CN110141182A - A microscopic endoscopic imaging method and system based on structured light illumination - Google Patents

Abstract

A microscopic endoscopic imaging method and system based on structured light illumination comprises controlling a first illumination light source and a second illumination light source to sequentially emit exciting light and respectively generate fluorescence signals; controlling a double-shutter camera to collect the respectively generated fluorescent signals and obtain two images; and processing the two collected images to obtain a frame of image, wherein the first illumination light source is a structured light illumination light source, and the second illumination light source is a wide-field illumination light source. The system comprises a first illumination light path, a second illumination light path, a beam combining light path and an imaging light path. According to the invention, a scanning device is not needed, fluorescence signals are sequentially acquired in a mode of combining structured light illumination and wide-field illumination imaging, high-quality fluorescence in-focus images can be obtained only by synthesizing two images, and the imaging speed is high; and no pinhole is used for blocking light, the light energy utilization rate is high, the generated fluorescent signals are more, the fluorescence imaging sensitivity is high, and the acquired fluorescent tissue image information is rich.

Description

A microscopic endoscopic imaging method and system based on structured light illumination

technical field

The invention belongs to the field of microscopic endoscopic imaging, in particular to a microscopic endoscopic imaging method and system based on structured light illumination.

Background technique

In the field of microendoscopic imaging, optical microscopic imaging has many advantages such as non-destructive, fast, and low-cost, and has been favored by people all the time. Optical microscopic imaging technology is mainly based on the principle of optical microscopy. By using optical lenses to magnify and image objects, the morphological and physiological characteristics of microscopic biological tissue cells can be observed, which is convenient for disease screening and diagnosis of physiological tissues.

High Resolution Fluorescence Microendoscopy (HRME) is a new type of microendoscopy imaging system, which enters the human body through a soft fiber optic bundle, which can simultaneously transmit illumination light and image signals to realize Examination of tissue lesions in the human body. The high-resolution fluorescent endoscopic microscope adopts LED as the light source, through the proprietary optical path structure and optical fiber bundle, the optical signal reaches the fluorescent dye on the surface of the tissue, and the excited fluorescent signal returns through the optical fiber bundle, is amplified by the imaging optical path and enters the CCD An image detection device that produces an image or video output. The internal structure of the high-resolution fluorescence endoscopic microscope is simple, the imaging speed is fast, and the operation is easy. It only needs to put the fiber bundle close to the tissue surface to obtain the image of the tissue surface. However, during the imaging process, due to the lack of suppression of background light signals, a large number of out-of-focus signals will also appear in the displayed image, which seriously interferes with the identification and observation of in-focus signals, and the image contrast is poor, which is not conducive to the detection of lesions. Precise judgment and diagnosis of tissue cells.

Laser confocal endomicroscopy (Laser Confocal Endomicroscopy, CLE) is an advanced endoscopic imaging technology that has been put into clinical use at present. Similarly, it also uses a soft optical fiber bundle to enter the human body to irradiate and transmit fluorescent signals at the same time. . The biggest feature of laser confocal microscopy imaging technology is that a pinhole device is added in front of the signal detector, which can effectively block the transmission of out-of-focus signals, suppress background light signals, and greatly improve image resolution and contrast. Laser confocal endoscopy has high accuracy and sensitivity in early cancer screening. The disadvantage is that the pinhole device is added, which requires high light source. At the same time, laser confocal technology requires scanning, imaging The speed is slow, and the structure of the internal optical path is complicated, and the cost is high, which increases the economic burden on medical institutions and patients.

Therefore, it is necessary to provide a new microendoscopic imaging method and system based on structured light illumination to meet the needs of the market.

Contents of the invention

In view of this, the object of the present invention is to provide a microendoscopic imaging method and system based on structured light illumination, which can improve the suppression of background light signals and enhance image contrast while ensuring imaging speed and image resolution.

To achieve the above object, the present invention provides the following technical solutions:

A microendoscopic imaging method based on structured light illumination, comprising steps:

controlling the first illuminating light source in the first illuminating light path and the second illuminating light source in the second illuminating light path to sequentially emit excitation light, and respectively generate fluorescent signals;

Control the dual-shutter camera to collect the fluorescent signals generated separately and obtain two images;

Process the collected two images to obtain one frame of image.

