Microscopic endoscopic imaging method and system based on structured light illumination

Abstract

A kind of microscopic endoscopic imaging method and system based on structured light illumination, the method includes controlling first illumination light source and second illumination light source emit excitation light in turn, and generate fluorescent signal respectively;Control double shutter camera to collect the fluorescent signal generated respectively and obtain two images;Two images collected are handled, and a frame of image is obtained, the first illumination light source is structured light illumination light source, and the second illumination light source is wide field illumination light source.The system includes first illumination light path, second illumination light path, beam combining light path and imaging light path.The present application does not need scanning device, by the way of combination of structured light illumination and wide field illumination imaging, fluorescent signal is collected in turn, only two images are synthesized to obtain high-quality fluorescent in-focus image, and imaging speed is fast;And there is no pinhole light blocking, and light energy utilization rate is high, and the fluorescent signal generated is more, and fluorescent imaging sensitivity is high, and the fluorescent tissue image information obtained is rich.

Description

Technical Field

[0001] This invention belongs to the field of microscopic endoscopic imaging, and particularly relates to a microscopic endoscopic imaging method and system based on structured light illumination. Background Technology

[0002] In the field of microscopic endoscopic imaging, optical microscopy has been highly sought after due to its numerous advantages, including being non-destructive, rapid, and low-cost. Optical microscopy technology is primarily based on the principle of optical microscopy. By using optical lenses to magnify and image the target object, it is possible to observe the morphological and physiological characteristics of microscopic biological tissue cells, facilitating the screening and diagnosis of lesions in physiological tissues.

[0003] High-resolution microendoscopy (HRME) is a novel microscopic endoscopic imaging system that enters the human body through a flexible fiber optic bundle. This bundle simultaneously transmits illumination light and image signals, enabling the examination of lesions in internal tissues. HRME uses LEDs as the light source. Through a proprietary optical path structure and fiber optic bundle, the light signal reaches the fluorescent dye on the tissue surface. The excited fluorescence signal returns through the fiber optic bundle, is amplified by the imaging optical path, and then enters the CCD image detection device, thus producing an image or video output. HRME boasts a simple internal structure, fast imaging speed, and easy operation; simply placing the fiber optic bundle close to the tissue surface is sufficient to acquire an image. However, during imaging, the lack of background light suppression results in a large amount of out-of-focus signals appearing in the displayed image, severely interfering with the identification and observation of in-focus signals. This leads to poor image contrast and hinders the accurate diagnosis and treatment of lesions and tissue cells.

[0004] Laser confocal endomicroscopy (CLE) is an advanced endoscopic imaging technology already in clinical use. Like other techniques, it uses a flexible fiber optic cable inserted into the body to simultaneously illuminate and transmit fluorescence signals. The most significant feature of CLE is the addition of a pinhole device in front of the signal detector, effectively blocking out-of-focus signals and suppressing background light, thus greatly improving image resolution and contrast. CLE offers high accuracy and sensitivity in early cancer screening. However, its drawbacks include the need for a high-quality light source due to the pinhole device, the slow imaging speed resulting from the scanning process, and the complex internal optical path structure, leading to higher costs and increasing the financial burden on medical institutions and patients.

[0005] Therefore, it is necessary to provide a new microscopic endoscopic imaging method and system based on structured light illumination to meet market demands. Summary of the Invention

[0006] In view of this, the purpose of the present invention is to provide a microscopic endoscopic imaging method and system based on structured light illumination, which improves the suppression of background light signals and enhances image contrast while ensuring imaging speed and image resolution.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] A microscopic endoscopic imaging method based on structured light illumination includes the following steps:

[0009] The first illumination source in the first illumination optical path and the second illumination source in the second illumination optical path are controlled to emit excitation light in sequence and generate fluorescence signals respectively;

[0010] The dual-shutter camera is controlled to acquire the separately generated fluorescence signals and obtain two images;

[0011] The two acquired images are processed to obtain a single image frame.

[0012] Furthermore, the step of controlling the first illumination source in the first illumination optical path and the second illumination source in the second illumination optical path to sequentially emit excitation light includes the following steps:

[0013] The first illumination source is controlled to emit excitation light at a first time, and only the first illumination source generates the light signal for excitation fluorescence during the first time.

