal pinhole 7 is a confocal pinhole with adjustable size, which can adjust parameters such as signal-to-noise ratio, contrast, and resolution to increase practicability. Then, the excitation light emerging from the confocal pinhole 7 is collected and collimated by a second objective lens 8 and enters a two-dimensional scanner 9. Then, the excitation light deflected by the two-dimensional scanner 9 enters a scanning lens 10 and then enters a tube lens 11 to form a parallel light beam. Then, the excitation light collimated by the tube lens 11 enters a third objective lens 12 and is converged and coupled into a certain optical fiber in an optical fiber bundle 13. Then, after emerging from the corresponding optical fiber at the other end of the optical fiber bundle 13, it is focused on a sample 15 by a microscopic lens 14. Then, according to the principle of reversibility of the optical path, the fluorescence excited by the sample 15 by the excitation light is coupled into the corresponding optical fiber in the optical fiber bundle 13 by the microscopic lens 14, and then transmitted through the third objective lens 12 to the tube lens 11. Then, it passes through the scanning lens 10, the two-dimensional scanner 9, and the second objective lens 8 in sequence and enters the confocal pinhole 7. The confocal pinhole 7 can block the fluorescence collected and transmitted by other optical fibers except the optical fiber corresponding to the incoming excitation light in the fluorescence beam. Then, the fluorescence filtered by the confocal pinhole 7 forms a collimated light by the first objective lens 6. This collimated light is reflected by the dichroic mirror 5 to an emission filter 16, and the residual excitation light is blocked by the emission filter 16 to filter out the fluorescence. Then, the filtered fluorescence is focused on a detector 18 by a first focusing lens 17, and a confocal fluorescence super-resolution microscopic image is obtained by the detector 18.

[0026] In the SHG confocal endoscopy imaging system based on structured light of the present invention, in order to obtain a confocal fluorescence super-resolution microscopic image of the sample 15, every time the two-dimensional scanner 9 deflects the beam of the excitation light by one step, the optical fiber that is focused and coupled into the optical fiber bundle 13 by the third objective lens 12 and into which the excitation light is introduced also changes accordingly, resulting in a corresponding change in the position of the excitation spot of the excitation light in the sample 15 to achieve the purpose of scanning the sample. The fluorescence collected by other optical fibers in the optical fiber bundle 13 is blocked by the confocal pinhole 7. In this way, compared with the traditional method that only uses the aperture of the optical fiber in the optical fiber bundle to realize the confocal optical path, this system introduces the confocal pinhole 7 to achieve true conjugate imaging, and can filter out the interfering fluorescence signals collected by other adjacent optical fibers in the optical fiber bundle, while the traditional method using the aperture of the optical fiber itself cannot achieve this effect. In addition, the confocal pinhole 7 is in a self-scanning circuit, which not only realizes the effect of an excitation spot light source but also realizes the control of point object luminescence. It has a simple structure and stable performance, which is conducive to the industrialization of the system.

[0027] In one embodiment, the two-dimensional scanner 9 can be a two-dimensional galvanometer, an acousto-optic device, or other scanners, and can achieve raster scanning or random scanning.

[0028] In one embodiment, the dichroic mirror 5 has the characteristics of totally reflecting the wide-field imaging laser and highly transmitting the excitation light and fluorescence.

[0029] In one embodiment, the detector 18 can be a photomultiplier tube (PMT) or other single-point detectors; In one embodiment, the electro-optic modulator 3, the two-dimensional scanner 9, and the detector 18 are all controlled, displayed, and stored by a mobile communication terminal. Specifically, the mobile communication terminal can be a computer.

[0030] The SHG confocal endoscopy imaging system based on structured light of the present invention combines SHG and SIM, and through optical devices such as an electro-optic modulator, realizes super-resolution microscopy imaging, which can make up for the high imaging resolution and optical sectioning ability for relatively thick samples that the existing fluorescence confocal microscopy imaging system does not have, solves the technical problem of being unable to image when encountering some small biological cells or relatively thick samples, and is conducive to the industrial application of the system.

[0031] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not limited to the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, for those skilled in the art, they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A SHG confocal endoscopy imaging system based on structured light, characterized in that: The system includes: A laser light source for generating excitation light; A half-wave plate placed on the optical path of the excitation light emitted by the excitation light source; An electro-optic modulator placed on the optical path of the excitation light after passing through the half-wave plate; An excitation filter placed on the optical path of the excitation light modulated by the electro-optic modulator; A dichroic mirror placed on the optical path of the excitation light after passing through the excitation filter; A first objective lens placed on the optical path of the excitation light transmitted through the dichroic mirror; A confocal pinhole placed on the optical path of the excitation light converged by the first objective lens; A second objective lens placed on the optical path of the excitation light after passing through the confocal pinhole; A two-dimensional scanner placed on the optical path of the excitation light collimated by the second objective lens; A scanning lens placed on the optical path of the excitation light two-dimensionally scanned by the two-dimensional scanner, and the two-dimensional scanner is placed on the focal plane of the scanning lens; A tube lens placed on the optical path of the excitation light after passing through the scanning lens, and the excitation light is collimated by the tube lens and then passes through a third objective lens; An optical fiber bundle placed on the optical path of the excitation light converged by the third objective lens; A microscopic lens placed on the optical path of the excitation light after exciting the optical fiber bundle; An emission filter placed on the optical path of the fluorescence obtained by exciting the sample with the excitation light and reflected by the dichroic mirror; A focusing lens placed on the optical path of the fluorescence filtered by the emission filter; A detector placed on the optical path of the fluorescence focused by the focusing lens, and the detector is placed on the rear focal plane of the focusing lens.

2. The SHG confocal endoscopy imaging system based on structured light according to claim 1, wherein: The electro-optic modulator is placed between the half-wave plate and the excitation filter.

3. The SHG confocal endoscopy imaging system based on structured light according to claim 2, wherein: The two opposite sides of the dichroic mirror are respectively the first objective lens and the emission filter.

4. The SHG confocal endoscopy imaging system based on structured light according to claim 3, characterized in that: The confocal pinhole is placed between the first objective lens and the second objective lens, and the tube lens is placed between the scanning lens and the third objective lens.

5. The SHG confocal endoscopic imaging system based on structured light according to claim 4, wherein: The optical fiber bundle connects the microscopic lens and the third objective lens.

6. The SHG confocal endoscopic imaging system based on structured light according to claim 5, wherein: It is placed between the emission filter and the detector.

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