Further, the step of controlling the first illumination light source in the first illumination light path and the second illumination light source in the second illumination light path to sequentially emit excitation light includes:

controlling the first illumination light source to emit excitation light at a first time, and only the first illumination light source generates an optical signal for exciting fluorescence within the first time;

The second illumination light source is controlled to emit excitation light at a second time after the first time, and at the second time only the light signal for exciting fluorescence is generated by the second illumination light source.

Further, processing the collected two images to obtain a frame of image further includes:

Using a synthesis algorithm, each pair of collected two images is processed, the out-of-focus signal is suppressed, and the in-focus fluorescence signal is extracted, so as to obtain a required frame of image.

Further, both the first lighting source and the second lighting source are LED light sources.

To achieve the above object, the present invention also provides the following technical solutions:

A micro-endoscopic imaging system based on structured light illumination, comprising:

a first illumination light path, configured to generate illumination light in a first direction;

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

A beam-combining optical path, used to combine the illumination lights of the first illuminating optical path and the second illuminating optical path;

The imaging optical path is used to collect and image the fluorescent signal.

Further, the first illumination light path sequentially includes a first illumination light source, a first collimator lens for collimating light emitted by the first illumination light source, a grating, and a lens for collecting diffracted light generated by the grating.

Further, the first illumination light source is an LED light source.

Further, the grating includes a number of periodic grooves along the same direction, and the light diffracted by the grating presents a stripe structure to realize stripe illumination.

Further, the grating is located at the front focal plane of the lens, and the lens is combined with the imaging optical path to project and image the surface of the grating, so that the periodic fringes of the grating are projected onto the sample surface.

Further, the second illumination light path includes a second illumination light source and a second collimator lens for collimating the light emitted by the second illumination light source.

Further, the second illumination light source is an LED light source.

Further, the beam combining optical path sequentially includes a first optical filter, a dichroic mirror for reflecting the spectrum filtered by the first optical filter, an objective lens for collecting the optical signal reflected by the dichroic mirror, and a The first optical filter is used for spectrally filtering the illumination light from the first illumination optical path and the second illumination optical path to block the spectrum that is invalid for the excitation process of the fluorescent dye.

Further, the objective lens includes an object space and an image plane space, the optical fiber bundle is located in the object space of the objective lens, and the first illumination light source, the second illumination light source and the grating surface of the grating are all located in the image plane space.

Further, the imaging optical path sequentially includes an optical fiber bundle for collecting and transmitting fluorescent signals, an objective lens for amplifying and transmitting the fluorescent signals, a dichroic mirror for processing the fluorescent signals transmitted by the objective lens, and a second filter for spectrally filtering the fluorescent signals. light sheet.

Further, the imaging optical path also includes an imaging lens and a double-shutter camera, and the imaging lens and the double-shutter camera are used together to collect and image the fluorescent signal filtered by the second filter, and the fluorescent signal projected onto the double-shutter camera After photoelectric conversion, an image with human tissue information is displayed.

Further, the microendoscopic imaging system based on structured light illumination also includes a beam splitter, the beam splitter is located at the intersection of the propagation direction of the first illumination light path and the propagation direction of the second illumination light path, and the beam splitter is from the first Light from the illumination optical path and the second illumination optical path enters the same optical path system after being reflected by the beam splitter mirror.

It can be seen from the above technical solutions that the present invention adds structured light to the optical path of the traditional wide-field fluorescence microscopic imaging, and at the same time connects the optical fiber bundle to construct a microscopic endoscopic imaging system. The system is based on the principle of fluorescent imaging , corresponding to the specific fluorescent dye, design the corresponding excitation light source and optical path system, and successfully collect the fluorescent signal and shield the stray light interference of other bands, and finally obtain the fluorescent image that can reflect the tissue information.

Compared with prior art, the beneficial effect of the present invention is:

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

2. Based on fluorescence microscopy imaging technology, with the corresponding fluorescent dye labeling, high-resolution histocytopathological images can be obtained, which is convenient for specific research on cell molecules.

