WO2012075860A1

WO2012075860A1 - Fluorescence endoscopic imaging method and system

Info

Publication number: WO2012075860A1
Application number: PCT/CN2011/081220
Authority: WO
Inventors: 邵永红; 屈军乐; 牛憨笨
Original assignee: 深圳大学
Priority date: 2010-12-09
Filing date: 2011-10-25
Publication date: 2012-06-14
Also published as: CN102525411A

Abstract

Disclosed are a fluorescence endoscopic imaging method and system. The fluorescence endoscopic imaging method comprises the following steps: generating excitation light (S101); dividing the excitation light into multiple sub-beams, the multiple sub-beams correspond to multiple sub-regions of a sample 12, and fluorescent material is distributed in the sample (S102); adjusting the multiple sub-beams so that each sub-beam is transmitted to the inside of the organism and focused on the sub-regions of the sample 12 (S103); scanning the sample 12 using the multiple sub-beams so that the fluorescent material in each sub-region emits fluorescent light (S104); and collecting the emitted fluorescent light in real time during scanning to generate a fluorescent image (S105). The fluorescence endoscopic imaging method and system perform two-dimensional scan on the sample 12 with multiple sub-beams, thereby obtain the fluorescent image of the entire sample 12, and have the advantages of shorter time, quick and less damages to the organism.

Description

Fluorescence endoscopic imaging method and system

Technical field

The invention belongs to the field of photoelectric detection, and in particular relates to a fluorescence endoscopic imaging method and system.

Background technique

Fluorescence microscopy has become an important tool in life sciences, especially in cell biology research. Multiphoton excited fluorescence microscopy has the advantages of small killing effect on living organisms, large penetration depth and chromatographic ability, and has become an important means of life science research. Fluorescent images provide structural and functional information for biomedical detection and analysis.

In recent years, with the rapid development of new fiber and micro-manufacturing technologies, The study of optical fiber two-photon fluorescence microscopy and endoscopy has made it possible to study two-photon fluorescence microscopy imaging in living organs and living animals. At present, two-photon fluorescence endoscopic microscopy has attracted great attention from the international community. A large number of research results have been made on this subject, endoscopic system design, scanning mechanism, optical conduction and high numerical aperture micro-objectives and their A lot of research results have been achieved in applications. Due to the limitations of the application conditions in vivo, the imaging time should not be too long. However, the current fluorescence endoscopic imaging is slow, inefficient, and time consuming, and has a great impact on living organisms.

technical problem

An object of the embodiments of the present invention is to provide a fluorescence endoscopic imaging method, which aims to solve the problem that the current fluorescence endoscopic imaging is slow and inefficient.

Technical solution

The embodiment of the present invention is implemented as follows. A fluorescence endoscopic imaging method includes the following steps:

Generating excitation light;

Dividing the excitation light into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, wherein the sample is distributed with a fluorescent substance;

Adjusting the plurality of sub-beams such that each sub-beam is conducted into a living body and focused on a sub-region of the sample;

Scanning the sample with the plurality of sub-beams to fluoresce the fluorescent substance in each sub-area;

The fluorescence emitted during the scan is acquired in real time to generate a fluorescent image.

Another object of embodiments of the present invention is to provide a fluorescence endoscopic imaging system, the system comprising:

Exciting a light source for generating excitation light;

a beam splitter, configured to divide the excitation light into a plurality of sub-beams, the plurality of sub-beams corresponding to a plurality of sub-regions of the sample, wherein the sample is distributed with a fluorescent substance;

a flexible medium for adjusting the plurality of sub-beams to conduct the plurality of sub-beams into the living body;

a focusing element for focusing each sub-beam to a sub-region of the sample;

a scanning element for scanning the sample by using the plurality of sub-beams to fluoresce the fluorescent substance in each sub-area;

a dichroic mirror and an imaging medium for directing the fluorescence from the living body;

a detector for real-time collecting fluorescence emitted during scanning to generate a fluorescent image;

The dichroic mirror is disposed between the scanning element and the focusing element.