[0014] The second illumination source is controlled to emit excitation light at a second time after the first time, and at the second time, only the second illumination source generates the light signal for excitation fluorescence.

[0015] Furthermore, processing the two acquired images to obtain a single image frame further includes:

[0016] Using a synthesis algorithm, each pair of acquired images is processed to suppress out-of-focus signals and extract in-focus fluorescence signals, thereby obtaining the desired frame image.

[0017] Furthermore, both the first and second lighting sources are LED light sources.

[0018] To achieve the above objectives, the present invention also provides the following technical solution:

[0019] A microscopic endoscopic imaging system based on structured light illumination, comprising:

[0020] The first illumination path is used to generate illumination light in the first direction;

[0021] The second illumination path is used to generate illumination light in a second direction;

[0022] A beam combining optical path is used to combine the illumination light from the first illumination optical path and the second illumination optical path.

[0023] The imaging optical path is used to collect fluorescence signals and image them.

[0024] Furthermore, the first illumination optical path sequentially includes a first illumination source, a first collimating lens for collimating the light emitted from the first illumination source, a grating, and a lens for collecting the diffracted light generated by the grating.

[0025] Furthermore, the first lighting source is an LED light source.

[0026] Furthermore, the grating includes several periodic grooves along the same direction, and the light diffracted by the grating presents a striped structure to achieve striped illumination.

[0027] Furthermore, the grating is located at the front focal plane of the lens, and the lens, together with the imaging optical path, projects an image onto the grating surface, thereby projecting the periodic stripes of the grating onto the sample surface.

[0028] Furthermore, the second illumination optical path includes a second illumination source and a second collimating lens for collimating the light emitted from the second illumination source.

[0029] Furthermore, the second lighting source is an LED light source.

[0030] Furthermore, the beam combining optical path sequentially includes a first filter, a dichroic mirror for reflecting the spectrum filtered by the first filter, an objective lens for collecting the light signal reflected by the dichroic mirror, and an optical fiber bundle for transmitting the illumination light wave. The first filter is used to perform spectral filtering on the illumination light from the first illumination optical path and the second illumination optical path, blocking the spectrum that is ineffective for the excitation process of the fluorescent dye.

[0031] Furthermore, the objective lens includes an object space and an image plane space, the fiber bundle is located in the object space of the objective lens, and the first illumination source, the second illumination source, and the grating surface of the grating are all located in the image plane space.

[0032] Furthermore, the imaging optical path sequentially includes an optical fiber bundle for collecting and transmitting fluorescence signals, an objective lens for amplifying and transmitting fluorescence signals, a dichroic mirror for processing the fluorescence signals transmitted by the objective lens, and a second filter for spectral filtering of the fluorescence signals.

[0033] Furthermore, the imaging optical path also includes an imaging lens and a dual-shutter camera. The imaging lens and the dual-shutter camera work together to collect and image the fluorescence signal filtered by the second filter. The fluorescence signal projected onto the dual-shutter camera is converted by photoelectric conversion to display an image containing human tissue information.

[0034] Furthermore, the structured light illumination-based microscopic endoscopic imaging system also includes a beam splitter, which is located at the intersection of the propagation directions of the first illumination light path and the second illumination light path. Light from the first illumination light path and the second illumination light path enters the same optical path system after being reflected by the beam splitter.

[0035] As can be seen from the above technical solution, the present invention adds a structured light illumination optical path to the traditional wide-field fluorescence microscopy imaging optical path, and simultaneously connects an optical fiber bundle to construct a microscopic endoscopic imaging system. This system is based on the principle of fluorescence imaging, and designs a corresponding excitation source and optical path system for a specific fluorescent dye, and successfully acquires fluorescence signals and shields stray light interference from other bands, ultimately obtaining a fluorescence image that can reflect tissue information.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] 1. Using LEDs as the light source is economical and environmentally friendly, with high light energy conversion efficiency, avoiding the potential hazards of laser light sources, and is safe, efficient, and has a long service life.

[0038] 2. Based on fluorescence microscopy and corresponding fluorescent dye labeling, high-resolution tissue and cell pathological images can be obtained, facilitating specific research on cells and molecules.

