WO2017104949A1

WO2017104949A1 - Confocal microscope and image processing method using same

Info

Publication number: WO2017104949A1
Application number: PCT/KR2016/010818
Authority: WO
Inventors: 김필한; 안진효
Original assignee: 한국과학기술원
Priority date: 2015-08-04
Filing date: 2016-09-27
Publication date: 2017-06-22
Also published as: KR101898220B1; KR20170016773A

Abstract

The present invention relates to a confocal microscope and an image processing method using the same. A confocal microscope according to an embodiment of the present invention comprises: a photographing unit for outputting at least two laser light sources having different wavelengths to a photographing region, scanning a light reflected from the photographing region in a confocal manner, and then obtaining a two-dimensional image; an optical probe which is inserted into the photographing region to transfer a light output by the photographing unit to the photographing region and transfer the light reflected from the photographing region to the photographing unit; and a coupling unit for coupling a lens of the photographing unit and the optical probe and controlling a movement of the optical probe.

Description

Confocal Microscope and Image Processing Method Using the Same

The present invention relates to a confocal microscope and an image processing method using the same.

A confocal microscope is a microscope having a resolution in the depth direction, and is widely used in various industries and bio fields because it can obtain three-dimensional information of a specimen. Confocal microscopes are known to have about 40% better resolution than conventional microscopes, and their utility is superior in that they can acquire three-dimensional images without the need for physical sections.

However, it is difficult to photograph and image the composition of internal tissues in a live animal model because of the limited size of the objective lens which cannot be accessed internally and the penetration depth of the laser light source.

Due to these limitations, cell imaging studies using confocal microscopy are mostly performed by observing the epidermal state of skin or by histological analysis of in vitro samples.

In order to solve this problem, an endoscopic microscope method is used to transmit a light source transmitted through an objective lens of a confocal microscope into a tissue through a small lens. It is difficult and there is a problem that the loss of the light source and the detection signal may occur in the process of transferring the light source from the objective lens to the small lens has been studied to improve this.

The technical problem to be achieved by the present invention is to provide a confocal microscope and an image processing method using the same that can be photographed and imaged finely inside the biological tissue.

The confocal microscope according to the embodiment of the present invention outputs at least two laser light sources having different wavelengths to a photographing site, and acquires a two-dimensional image by scanning light reflected from the photographing site in a confocal manner. And an optical probe which invades the photographing part, transmits the light output from the photographing part to the photographing part, and transmits the light reflected from the photographing part to the photographing part, and combines the lens of the photographing part and the optical probe. And a coupling for controlling movement of the optical probe.

The optical probe may include a needle tip invading into the living body, and a lens unit positioned inside the needle tip.

The lens unit may include a coupling lens positioned on the photographing unit side, an image lens positioned on the photographing part side, and a relay lens positioned between the coupling lens and the image lens.

The coupling lens, the image lens and the relay lens may be formed of a gradient-index (GRIN) lens.

The optical probe may further include a mirror unit positioned at an end of the lens unit and reflecting the light to change a traveling direction of the light.

The coupling part may include an x and y axis moving part for controlling the movement of the optical probe in the x and y axis directions, and a z axis moving part for controlling the movement of the optical probe in the z axis direction.

The needle tip may include a tubular body, and one end of the body may include an opening that exposes at least a portion of the lens unit.

The front end portion of the main body may have an incline in cross section.

The inclination angle of the tip portion may be 1 to 20 degrees.

A protective layer surrounding the needle tip may be further included.

The protective layer may be made of an optical adhesive film.

The photographing unit may include a light source unit for outputting at least two laser light sources having different wavelengths, an objective lens adjusted to focus the laser light source at the photographing site, and reading an image observed from the photographing site in a two-dimensional array form. It may include a scanning unit for generating a scanning image, and an image acquisition unit for passing the scanning image to a slit portion installed in a confocal plane to generate an image excited by the laser light source.

An image processing method using a confocal microscope according to an embodiment of the present invention comprises the steps of invading the optical probe to the in vivo imaging area through the skin, irradiating a laser light source to the imaging area, and the light reflected from the imaging site And receiving the received image.

According to the exemplary embodiment of the present invention, the inside of the living tissue may be finely approached and photographed to image it. According to the exemplary embodiment of the present invention, the loss of the light source and the detection signal can be reduced.

1 is a view showing a schematic configuration of a confocal microscope according to an embodiment of the present invention.

2 is a view showing a detailed configuration of the confocal microscope according to an embodiment of the present invention.

3 is a view showing the structure of a confocal microscope optical probe according to an embodiment of the present invention.

4 is a diagram illustrating a confocal microscope optical probe according to an embodiment of the present invention.

5 is an exploded cross-sectional view of a structure of a confocal microscope optical probe according to one embodiment of the present invention.

6 is a view showing the structure of the confocal microscope coupling portion according to an embodiment of the present invention.

7 is a view showing the flow of an image processing method using a confocal microscope according to an embodiment of the present invention.

8 to 10 are diagrams showing photographing results obtained by using a confocal microscope according to an embodiment of the present invention.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art may easily implement the present invention. As those skilled in the art would realize, the described embodiments may be modified in various different ways, all without departing from the spirit or scope of the present invention. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and like reference numerals designate like parts throughout the specification.

Throughout the specification, when a part is said to "include" a certain component, it means that it can further include other components, without excluding other components unless specifically stated otherwise.

A confocal microscope according to an embodiment of the present invention will now be described in detail with reference to the drawings.

1 is a view showing a schematic configuration of a confocal microscope according to an embodiment of the present invention, Figure 2 is a view showing a detailed configuration of a confocal microscope according to an embodiment of the present invention.