Beneficial effect

In the embodiment of the invention, the excitation light is divided into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, and the plurality of sub-beams are transmitted to the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, which is fluorescent. Export and multi-beam scanning of the sample to obtain the fluorescence image of the whole sample, the time is short, the speed is fast, and the damage to the organism is small, which is beneficial to biomedical research, especially for early diagnosis of cancer. significance.

DRAWINGS

1 is a flowchart of an implementation of a fluorescence endoscopic imaging method according to an embodiment of the present invention;

2 is a schematic diagram of a structure and an optical path diagram of a fluorescence endoscopic imaging system according to an embodiment of the present invention;

FIG. 3 is a dot diagram of a laser array according to an embodiment of the present invention.

Embodiments of the invention

The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It is understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In the embodiment of the invention, the excitation light is divided into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, and the plurality of sub-beams are transmitted to the living body, and each sub-beam is focused on a sub-region of the sample to form multi-point excitation fluorescence, which is fluorescent. The two-beam scanning of the sample is performed by two sub-beams, so that the fluorescence image of the whole sample is obtained, and the time is short, the speed is fast, and the damage to the organism is small, which is beneficial to biomedical research.

The fluorescence endoscopic imaging method provided by the embodiment of the invention includes the following steps:

Generating excitation light;

The fluorescence endoscopic imaging system provided by the embodiment of the invention includes:

Exciting a light source for generating excitation light;

a focusing element for focusing each sub-beam to a sub-region of the sample;

The implementation of the present invention will be described in detail below with reference to specific embodiments.

FIG. 1 is a flowchart showing an implementation process of a fluorescence endoscopic imaging method according to an embodiment of the present invention, which is described in detail as follows:

In step S101, excitation light is generated;

In the embodiment of the present invention, a femtosecond (ultra-short) pulsed laser with a working frequency of 76 MHz, a period of 120 fs, and a center wavelength of 800 nm is preferably used as the excitation light, and the excitation light can realize two-photon excitation of the fluorescent substance. Generally, the pulsed laser is subjected to beam expansion collimation and its intensity distribution is adjusted to form a flat-top beam having an evenly distributed intensity.

In step S102, the excitation light is divided into a plurality of sub-beams, and the plurality of sub-beams correspond to a plurality of sub-regions of the sample, and a fluorescent substance is distributed in the sample;

In the embodiment of the invention, the excitation light uniformly distributed in intensity is divided into a plurality of sub-beams, and the plurality of sub-beams correspond one-to-one to a plurality of sub-regions of the sample having the fluorescent substance.

In step S103, the plurality of sub-beams are adjusted such that each sub-beam is conducted into the living body and focused on a sub-region of the sample;

Embodiments of the present invention cause a plurality of sub-beams to be conducted in parallel to a living body, each focusing on a sub-region of the sample. Specifically, a plurality of sub-beams are first coupled into the flexible medium, and the plurality of sub-beams enter the living body in parallel via the flexible medium, and the plurality of sub-beams are focused in the living body to form the excitation light array points and respectively projected to the corresponding sub-regions. The flexible medium is a photonic crystal fiber array, the input end of which is located outside the organism, and the output end is located in the living body.

Before multiple sub-beams are coupled into the flexible medium, each sub-beam is collimated such that each sub-beam becomes parallel. After the plurality of sub-beams are output from the flexible medium, they need to be collimated, so that each sub-beam is focused on the corresponding sample sub-area.

In step S104, the sample is scanned by using a plurality of sub-beams to fluoresce the fluorescent substance in each sub-area;

In the embodiment of the invention, the scanning is divided into line scanning and step scanning, and the specific process is as follows:

1, line scan

The plurality of sub-beams are focused to form an excitation light array point to be projected onto the sample, and each sub-area is linearly scanned along the longitudinal direction of the sample, and the fluorescent substance in each sub-area emits fluorescence under the action of the excitation light array point. This line scans quickly and for a short time.

2, step scan

After the end of the vertical line scan of each sub-area, step-scanning of each sub-area in the lateral direction of the sample adjusts the position of the excitation light array point in the lateral direction of the sample.