[0039] 3. By introducing a structured light illumination path, a structured light illumination imaging system is formed. The stripes generated by the grating illuminate the in-focus signal, suppress the out-of-focus signal, and effectively improve the image contrast.

[0040] 4. No scanning device is required. Fluorescence signals are acquired sequentially by combining structured light illumination and wide-field illumination imaging. Only two images need to be combined to obtain a high-quality fluorescence in-focus image, and the imaging speed is fast.

[0041] 5. There are no pinholes to block light, resulting in high light energy utilization, abundant fluorescence signals, high fluorescence imaging sensitivity, and rich information in the acquired fluorescent tissue images.

[0042] 6. Using a dual-shutter camera to acquire fluorescence images in pairs, the acquisition interval between the two fluorescence signals excited by different structured light illuminations is shorter, reducing motion artifacts and enhancing the ability to acquire tissue motion information.

[0043] 7. Modular design: The illumination and imaging optical paths of the microscopic endoscopy system are separated, as are the structured light illumination path and the wide field illumination path, resulting in a simple structure and good stability. Attached Figure Description

[0044] Figure 1 This is a flowchart of the microscopic endoscopic imaging method based on structured light illumination according to the present invention.

[0045] Figure 2 This is a schematic diagram of the structure of the microscopic endoscopic imaging system based on structured light illumination according to the present invention.

[0046] Figure 3 These are schematic diagrams of two images obtained by the microscopic endoscopic imaging system based on structured light illumination according to the present invention, wherein (a) is a schematic diagram of fluorescence imaging generated by structured light illumination, and (b) is a schematic diagram of fluorescence imaging generated by wide field illumination.

[0047] Among them: 100-beam splitter, 11-first illumination source, 12-first collimating lens, 13-grating, 14-lens, 21-second illumination source, 22-second collimating lens, 31-first filter, 32-second filter, 33-objective lens, 34-fiber bundle, 35-dichroic mirror, 36-imaging lens, 37-dual shutter camera. Detailed Implementation

[0048] This invention discloses a microscopic endoscopic imaging method and system based on structured light illumination, which improves the suppression of background light signals and enhances image contrast while ensuring imaging speed and image resolution.

[0049] like Figure 1 As shown, Figure 1 This is a flowchart of the microscopic endoscopic imaging method based on structured light illumination according to the present invention. The microscopic endoscopic imaging method based on structured light illumination includes the following steps:

[0050] S101: Control the first illumination source in the first illumination optical path and the second illumination source in the second illumination optical path to emit excitation light in sequence and generate fluorescence signals respectively.

[0051] Specifically, controlling the first illumination source in the first illumination optical path and the second illumination source in the second illumination optical path to sequentially emit excitation light includes the following steps:

[0052] The first illumination source is controlled to emit excitation light at a first time, and only the first illumination source generates the light signal for excitation fluorescence during the first time.

[0053] The second illumination source is controlled to emit excitation light at a second time after the first time, and at the second time, only the second illumination source generates the light signal for excitation fluorescence.

[0054] In one embodiment, the first illumination path is a structured light illumination path, and the second illumination path is a wide-field illumination path.

[0055] In one embodiment, both the first and second lighting sources are LED light sources. Using LEDs as light sources is economical and environmentally friendly, has high light energy conversion efficiency, avoids the potential hazards of laser light sources, and is safe, efficient, and has a long service life.

[0056] S102: Controls the dual-shutter camera to acquire the separately generated fluorescence signals and obtain two images.

[0057] The two images produced by the first and second lighting sources emitting excitation light in sequence are inconsistent.

[0058] S103: Process the two acquired images to obtain a single image frame.

[0059] The process of processing the two acquired images to obtain a single image frame further includes:

[0060] Using a synthesis algorithm, each pair of acquired images is processed to suppress out-of-focus signals and extract in-focus fluorescence signals, thereby obtaining the desired frame image.

[0061] This method requires no scanning device and acquires fluorescence signals sequentially by combining structured light illumination and wide-field illumination imaging. Only two images need to be combined to obtain a high-quality fluorescence in-focus image, resulting in fast imaging speed.