3. Introduce the structured light illumination optical path and have a structured light illumination imaging system, use the stripe illumination generated by the grating to mark the in-focus signal, suppress the out-of-focus signal, and effectively improve the image contrast.

4. Without a scanning device, the fluorescence signals are collected sequentially through the combination of structured light illumination and wide-field illumination imaging. Only two images are combined to obtain high-quality fluorescence in-focus images, and the imaging speed is fast.

5. There is no pinhole to block the light, the utilization rate of light energy is high, more fluorescent signals are generated, the sensitivity of fluorescent imaging is high, and the obtained fluorescent tissue images are rich in information.

6. Using dual-shutter cameras to collect fluorescence images in pairs, the acquisition interval of fluorescence signals excited by two different structured light illuminations is shorter, which reduces motion artifacts and has a stronger ability to acquire tissue motion information.

7. Modular design, separation of illumination light path and imaging light path in the microendoscopy system, separation of structured light illumination light path and wide-field illumination light path, simple structure and good stability.

Description of drawings

Fig. 1 is a flow chart of the microendoscopic imaging method based on structured light illumination of the present invention.

Fig. 2 is a schematic structural diagram of the microendoscopic imaging system based on structured light illumination of the present invention.

Figure 3 is a schematic diagram of two images obtained by the microendoscopic imaging system based on structured light illumination of the present invention, wherein, (a) is a schematic diagram of fluorescence imaging produced by structured light illumination, and (b) is a schematic diagram of fluorescence imaging produced by wide-field illumination Schematic diagram of fluorescence imaging.

Among them: 100-beam splitter, 11-first illumination source, 12-first collimator lens, 13-grating, 14-lens, 21-second illumination source, 22-second collimator lens, 31-first Optical filter, 32-second optical filter, 33-objective lens, 34-fiber bundle, 35-dichroic mirror, 36-imaging lens, 37-double shutter camera.

Detailed ways

The invention discloses a microscopic endoscopic imaging method and system based on structured light illumination, which improves suppression of background light signals and enhances image contrast under the condition of ensuring imaging speed and image resolution.

As shown in FIG. 1 , FIG. 1 is a flow chart of the microendoscopic imaging method based on structured light illumination of the present invention. The microendoscopic imaging method based on structured light illumination comprises the following steps:

S101: Control the first illuminating light source in the first illuminating light path and the second illuminating light source in the second illuminating light path to sequentially emit excitation light, and generate fluorescence signals respectively.

Specifically, the step of controlling the first illumination light source in the first illumination optical path and the second illumination light source in the second illumination optical path to sequentially emit excitation light includes:

controlling the first illumination light source to emit excitation light at a first time, and only the first illumination light source generates an optical signal for exciting fluorescence within the first time;

The second illumination light source is controlled to emit excitation light at a second time after the first time, and at the second time only the light signal for exciting fluorescence is generated by the second illumination light source.

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

In one embodiment, both the first lighting source and the second lighting source are LED light sources, and LEDs are used as light sources, which are economical and environmentally friendly, have high light energy conversion efficiency, avoid potential hazards of laser light sources, are safe and efficient, and have a long service life. long.

S102: controlling the dual-shutter camera to collect the respectively generated fluorescence signals and obtain two images.

Wherein, the two images generated by sequentially emitting excitation light from the first illumination light source and the second illumination light source are inconsistent.

S103: Process the two collected images to obtain one frame of image.

Wherein, processing the two images collected to obtain a frame of image further includes:

Using a synthesis algorithm, each pair of collected two images is processed, the out-of-focus signal is suppressed, and the in-focus fluorescence signal is extracted, so as to obtain a required frame of image.

This method does not require a scanning device, and sequentially collects fluorescence signals through the combination of structured light illumination and wide-field illumination imaging. Only two images are synthesized to obtain high-quality fluorescence in-focus images, and the imaging speed is fast.