The above line scan and step scan are performed cyclically until the scanning of each sub-area of the sample is completed. It should be understood that the direction of the line scan and the step scan can also be changed in the specific implementation. In addition, the sample can be scanned randomly, and the sub-areas of the sample can be scanned.

In step S105, fluorescence emitted during scanning is acquired in real time to generate a fluorescent image.

In the embodiment of the present invention, the fluorescence emitted by the fluorescent substance in each sub-area is collected while scanning the sub-areas to generate a fluorescent image. Specifically, the fluorescence emitted by the fluorescent substance in each sub-area is first derived from the imaging fiber, and then the intensity information of the fluorescence and the position information of the fluorescence in the sample are acquired by the detector, and finally the fluorescence image is generated from the position and intensity information of the fluorescence.

It should be understood by those skilled in the art that all or part of the steps of the above embodiments may be implemented by a program to instruct related hardware, and the program may be stored in a computer readable storage medium such as a ROM/RAM or a disk. , CD, etc.

FIG. 2 shows the structure of a fluorescence endoscopic imaging system according to an embodiment of the present invention. For the convenience of description, only parts related to the embodiment of the present invention are shown.

The fluorescence endoscopic imaging system provided by the embodiment of the invention has an excitation light path and a detection light path. The excitation light path includes an excitation light source, a beam expanding collimator, a shaper, a beam splitter, a collimating lens, a first coupling lens, a photonic crystal fiber array, a first self-focusing lens, a scanning mirror, and a micro objective lens. The detection optical path includes a micro objective lens, a dichroic mirror, a second autofocus lens, a imaging fiber bundle, a second coupling lens, a filter element, an imaging lens, and a detector. The micro objective lens is shared by the excitation light path and the probe light path.

The structure of the excitation light path will be described in detail below.

As shown in FIG. 2, in the embodiment of the present invention, a titanium gemstone femtosecond laser 1 is preferably used as an excitation light source, which can generate a pulsed laser having a center wavelength of 800 nm, a frequency of 76 MHz, and a period of 120 fs, and the pulsed laser can realize two-photon of a fluorescent substance. excitation. The pulsed laser light is converted into collimated light of a desired size via the beam expanding collimator 2.

In the embodiment of the present invention, the shaper is a beam shaper 3, and the collimated pulse laser is shaped by the beam shaper 3 to form a flat top beam with uniform intensity distribution. The spectroscope may be a microlens array, a diffractive optical element or a beam splitter. In this embodiment, the microlens array 4 is preferred. The flat top distributed pulsed laser light is divided into a plurality of sub-beams via the microlens array 4, and the plurality of sub-beams correspond to the sample 12 In the sub-region, the microlens array 4 in this embodiment is a 3×3 microlens array, that is, the microlens array has nine micro objective lenses.

The back focal plane of the collimating lens 5 coincides with the front focal plane of the microlens array 4, and the sub-beams are focused on the front focal plane of the microlens array 4, that is, on the back focal plane of the collimating lens 5, and the sub-beams are collimated through the collimating lens. 5 all become sub-beams of parallel light.

In an embodiment of the invention, a plurality of sub-beams are coupled and coupled into the photonic crystal fiber array 7 via the first coupling lens 6. The photonic crystal fiber array 7 is formed by equally arranging a plurality of photonic crystal fibers, and the number of photonic crystal fibers and their arrangement are the same as those of the microlens array 4. A plurality of sub-beams are conducted through the photonic crystal fiber array 7 into the living body, and a plurality of sub-beams emerging from the photonic crystal fiber array 7 are projected to the scanning mirror 9 via the first autofocus lens 8. The self-focusing lens is a rod lens whose refractive index is gradually gradual, and each of the sub-beams becomes parallel light via the first self-focusing lens 8. Each of the sub-beams is projected by a scanning mirror 9 to a sample 12 having a fluorescent substance, and a micro-objective 11 for converging is provided between the sample 12 and the scanning mirror 9. The scanning mirror 9 is preferably a MEMS (Micro-Electro-Mechanical) Systems, MEMS) scanning mirrors, MEMS scanning mirrors are two-dimensional scanning mirrors for line scanning and step scanning of samples.