[0062] like Figure 2 As shown, Figure 2 This is a schematic diagram of the structure of the structured light illumination-based microscopic endoscopic imaging system of the present invention. The system includes a first illumination optical path, a second illumination optical path, a beam combining optical path, and an imaging optical path. The first illumination optical path generates illumination light in a first direction. The second illumination optical path generates illumination light in a second direction. The beam combining optical path combines the illumination light from the first and second illumination optical paths. The imaging optical path collects the generated fluorescence signal and images it. The system adopts a modular design, with separate illumination and imaging optical paths, resulting in a simple structure and good stability. This structured light illumination-based microscopic endoscopic imaging system is based on fluorescence microscopy and, with corresponding fluorescent dye labeling, can acquire high-resolution tissue and cellular pathological images, facilitating specific studies of cells and molecules.

[0063] In one embodiment, the structured light illumination-based microscopic endoscopy imaging system further includes a beam splitter located at the intersection of the propagation directions of the first and second illumination light paths. Light from the first and second illumination light paths is reflected by the beam splitter and enters the same optical path system. The beam combining optical path is located in the optical path after the beam splitter. The modular design separates the illumination and imaging optical paths, as well as the structured light illumination and wide-field illumination optical paths, resulting in a simple structure and good stability. Furthermore, the absence of pinholes obstructing light leads to high light energy utilization, abundant fluorescence signals, high fluorescence imaging sensitivity, and rich information in the acquired fluorescent tissue images.

[0064] In one embodiment, the first illumination path is a structured light illumination path, and the second illumination path is a wide-field illumination path.

[0065] In one embodiment, both the first and second lighting sources are LED light sources. Using LEDs as the light source is economical and environmentally friendly, has high light energy conversion efficiency, avoids the potential hazards of laser light sources, and is safe, efficient, and has a long service life. In another embodiment, because it is necessary to correspond to a specific fluorescent dye excitation wavelength, both the first and second lighting sources use LED lighting of a specific wavelength, which is economical and inexpensive.

[0066] like Figure 2 As shown, in one embodiment, in the structured light illumination path (i.e., the first illumination path), with a beam splitter as the confluence point, the first illumination path sequentially includes a first illumination source 11, a first collimating lens 12 for collimating the light emitted from the first illumination source 11, a grating 13, and a lens 14 for collecting the diffracted light generated by the grating 13. The first illumination source 11 specifically includes LED beads. The grating 13 includes several periodic grooves along the same direction. The light diffracted by the grating 13 presents a striped structure, thus achieving striped illumination. The grating 13 is located at the front focal plane of the lens. The lens 14, in conjunction with the imaging path, projects an image onto the grating surface, thereby projecting the periodic stripes of the grating onto the sample surface. This invention introduces a structured light imaging system, utilizing the striped illumination generated by the grating 13 to mark the in-focus signal, suppress the out-of-focus signal, and effectively improve image contrast.

[0067] like Figure 2As shown, due to the large divergence angle of the LED beads used, a first collimating lens 12 is added in front of the LED to collimate the light emitted by the LED beads and collect more illumination light into the main optical path in order to improve the utilization rate of light energy. The collimated illumination light is incident on the surface of the grating 13, and diffracted light is generated after passing through the grating 13 to realize structured light illumination. The grating 13 is composed of at least a number of periodic grooves along the same direction. Therefore, the light diffracted by the grating 13 exhibits a specific fringe structure, thereby realizing fringe illumination. The diffracted light generated by the grating 13 is collected by the lens 14 placed behind it. The grating 13 is located at the front focal plane of the lens 14. The lens 14, together with the components in the imaging optical path, can project an image onto the surface of the grating 13, thereby projecting the periodic fringes of the grating 13 onto the sample surface. The fringe structured light generated by the structured light path reaches a beam splitter 10. After being reflected by the beam splitter 10, it enters the same optical path system as the light from the wide-field illumination. The function of the beam splitter 10 is to separate or combine the light waves. The beam splitter 10 is located at the intersection of the propagation direction of the structured light path and the illumination direction of the wide-field light path.