As shown in FIG. 2 , FIG. 2 is a schematic structural diagram of a microendoscopic imaging system based on structured light illumination according to the present invention. The microendoscopic imaging system based on structured light illumination includes a first illumination optical path, a second illumination optical path, a combined beam optical path and an imaging optical path. The first illumination light path is used to generate illumination light in a first direction. The second illumination light path is used to generate illumination light in a second direction. The beam combining optical path is used to combine the illumination lights of the first illuminating optical path and the second illuminating optical path. The imaging light path is used to collect the generated fluorescent signal and image the fluorescent signal. The system adopts modular design, the illumination light path and imaging light path are separated in the system, the structure is simple, and the stability is good. The microendoscopic imaging system based on structured light illumination of the present invention is based on fluorescence microscopic imaging technology, and can obtain high-resolution histocytopathological images with corresponding fluorescent dye labels, which is convenient for specific research on cell molecules.

In one embodiment, the microendoscopic imaging system based on structured light illumination further includes a beam splitter, the beam splitter is located at the intersection of the propagation direction of the first illumination light path and the propagation direction of the second illumination light path, from the The light in the first illumination optical path and the second illumination optical path enters the same optical path system after being reflected by the beam splitter mirror. The beam combining optical path is located in the optical path behind the beam splitter. Modular design, separation of illumination light path and imaging light path in the microendoscopy system, separation of structured light illumination light path and wide-field illumination light path, simple structure, good stability; moreover, there is no pinhole to block light, high light energy utilization rate, and There are many fluorescent signals, the sensitivity of fluorescence imaging is high, and the obtained fluorescent tissue images are rich in information.

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

In one embodiment, both the first lighting source and the second lighting source are LED light sources, and LEDs are used as light sources, which are economical and environmentally friendly, have high light energy conversion efficiency, avoid potential hazards of laser light sources, are safe and efficient, and have a long service life. long. In one embodiment, since the first illumination light source and the second illumination light source both use LED illumination of a specific wavelength band to correspond to a special excitation wavelength band of the fluorescent dye, the economy is low.

As shown in Figure 2, in one embodiment, in the structured light illumination optical path (i.e. the first illumination optical path), the beam splitter is used as the meeting point, and the first illumination optical path includes the first illumination light source 11, the first A first collimator lens 12 for collimating the light emitted by the illumination source 11 , a grating 13 and a lens 14 for collecting diffracted light generated by the grating 13 . The first illumination light source 11 specifically includes LED lamp beads. The grating 13 includes a number of periodic grooves along the same direction, and the light diffracted by the grating 13 presents a stripe structure to realize stripe illumination. The grating 13 is located at the front focal plane of the lens, and the lens 14 is combined with the imaging optical path to project and image the surface of the grating, so that the periodic fringes of the grating are projected onto the sample surface. The present invention introduces a structured light imaging system, uses the stripe illumination generated by the grating 13 to mark out-of-focus signals, suppresses out-of-focus signals, and effectively improves image contrast.

As shown in Figure 2, since the LED lamp bead used has a large divergence angle, in order to improve the utilization rate of light energy, a first collimating lens 12 is added in front of the LED to collimate the light emitted by the LED lamp bead to collect more More illumination light enters the main light path. The collimated illumination light is incident on the surface of the grating 13, and diffracted light is generated 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, so the The light appears in a specific fringe structure, thus realizing fringe lighting. 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 and the component structure in the imaging optical path can project and image the surface of the grating 13, so that the grating 13 Periodic fringes are projected onto the sample surface. The fringe structured light generated by the structured light path reaches a beam splitter 10, and after being reflected by the beam splitter 10, enters the same optical path system as the light from the wide-field illumination. The function of the beam splitter 10 is to split or combine 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.

In one embodiment, the second illumination light path includes a second illumination light source 21 and a second collimator lens 22 for collimating the light emitted by the second illumination light source 21 . The second illumination light source 21 specifically includes LED lamp beads. The wide-field illumination optical path (that is, the second illumination optical path) only includes the second illumination light source 21 and the matching second collimator lens 22, and the incident direction of the light emitted by it and the direction of the structured light signal are vertical to each other in space, so that Without interference, the collimated light wave also enters the beam splitter smoothly from another direction.