As shown in FIG. 3, each sub-beam is conducted through the excitation light path to form an excitation light array point and is focused on a sub-area of the sample 12, and the fluorescent substance in the excitation sample 12 emits fluorescence. Correspondingly, the fluorescence also has a plurality of sub-beams.

The structure of the probe light path will be described in detail below.

In the embodiment of the present invention, the dichroic mirror 10 is disposed between the scanning mirror 9 and the micro objective lens 11. The dichroic mirror 10 is highly transparent to a pulsed laser having a center wavelength of 800 nm, and is highly reflective to a wavelength of 400 to 700 nm. The dichroic mirror 10 The angle between the fluorescence and the fluorescence is 45 or 135. The above fluorescence is collected by the micro objective lens 11 to form a plurality of collimated fluorescent sub-beams, and the dichroic mirror 10 reflects the fluorescence from the excitation light path and is focused by the second autofocus lens 13 on the inner end of the imaging fiber bundle 14. The fluorescent sub-beam is derived from the living body by the imaging fiber bundle 14 and converted into multiple parallel light by the second coupling lens 13, and the excitation light and other stray light are filtered by the filter element 16, and focused by the imaging lens 17 to the detector 18. Sensitive face. The detector 18 collects the fluorescence emitted by the scanning mirror 9 in real time, thereby obtaining the intensity information of the fluorescence and the positional information of the fluorescence in the sample 12. Finally, the fluorescence image is generated from the position and intensity information of the fluorescence.

The focal plane of the excitation light array point in the sample 12 and the inner end surface of the imaging fiber bundle 14 are mutually conjugated, and the outer end surface of the image fiber bundle 14 and the sensitive surface of the detector 18 are mutually conjugated surfaces, that is, Fluorescence excited from different positions in the sample 12 is focused by the micro objective lens 11 and the second self-focusing lens 13 to corresponding positions on the inner end surface of the image fiber bundle 14, and transmitted from the image fiber bundle 14 to the corresponding position on the outer end surface of the body, and then The second coupling lens 15 and the imaging lens 17 are focused to corresponding positions of the detector 18. In this way, the detector 18 can detect the fluorescence of the focal plane in the sample 12 to generate a fluorescent image.

In the embodiment of the present invention, the detector 18 is an area array detector for acquiring intensity and spatial information of fluorescence, and the area array detector is preferably a CCD camera or a CMOS camera. The area array detector is coupled to a computer 21 which stores, processes and reads the fluorescence intensity and position information detected by the detector 18, and the computer 21 controls the exposure of the area array detector.

Typically, scanning mirror 9 begins scanning and detector 18 begins to expose; when scanning of sample 12 is complete, detector 18 is exposed. The exposure time of the detector 18 is the same as the scanning time of the scanning mirror 9, and the scanning time may be the time when the sample is scanned once, or may be an integral multiple of the scanning time.

The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. Within the scope.

Claims

A fluorescence endoscopic imaging method, characterized in that the method comprises the following steps:

Generating excitation light;

Dividing the excitation light into a plurality of sub-beams corresponding to a plurality of sub-regions of the sample, wherein the sample is distributed with a fluorescent substance;

Adjusting the plurality of sub-beams such that each sub-beam is conducted into a living body and focused on a sub-region of the sample;

Scanning the sample with the plurality of sub-beams to fluoresce the fluorescent substance in each sub-area;

The fluorescence emitted during the scan is acquired in real time to generate a fluorescent image.
The fluorescence endoscopic imaging method according to claim 1, wherein said scanning is divided into a line scan and a step scan, and said plurality of sub-beams are used to scan said sample so that each sub-area The step of emitting fluorescence by the fluorescent substance is specifically as follows:

Each sub-beam scans a corresponding sub-area along the longitudinal line of the sample, so that the fluorescent substance in each sub-area emits fluorescence;

After the line scanning is finished, the corresponding sub-areas are step-scanned along the lateral direction of the sample, that is, the position of each sub-beam in the lateral direction of the sample is adjusted;