[0068] In one embodiment, the second illumination optical path includes a second illumination source 21 and a second collimating lens 22 for collimating the light emitted from the second illumination source 21. The second illumination source 21 specifically includes LED beads. The wide-field illumination optical path (i.e., the second illumination optical path) only includes the second illumination source 21 and the matching second collimating lens 22, and the incident direction of the light emitted by it is spatially perpendicular to the direction of the structured light signal. In this way, without interference, the collimated light wave can also smoothly enter the beam splitter from another direction.

[0069] As can be seen from the above description, the microscopic endoscopic imaging system based on structured light illumination is mainly divided into an illumination optical path and an imaging optical path. The illumination optical path is equipped with two illumination sources, which are located in different positions. The two illumination lights from different directions are introduced into the same optical path system through a beam splitter.

[0070] like Figure 2As shown, the optical path system after beam combining includes a beam combining optical path, which sequentially includes a first filter 31, a dichroic mirror 35 for reflecting the spectrum filtered by the first filter 31, an objective lens 33 for collecting the light signal reflected from the dichroic mirror 35, and an optical fiber bundle 34 for transmitting the illumination light wave. The first filter 31 is used to perform spectral filtering on the illumination light from the first illumination optical path and the second illumination optical path, blocking spectra that are ineffective for the excitation process of fluorescent dyes. In this illumination optical path system, only one filter is used, that is, the beam combining optical path uses only one filter and is located in the optical path after the beam splitter 10, which can simultaneously perform spectral filtering on the light from the structured optical path and the wide-field illumination optical path, blocking spectra that are ineffective for the excitation process of fluorescent dyes. The dichroic mirror 35 is used in conjunction with the first filter 31. The spectrum filtered by the first filter 31 is completely reflected by the dichroic mirror 35 and enters the objective lens. After the objective lens 33 collects the light signal, the two types of illumination beams are successfully coupled into the fiber bundle 34. The objective lens is a key component in the light processing process.

[0071] In one embodiment, the objective lens 33 includes an object space and an image plane space. The fiber bundle 34 is located in the object space of the objective lens, while the first illumination source 11, the second illumination source 21, and the grating surface of the grating 13 are all located in the image plane space. Specifically, in this illumination optical path, the fiber is located in the object space of the objective lens, and the light sources of both structures and the grating surface in the structured light path are all located in the image plane space of the objective lens. For wide-field illumination, the objective lens 33 mainly collects and transmits light signals. For structured light illumination, the objective lens 33 reduces the light signal containing the grating fringe structure and images it onto the fiber target surface. The fiber target surface and the grating surface are in an optically conjugate position, thus the objective lens has good aberration correction and flat-field imaging capabilities. The fiber bundle mainly serves to transmit illumination light waves, transmitting the excitation light signal to the surface of human tissue.

[0072] like Figure 2 As shown, in one embodiment, the imaging optical path sequentially includes an optical fiber bundle 34 for collecting and transmitting fluorescence signals, an objective lens 33 for amplifying and transmitting fluorescence signals, a dichroic mirror 35 for processing the fluorescence signals transmitted by the objective lens 33, and a second filter 32 for spectral filtering of the fluorescence signals. The optical fiber bundle 34, objective lens 33, and dichroic mirror 35 can be shared by both the beam combining optical path and the imaging optical path.