It can be known from the above description that the microendoscopic imaging system based on structured light illumination is mainly divided into an illumination optical path and an imaging optical path. Directional illumination light is introduced into the same optical path system.

As shown in Figure 2, the beam combining optical path is included in the optical path system after the beam combining, and the beam combining optical path includes the first optical filter 31 in turn, the dichroic light for reflecting the spectrum filtered by the first optical filter 31 A mirror 35, an objective lens 33 for collecting the light signal reflected by the dichroic mirror 35, and an optical fiber bundle 34 for transmitting the light wave of the illumination light. The first optical filter 31 is used for spectrally filtering the illumination light from the first illumination light path and the second illumination light path, and blocking the spectrum that is invalid for the excitation process of the fluorescent dye. In this illumination optical path system, only one optical filter is used, that is, only one optical filter is used in the beam combining optical path and the beam combining optical path is located in the optical path behind the beam splitter 10, which can simultaneously illuminate light from the structured optical path and wide-field illumination. The light in the optical path is spectrally filtered to block the spectrum that is invalid for the excitation process of the fluorescent dye. The dichroic mirror 35 is used in conjunction with the first optical filter 31, and the spectrum filtered by the first optical filter 31 can be completely reflected by the dichroic mirror 35 and enters the objective lens. After the optical signal is collected by the objective lens 33, two types The illuminating light beam is smoothly coupled into the fiber bundle 34. Objective lenses are key elements in the light processing process.

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, and the first illumination light source 11 , the second illumination light source 21 and the grating surface of the grating 13 are all located in the image plane space. Specifically, in this illumination light path, the optical fiber is located in the object space of the objective lens, and the light sources of the two 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 optical signals , for structured light illumination, the objective lens 33 reduces the optical signal containing the grating stripe structure and images it on the fiber target surface, and the fiber target surface and the grating surface are in an optical conjugate position, so the objective lens has good aberration correction and flat-field imaging functions. The optical fiber bundle mainly plays the role of transmitting illumination light waves, and transmits excitation light signals to the surface of human tissue.

As shown in FIG. 2 , in one embodiment, the imaging optical path sequentially includes an optical fiber bundle 34 for collecting and transmitting fluorescent signals, an objective lens 33 for amplifying and transmitting the fluorescent signals, and a dichroic lens for processing the fluorescent signals transmitted by the objective lens 33 A mirror 35 and a second filter 32 for spectrally filtering the fluorescence signal. Wherein, the optical fiber bundle 34, the objective lens 33 and the dichroic mirror 35 can be shared by the beam combining optical path and the imaging optical path.

As shown in Figure 2, the imaging optical path also includes an imaging lens 36 and a double-shutter camera 37. The imaging lens 36 and the double-shutter camera 37 are used together to collect and image the fluorescent signal filtered by the second filter 32 and project it to the double-shutter camera. After the fluorescent signal on the shutter camera 37 undergoes photoelectric conversion, an image with human tissue information is displayed. The double-shutter camera 37 is used to collect fluorescence images in pairs, and the collection interval of fluorescence signals excited by two different structured illuminations is shorter, which reduces motion artifacts and has a stronger ability to acquire tissue motion information. Specifically, the imaging optical path is a series of optical transmission systems designed and built 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 Dual shutter cameras37. The fluorescent signal is mainly generated by the excitation of the fluorescent dye by the illumination light, so the excited fluorescent signal is also collected and transmitted by the optical fiber bundle 34 at the same time, and exits from the other end of the optical fiber bundle 34 , that is, the port located at the front focal plane of the objective lens 33 . The objective lens 33 plays the role of amplifying and transmitting the fluorescent signal, and the fluorescent signal generated at this time may contain the illumination spectrum from the excitation light, so the amplified optical signal needs to be filtered by the dichroic mirror 35 and the second filter 32 Invalid spectrum. Since the excitation spectrum and the fluorescence spectrum have a certain spectral difference, the excitation spectrum and the fluorescence spectrum can be effectively separated by using a dichroic mirror 35 corresponding to the spectrum difference. Here, the function of the dichroic mirror 35 is to perform Reflect and transmit the fluorescent signal, so in the imaging optical path, the fluorescent signal can pass through the dichroic mirror 35 smoothly, and another second filter 32 is set behind the dichroic mirror 35 to further block stray light Elimination, to ensure the purity of the fluorescent signal without interference. The imaging lens 36 and the double-shutter camera 37 are used together to collect and image the filtered fluorescent signals, and the fluorescent signals projected onto the CCD target surface of the camera are photoelectrically converted to display images with human tissue information. The transmission imaging process of the entire fluorescent light path is completed above.