The line scan and step scan are performed cyclically until the scanning of each sub-area is completed.
The fluorescence endoscopic imaging method according to claim 1, wherein the step of real-time collecting fluorescence emitted during scanning to generate a fluorescent image is specifically:

Fluorescence emitted by the fluorescent substance in each sub-area is derived from the image-forming optical fiber;

Obtaining intensity information of the fluorescence and position information of the fluorescence in the sample by the detector;

A fluorescent image is generated from the position and intensity information of the fluorescence.
The fluorescence endoscopic imaging method according to claim 1, wherein the step of generating excitation light further comprises the following steps:

Performing beam expansion and collimation on the excitation light;

Adjusting an intensity distribution of the excitation light to make the intensity distribution of the excitation light uniform;

The step of adjusting the plurality of sub-beams such that each sub-beam is conducted into a living body and focused on a sub-region of the sample is specifically:

The plurality of sub-beams are coupled into the photonic crystal fiber array and enter the living body via the photonic crystal fiber array;

A plurality of sub-beams are focused in the living body to form an array of excitation light to be projected onto the sample.
A fluorescent endoscopic imaging system, the system comprising:

Exciting a light source for generating excitation light;

a beam splitter, configured to divide the excitation light into a plurality of sub-beams, the plurality of sub-beams corresponding to a plurality of sub-regions of the sample, wherein the sample is distributed with a fluorescent substance;

a flexible medium for adjusting the plurality of sub-beams to conduct the plurality of sub-beams into the living body;

a focusing element for focusing each sub-beam to a sub-region of the sample;

a scanning element for scanning the sample by using the plurality of sub-beams to fluoresce the fluorescent substance in each sub-area;

a dichroic mirror and an imaging medium for directing the fluorescence from the living body;

a detector for real-time collecting fluorescence emitted during scanning to generate a fluorescent image;

The dichroic mirror is disposed between the scanning element and the focusing element.
The fluorescent endoscopic imaging system according to claim 5, wherein the excitation light source and the optical splitter are further provided with:

a beam expanding collimating device for adjusting the size of the excitation light and performing collimation;

a shaper for adjusting an intensity distribution of the excitation light to make the intensity distribution of the excitation light uniform;

The optical splitter and the flexible medium are further provided with:

a collimating lens for collimating the sub-beams such that the sub-beams become parallel light;

a first coupling lens for coupling each sub-beam into the flexible medium;

The flexible medium and the scanning mirror are further provided with:

a first self-focusing lens for collimating respective sub-beams output from the flexible medium;

The dichroic mirror and the imaging medium are further provided with:

a second self-focusing lens for focusing the fluorescent light on an inner end of the imaging medium;

The imaging medium and the detector are further provided with:

a second coupling lens for collimating fluorescence output from the imaging medium;

An imaging lens for imaging the fluorescence to the detector;

The focusing element is a micro objective.
The fluorescence endoscopic imaging system according to claim 6, wherein the spectroscope is a microlens array, a diffractive optical element or a beam splitter, a front focal plane of the microlens array and the collimating lens The back focal planes are coincident; the flexible medium is a photonic crystal fiber array, and the image medium is an image fiber bundle.
A fluorescent endoscopic imaging system according to claim 7 wherein said fluorescence is collected by said micro objective and forms a plurality of collimated fluorescent sub-beams; said dichroic mirror exciting said fluorescent sub-beams The optical path is reflected and focused by the second autofocus lens at the inner end of the imaging fiber bundle.
The fluorescent endoscopic imaging system according to claim 8, wherein a focal plane of the excitation light array point in the sample and a body end surface of the imaging fiber bundle are mutually conjugated, the image fiber bundle The outer surface of the outer surface and the sensitive surface of the detector are mutually conjugated.
The fluorescence endoscopic imaging system according to any one of claims 5 to 9, wherein the exposure time of the detector is the same as the scanning time of the scanning element, and the scanning time of the scanning element is the sample The time to complete a scan, or the time to complete multiple scans of the sample.