[0073] like Figure 2As shown, the imaging optical path also includes an imaging lens 36 and a dual-shutter camera 37. The imaging lens 36 and the dual-shutter camera 37 work together to collect and image the fluorescence signal filtered by the second filter 32. The fluorescence signal projected onto the dual-shutter camera 37 is converted by photoelectric conversion to display an image containing human tissue information. Using the dual-shutter camera 37, fluorescence images are acquired in pairs, and the acquisition interval between the two fluorescence signals excited by different structure illuminations is shorter, reducing motion artifacts and enhancing the ability to acquire tissue motion information. Specifically, the imaging optical path is a series of optical transmission systems designed and constructed from the fluorescence generation position to the fluorescence collection position, which sequentially includes an optical fiber bundle 34, an objective lens 33, a dichroic mirror 35, a second filter 32, an imaging lens 36, and a dual-shutter camera 37. The fluorescence signal is mainly generated by the excitation of fluorescent dyes by illumination light. Therefore, the excited fluorescence signal is also simultaneously collected and transmitted by the optical fiber bundle 34 and emitted from the other end of the optical fiber bundle 34, i.e., the port located at the front focal plane of the objective lens 33. Objective lens 33 amplifies and transmits the fluorescence signal. The generated fluorescence signal may contain the illumination spectrum from the excitation light; therefore, the amplified light signal needs to be filtered by a dichroic mirror 35 and a second filter 32 to remove invalid spectra. Since the excitation and fluorescence spectra have certain spectral differences, the dichroic mirror 35, which corresponds to these spectral differences, can effectively separate the excitation and fluorescence spectra. The dichroic mirror 35 reflects the excitation light signal while transmitting the fluorescence signal. Therefore, in the imaging optical path, the fluorescence signal can pass smoothly through the dichroic mirror 35. Simultaneously, a second filter 32 is placed after the dichroic mirror 35 to further block and remove stray light, ensuring the purity of the fluorescence signal. Imaging lens 36 and dual-shutter camera 37 work together to collect and image the filtered fluorescence signal. The fluorescence signal projected onto the camera's CCD target surface undergoes photoelectric conversion, displaying an image containing human tissue information. This completes the entire fluorescence optical path transmission and imaging process.

[0074] In this technical solution, the structured light illumination source and the wide-field illumination source are sequentially activated. Specifically, while the structured light illumination source is illuminated, a structured light-excited fluorescence image is acquired. Then, the structured light illumination source is turned off and the wide-field illumination source is activated, and another wide-field illumination-excited fluorescence image is acquired. This acquisition process is achieved using a dual-shutter camera, meaning that fluorescence images are acquired in pairs within a single exposure, resulting in high acquisition speed and ensuring a high frame rate. Since the fluorescence image generated by wide-field illumination contains both in-focus and out-of-focus tissue information, while the fringe structure in the structured light-excited fluorescence image already marks the in-focus information, by performing algorithmic synthesis processing on these two images, the in-focus signal can be extracted while the out-of-focus signal is discarded, thereby suppressing background light signals and improving image contrast. Furthermore, the entire imaging process does not require a scanning device or a pinhole to block light, resulting in a fast imaging speed.

[0075] The technical solution of this method is described below with reference to specific embodiments.

[0076] like Figure 3 As shown, both structured light illumination paths use blue LEDs with a center wavelength of 450nm and the same power. First, in the structured light illumination path, the light emitted by LED light source 11 (i.e., the first illumination source 11) has a large divergence angle. It is converged by the first collimating lens 12, reflected by the grating 13, lens 14, and beam splitter 10, and then reaches the first filter 31. The first collimating lens 12 is located before the LED beads of LED light source 11, and the LED beads of LED light source 11 are positioned at the front focal plane of the first collimating lens 12. The emitted illumination light propagates parallel to the space behind the first collimating lens 12. The transmission-type diffraction grating 13 is located between the first collimating lens 12 and lens 14, at the front focal plane of lens 14. It is used to shape the collimated beam and generate an illumination beam with a specific structure. The structured beam is collected by the lens 14 placed behind it and reflected from the reflecting surface in the beam splitter 10 to the corresponding propagation direction. Beam splitter 10 has a two-way beam combining function, enabling beams from different directions to be guided into the same optical path. Beam splitter 10 is located at the intersection of the structured light path propagation direction and the wide-field light path illumination direction. Perpendicular to the structured light path is the wide-field illumination path composed of LED light source 21 (i.e., the second illumination source 21) and the second collimating lens 22. Similarly, the LED beads of LED light source 21 are located at the front focal plane of the second collimating lens 22, which is close to the beam splitter 10. After the structured light illumination and wide-field illumination pass through the intersection point of beam splitter 10, they first pass through the first filter 31, and specific wavelengths of the spectrum pass smoothly through the first filter 31. After reflection by the dichroic mirror 35 placed at a 45-degree angle, they enter the objective lens 33. The dichroic mirror 35 has short-wave reflection and long-wave transmission functions, reflecting light waves around 450nm and collimating them without deviation to the objective lens 33, while also allowing undamaged transmission of wavelengths above 500nm. Objective lens 33 is located between fiber bundle 34 and dichroic mirror 35, and performs imaging and compression of the light beam. The plan-field achromatic microscope objective lens used typically has a magnification of 10 or 20, which compresses the beam spot emanating from its image-side spatial aperture, reducing its beam radius to a certain width to minimize loss and ensure smooth coupling into the fiber bundle. One end of fiber bundle 34 is located at the front focal plane of objective lens 33, and the other end contacts the human tissue through a channel. The illumination beam reaches the surface of the tissue stained with fluorescent dye through this fiber bundle, exciting fluorescence in the corresponding wavelength band, and the fluorescence returns along the original path of the fiber bundle.