In this technical solution, the structured light illumination source and the wide-field illumination source are sequentially turned on for excitation, that is, when the structured light illumination source is turned on, a structured light-excited fluorescence image is collected, and then the structured light illumination source is turned off and clicked. Bright wide-field illumination light source, and then collect a wide-field illumination excitation fluorescence image, the acquisition process is realized by a double shutter camera, that is, in the case of one exposure, fluorescence images are collected in pairs, the acquisition speed is fast, and the imaging frame rate is guaranteed . Since the fluorescence image produced by wide-field illumination contains both in-focus and out-of-focus tissue information, and the fringe structure in the fluorescence image excited by structured light has already marked the in-focus information, the two images are synthesized by algorithm , the in-focus signal can be extracted while the out-of-focus signal is discarded, so as to suppress the background light signal and improve the contrast of the image. At the same time, there is no scanning device in the entire imaging process, and no pinhole blocking light is required, so the imaging speed is faster.

Below in conjunction with specific embodiment the technical scheme of this law is described,

As shown in Figure 3, the light sources of the two structured light illumination paths both use blue LED light sources with a center wavelength of 450nm, with the same power. The first is the structured light illumination optical path. The light emitted by the LED light source 11 (that is, the first illumination light source 11) has a large divergence angle, which is converged by the first collimator lens 12 in turn, and reflected by the grating 13, the lens 14 and the beam splitter 10. After that, it reaches the first optical filter 31 . The first collimating lens 12 is located in front of the LED lamp bead of the LED light source 11, and the LED lamp bead of the LED light source 11 is at the front focal plane position of the first collimating lens 12, and the emitted illumination light is parallel to the space behind the first collimating lens 12. spread. The transmissive diffractive grating 13 is located between the first collimating lens 12 and the lens 14 and at the front focal plane of the lens 14, and is used to shape the collimated light beam and generate an illuminating light beam with a specific structure, and the structured light beam is placed behind The 14 lenses collect and reflect from the reflective surface in the beam splitter 10 to the corresponding propagation direction. The beam splitter 10 has the function of combining beams in two directions, and can transmit light beams from different directions into the same optical path. 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, and the vertical position to the direction of the structured light path is determined by The LED light source 21 (ie the second illumination light source 21) and the wide-field illumination optical path formed by the second collimator lens 22, similarly, the lamp bead of the LED of the LED light source 21 is also positioned at the front focal plane position of the second collimator lens 22, The second collimating lens 22 is close to the beam splitter 10 . After the structured light illumination light and the wide-field illumination light pass through the intersection point of the beam splitter 10, they will first pass through the first filter 31 and the specific band spectrum will pass through the first filter 31 smoothly, and then pass through the two-way beam placed at an angle of 45 degrees. After being reflected by the color mirror 35, it enters the objective lens 33. The dichroic mirror 35 has the function of short-wave reflection and long-wave transmission, that is, it reflects light waves around 450nm and collimates them into the objective lens 33 without deviation, and can also pass through the spectral band above 500nm without loss. The objective lens 33 is located between the fiber bundle 34 and the dichroic mirror 35, and performs imaging and conduction compression on the light beam. The used plan achromatic microscope objective lens usually has a magnification of 10 or 20, so that the spatial aperture of the image space The irradiated beam spot is compressed, and the beam radius is reduced to a certain width to maximize low loss and smoothly couple into the fiber bundle. One end of the optical fiber bundle 34 is located at the front focal plane of the objective lens 33, and the other end is in contact with human tissue through a channel. The illumination beam passes through the fiber bundle to reach the surface of the tissue dyed by the fluorescent dye, and is excited to generate fluorescence of the corresponding band, and at the same time the fluorescence will return along the original path of the fiber bundle.