[0077] Since this embodiment uses 450nm excitation light, the generated fluorescence wavelength is generally after 500nm. After magnification by the microscope objective, the fluorescence passes smoothly through the dichroic mirror. The second filter 32 is located between the dichroic mirror 35 and the imaging lens 36. The second filter 32 only allows light with a center wavelength of 510nm to pass smoothly while blocking other wavelengths, especially those below 500nm. The dual-shutter camera 37 typically uses a CCD or CMOS sensor and works in conjunction with the imaging lens to collect the filtered fluorescence signal, convert it through photoelectric conversion, and then image it onto a display.

[0078] In the specific implementation process, the two light sources with different illumination structures are lit sequentially in chronological order. That is, when LED light source 11 is lit, the light signal that excites fluorescence on the biological tissue surface through the fiber optic bundle is only the structured light signal. Similarly, when LED light source 21 is lit, the light signal that excites fluorescence is the wide-field illumination beam. When these two light sources are lit sequentially to generate excitation light, a dual-shutter camera captures each fluorescence signal and outputs an image. Using a synthesis algorithm, each pair of acquired images is processed to suppress out-of-focus signals, extract the in-focus fluorescence signal, and perform algorithmic preprocessing to calculate the mean square factor. Based on the processing results, low-frequency and high-frequency filtering is applied to the structured light illumination fluorescence image and the wide-field illumination fluorescence image, respectively. Finally, the high and low frequency information is recombined to obtain the desired frame image. The fluorescence images produced by the sequential illumination of the two light sources are inconsistent, such as... Figure 3 As shown, (a) is a schematic diagram of fluorescence imaging generated by structured light illumination, where the stripes are the illumination structure generated by structured light. For each structured light illumination, the frequency and position of the stripes remain unchanged. (b) is a schematic diagram of fluorescence imaging generated by wide field illumination, without structured light illumination stripe structure. These two images were acquired by a dual-shutter camera in a single exposure. The two images were processed by an algorithm to synthesize a fluorescence image with effective suppression of defocus signals.

[0079] As can be seen from the above technical solution description, the present invention adds a structured light illumination optical path to the traditional wide-field fluorescence microscopy imaging optical path, and simultaneously connects an optical fiber bundle to construct a microscopic endoscopic imaging system. This system is based on the principle of fluorescence imaging, and designs a corresponding excitation source and optical path system for a specific fluorescent dye, and successfully acquires fluorescence signals and shields stray light interference from other bands, ultimately obtaining a fluorescence image that can reflect tissue information.

[0080] Compared with the prior art, the beneficial effects of the present invention are:

[0081] 1. Using LEDs as the light source is economical and environmentally friendly, with high light energy conversion efficiency, avoiding the potential hazards of laser light sources, and is safe, efficient, and has a long service life.

[0082] 2. Based on fluorescence microscopy and corresponding fluorescent dye labeling, high-resolution tissue and cell pathological images can be obtained, facilitating specific research on cells and molecules.

[0083] 3. By introducing a structured light illumination path, a structured light illumination imaging system is formed. The stripes generated by the grating illuminate the in-focus signal, suppress the out-of-focus signal, and effectively improve the image contrast.

[0084] 4. No scanning device is required. Fluorescence signals are acquired sequentially by combining structured light illumination and wide-field illumination imaging. Only two images need to be combined to obtain a high-quality fluorescence in-focus image, and the imaging speed is fast.

[0085] 5. There are no pinholes to block light, resulting in high light energy utilization, abundant fluorescence signals, high fluorescence imaging sensitivity, and rich information in the acquired fluorescent tissue images.

[0086] 6. Using a dual-shutter camera to acquire fluorescence images in pairs, the acquisition interval between the two fluorescence signals excited by different structured light illuminations is shorter, reducing motion artifacts and enhancing the ability to acquire tissue motion information.

[0087] 7. Modular design: The illumination and imaging optical paths of the microscopic endoscopy system are separated, as are the structured light illumination path and the wide field illumination path, resulting in a simple structure and good stability.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A microscopic endoscopic imaging system, employing a microscopic endoscopic imaging method based on structured light illumination, characterized in that, It includes: The first illumination path is used to generate illumination light in the first direction; The second illumination path is used to generate illumination light in a second direction; A beam combining optical path is used to combine the illumination light from the first illumination optical path and the second illumination optical path. The imaging optical path is used to collect fluorescence signals and image them. The combined optical path sequentially includes a first filter, a dichroic mirror for reflecting the spectrum filtered by the first filter, an objective lens for collecting the light signal reflected by the dichroic mirror, and an optical fiber bundle for transmitting the illumination light wave. The first filter is used to perform spectral filtering on the illumination light from the first illumination optical path and the second illumination optical path, blocking the spectrum that is ineffective for the excitation process of the fluorescent dye. The structured light illumination-based microscopic endoscope imaging system also includes a beam splitter, which is located at the intersection of the propagation directions of the first illumination light path and the second illumination light path. Light from the first illumination light path and the second illumination light path enters the same optical path system after being reflected by the beam splitter. The steps of the microscopic endoscopic imaging method include: The system controls the first illumination source in the first illumination optical path and the second illumination source in the second illumination optical path to emit excitation light sequentially and generate fluorescence signals respectively; the system controls the first illumination source to emit excitation light at a first time, during which only the first illumination source generates fluorescence-exciting light signals; the system controls the second illumination source to emit excitation light at a second time after the first time, during which only the second illumination source generates fluorescence-exciting light signals. The dual-shutter camera is controlled to acquire two images by collecting the fluorescence signals generated separately in a single exposure. Using a synthesis algorithm, each pair of acquired images is processed to suppress out-of-focus signals and extract in-focus fluorescence signals, thereby obtaining the desired frame image.

2. The microscopic endoscopic imaging system according to claim 1, characterized in that, The first illumination optical path includes, in sequence, a first illumination source, a first collimating lens for collimating the light emitted by the first illumination source, a grating, and a lens for collecting the diffracted light generated by the grating.

3. The microscopic endoscopic imaging system according to claim 1, characterized in that, The first lighting source is an LED light source.

4. The microscopic endoscopic imaging system according to claim 2, characterized in that, The grating includes several periodic grooves along the same direction. Light diffracted by the grating presents a striped structure, thus achieving striped illumination.

5. The microscopic endoscopic imaging system according to claim 2, characterized in that, The grating is located at the front focal plane of the lens, and the lens, together with the imaging optical path, projects an image onto the surface of the grating, thereby projecting the periodic stripes of the grating onto the sample surface.

6. The microscopic endoscopic imaging system according to claim 1, characterized in that, The second illumination optical path includes a second illumination source and a second collimating lens for collimating the light emitted from the second illumination source.

7. The microscopic endoscopic imaging system according to claim 6, characterized in that, The second lighting source is an LED light source.

8. The microscopic endoscopic imaging system according to claim 2, characterized in that, The objective lens includes an object space and an image plane space. The fiber bundle is located in the object space of the objective lens, and the first illumination source, the second illumination source, and the grating surface of the grating are all located in the image plane space.

9. The microscopic endoscopic imaging system according to claim 1, characterized in that, The imaging optical path sequentially includes an optical fiber bundle for collecting and transmitting fluorescence signals, an objective lens for amplifying and transmitting fluorescence signals, a dichroic mirror for processing the fluorescence signals transmitted by the objective lens, and a second filter for spectral filtering of the fluorescence signals.

10. The microscopic endoscopic imaging system according to claim 1, characterized in that, The imaging optical path also includes an imaging lens and a dual-shutter camera. The imaging lens and the dual-shutter camera work together to collect and image the fluorescence signal filtered by the second filter. The fluorescence signal projected onto the dual-shutter camera is converted by photoelectric conversion to display an image containing human tissue information.

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