Since the excitation light of 450nm is used in this embodiment, the wavelength band of the generally generated fluorescence is after 500nm, so that the fluorescence passes through the dichroic mirror smoothly after being enlarged by the microscope objective lens. The second optical filter 32 is located between the dichroic mirror 35 and the imaging lens 36, and the second optical filter 32 can only pass light waves with a central wavelength of 510nm and shield light of other wavelength bands, especially the spectrum below 500nm. The dual-shutter camera 37 generally adopts CCD or CMOS, and is used in conjunction with an imaging lens to collect filtered fluorescent signals and image them on a display after photoelectric conversion.

In the specific implementation process, the light sources of two different lighting structures are sequentially lit according to the time sequence, that is, when the LED light source 11 is lit, only the structured light signal can be used to stimulate the fluorescence on the surface of the biological tissue through the optical fiber bundle. Similarly, When the LED light source 21 is turned on, the light signal that excites the fluorescence is the wide-field illumination light beam. When the two light sources are turned on sequentially to generate excitation light, the double-shutter camera collects each fluorescent signal generated and outputs an image. Use the synthesis algorithm to process each pair of collected two images, suppress the out-of-focus signal, extract the in-focus fluorescence signal, and perform algorithmic preprocessing on the two collected fluorescence images to find the mean square factor , based on the processing results, respectively perform low-frequency and high-frequency filtering on the fluorescent image illuminated by structured light and the fluorescent image illuminated by wide-field illumination, and finally the high and low-frequency information is recombined to obtain the required frame of image. The fluorescence images produced by the two light sources sequentially turned on and illuminated are inconsistent, as shown in Figure 3, (a) is a schematic diagram of fluorescence imaging produced by structured light illumination, in which the stripes are the illumination structures produced by structured light, for each structured light illumination In other words, the frequency and position of the fringes are unchanged. (b) is a schematic diagram of fluorescence imaging produced by wide-field illumination, and there is no structured light illumination fringe structure. The image is processed by an algorithm to synthesize a fluorescent image with effectively suppressed out-of-focus signals.

From the description of the above technical solution, it can be seen that the present invention adds structured light to the optical path of the traditional wide-field fluorescence microscopy imaging, and at the same time connects the optical fiber bundle to construct a microscopic endoscopic imaging system. The system is based on fluorescence imaging. According to the principle, corresponding to a specific fluorescent dye, the corresponding excitation light source and optical path system are designed, and the fluorescent signal is successfully collected and the stray light interference of other bands is shielded, and finally a fluorescent image that can reflect tissue information is obtained.

Compared with prior art, the beneficial effect of the present invention is:

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

2. Based on fluorescence microscopy imaging technology, with the corresponding fluorescent dye labeling, high-resolution histocytopathological images can be obtained, which is convenient for specific research on cell molecules.

3. Introduce the structured light illumination optical path and have a structured light illumination imaging system, use the stripe illumination generated by the grating to mark the in-focus signal, suppress the out-of-focus signal, and effectively improve the image contrast.

4. Without a scanning device, the fluorescence signals are collected sequentially through the combination of structured light illumination and wide-field illumination imaging. Only two images are combined to obtain high-quality fluorescence in-focus images, and the imaging speed is fast.

5. There is no pinhole to block the light, the utilization rate of light energy is high, more fluorescent signals are generated, the sensitivity of fluorescent imaging is high, and the obtained fluorescent tissue images are rich in information.

6. Using dual-shutter cameras to collect fluorescence images in pairs, the acquisition interval of fluorescence signals excited by two different structured light illuminations is shorter, which reduces motion artifacts and has a stronger ability to acquire tissue motion information.

7. Modular design, separation of illumination light path and imaging light path in the microendoscopy system, separation of structured light illumination light path and wide-field illumination light path, simple structure and good stability.

The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements can be made without departing from the technical principle of the present invention. and modifications, these improvements and modifications should also be considered as the protection scope of the present invention.

Claims (16)

1. a kind of microscopy endoscopic imaging method based on Structured Illumination, which is characterized in that itself comprising steps of

Control the second lighting source in the first lighting source and the second illumination path in the first illumination path successively emit swash It shines, and generates fluorescence signal respectively；

Control double-shutter camera is acquired the fluorescence signal generated respectively and obtains two images；

Collected two images are handled, a frame image is obtained.

2. the microscopy endoscopic imaging method according to claim 1 based on Structured Illumination, which is characterized in that the control The second lighting source in the first lighting source and the second illumination path in first illumination path successively emits exciting light and includes Step:

It controls the first lighting source and emits exciting light in first time, only generated by the first lighting source within the first time Excite the optical signal of fluorescence；

The the second time transmitting exciting light of the second lighting source after the first time is controlled, in second time only by second Lighting source generates the optical signal of excitation fluorescence.

3. the microscopy endoscopic imaging method according to claim 1 based on Structured Illumination, which is characterized in that collecting Two images handled, obtain a frame image further comprise:

Using composition algorithm, every collected two images of a pair are all handled, restrain signal out of focus therein, are extracted Out in burnt fluorescence signal, thus a frame image required for obtaining.

4. the microscopy endoscopic imaging method according to claim 1 based on Structured Illumination, which is characterized in that described first Lighting source and the second lighting source are LED light source.

5. a kind of microscopy endoscopic imaging system based on Structured Illumination, characterized in that it comprises:

First illumination path, for generating the illumination light of first direction；

Second illumination path, for generating the illumination light of second direction；

Combined beam light road carries out conjunction beam for the illumination light to the first illumination path and the second illumination path；

Imaging optical path is imaged for collecting fluorescence signal, and to fluorescence signal.

6. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that described first Light the first collimation lens, the light that are collimated that illumination path successively includes the first lighting source, is issued to the first lighting source The lens of grid and the diffraction light for collecting grating generation.

7. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that described first Lighting source is LED light source.

8. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the grating Including several along the periodic groove of same direction, the light after optical grating diffraction is presented striated structure and realizes striped and shine It is bright.

9. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the grating Positioned at the front focal plane position of lens, the lens joint imaging optical path carries out projection imaging to grating surface, thus by grating Periodic stripe projects sample surfaces.

10. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that described Two illumination paths include the second collimation lens that the second lighting source and the light issued to the second lighting source are collimated.

11. the microscopy endoscopic imaging system according to claim 10 based on Structured Illumination, which is characterized in that described Two lighting sources are LED light source.

12. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the conjunction Beam optical path successively includes the first optical filter, the dichroscope for reflecting the spectrum after the first optical filter filters, for collecting The object lens for the optical signal that dichroscope reflects back and the fiber optic bundle for being used for transmission illumination light light wave, first optical filter are used In carrying out spectral filtering to from the illumination light of the first illumination path and the second illumination path, stop to fluorescent dye excitation process Invalid spectrum.

13. the microscopy endoscopic imaging system according to claim 12 based on Structured Illumination, which is characterized in that the object Mirror includes object space and Image space, and the fiber optic bundle is located at the object space of object lens, first lighting source, the second illumination light Source and the grating face of grating are respectively positioned on Image space.

14. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that it is described at As optical path successively includes collecting the fiber optic bundle of transmission fluorescence signal, the object lens that transmission is amplified to fluorescence signal, to object lens biography The dichroscope that defeated fluorescence signal is handled and the second optical filter to fluorescence signal progress spectral filtering.

15. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that it is described at As optical path further includes imaging lens and double-shutter camera, the imaging lens and double-shutter camera are used cooperatively, and are filtered to second The filtered fluorescence signal collection imaging of piece, projects the fluorescence signal on double-shutter camera after photoelectric conversion, shows Image with tissue information.

16. the microscopy endoscopic imaging system according to claim 5 based on Structured Illumination, which is characterized in that the base It further include beam splitter in the microscopy endoscopic imaging system of Structured Illumination, the beam splitter is located at the first illumination path direction of propagation With the position that crosses of the second illumination path direction of propagation, the light from first illumination path and the second illumination path is through beam splitting Enter same light path system after mirror reflection.