Referring to FIG. 1, the confocal microscope 1 according to the present embodiment includes a photographing unit 10, an optical probe 20, a coupling unit 30, and a computing unit 40.

As shown in FIG. 2, the photographing unit 10 includes a light source unit 110,

beam splitters

120a, 120b, 120c and 120d, a scan unit 130, an objective lens 140, and an image acquisition unit 150. It may include.

The light source unit 110 is an excitation light source of the image capturing unit 10 and irradiates light to excite the image capturing portion. The light source unit 110 includes two or more light sources having different wavelengths in order to photograph the observation targets labeled with different fluorescent samples. In the present exemplary embodiment, the light source unit 110 may include four laser light sources having a visible light band, and the laser light sources may have wavelength bands of 405 nm, 488 nm, 561 nm, and 640 nm, respectively. However, the present invention is not limited thereto, and the number and wavelength of the light sources may vary.

The light irradiated from the light source unit 110 passes through the scan unit 130 through at least one or

more beam splitters

120a, 120b, 120c, and 120d, and then passes through the objective lens 140 and the optical probe 20. Investigate the internal filming site.

The optical probe 20 invades the inside of the living tissue through the skin of the living tissue, transfers the light irradiated from the light source unit 110 to the imaging area inside the living tissue, and retransmits the reflected light from the inside of the living tissue 10. To pass). The shape of the optical probe 20 may vary, but in the present invention, a needle-shaped optical probe that is easy to invade will be described as an example.

At this time, the coupling unit 30 serves to couple the optical probe 20 to the imaging unit 10 to be fixed, and the detailed configuration of the optical probe 20 and the coupling unit 30 will be described in more detail below. Do it.

The light reflected from inside the biological tissue is returned to the imaging unit 10 through the optical probe 20. The reflected light passes through the objective lens 140 and the scan unit 130 in order, and is transmitted to the image acquisition unit 150.

The objective lens 140 is a lens into which the light of the fluorescent material excited by the light source unit 110 enters, and outputs an image signal in which an image of a photographing part labeled with the fluorescent material is formed to the scanning unit 130. In the present embodiment, the objective lens 140 may be set to have a field of view of 250 × 250um ² using a 40x objective lens or to have a field of view of 500 × 500um ² using a 20x objective lens. The present invention is not limited thereto and may be set to have various fields of view by setting a lens having an appropriate magnification.

The scan unit 130 reads an image signal incident through the objective lens 140 and configures the pixel in a 2D array form. In this case, the scan unit 130 may include a polygonal rotation mirror and a galvanometer mirror. The rotating polygon mirror scans the X-line, and the galvano mirror scans the Y-line.

The image acquisition unit 150 passes the scanning image signal generated by the scan unit 130 to at least one

beam splitter

151a, 151b, or 151c and separates each wavelength band. The separated scanning image signal passes through a band pass filter (BPF) 152 and a condensing lens 153 to a slit portion 154 installed in a confocal plane and is a photomultiplier pipe. tube, PMT) (155).

The

beam splitters

151a, 151b, and 151c separate and arrange the scanning images generated from the scan unit 130, and may be formed of a dichroic beam splitter (DBS).

The bandpass filter unit 152 is positioned in a path of light split from the

beam splitters

151a, 151b, and 151c, acquires the separated light, and passes the light in the spectral region of the designated visible light region.

The photomultiplier tube 155 is positioned in the path of the light passed from the bandpass filter unit 152, detects the fluorescent signal passing through the bandpass filter unit 152, generates an electric signal, and generates the computing signal 40. )

The computing unit 40 acquires an image signal photographed by the photographing unit 10. In this case, the computing unit 40 may correct the photographing result in order to obtain a physically meaningful result.

As described above, the confocal microscope 1 including the optical probe 20 can directly invade the internal organ tissue through the skin of the living body, and can access internal organ tissues. Shooting can be freely controlled and the inside of the photographed tissue can be imaged. In addition, the confocal microscope 1 including the optical probe 20 can invade the exact position and reduce the loss of light source and detection signal that can occur due to the use of the optical fiber.

Hereinafter, the optical probe 20 will be described in detail with reference to FIGS. 3 to 5.

3 is an exploded cross-sectional view of a structure of a confocal microscope optical probe according to an embodiment of the present invention, and FIG. 4 is a view showing an example of a confocal microscope optical probe according to an embodiment of the present invention. 5 is a view showing the structure of a confocal microscope optical probe according to an embodiment of the present invention.

As shown in FIG. 3, the optical probe 20 includes a needle tip 210 and a lens unit 220.

The needle tip 210 has a tubular body 211 having a circular or elliptical cross section in which the needle tip 210 is infiltrated to the imaging part, that is, the core of the biological tissue, and the inside thereof may be hollow because the hollow is formed. The lens unit 220 is positioned inside the main body 211.

In this embodiment, the tip 212 of the needle tip 210 main body 211 may be formed to have a predetermined slope as shown in FIG. 3 to facilitate invasion into the living body. At this time, the inclination angle may be 1 degree to 20 degrees.

One end of the body 211 may include an opening 213 exposing at least a portion of the lens unit 220. In this embodiment, the opening portion 213 may be formed by removing one side of the side portion of the main body 211 so that its cross section has a semi-circular shape in order to capture the image closer to the photographing portion inside the biological tissue.

Meanwhile, the needle tip 210 may have a diameter of 0.5 mm to 1.0 mm and may be invasive to the inside of the living body, and its length may be 10 mm to 30 mm.

In addition, to prevent the inflow of foreign matter when invading the inside of the biological tissue, it may further include a protective layer surrounding the surface of the needle tip (210). At this time, the protective layer may be composed of an optical adhesive film.

The lens unit 220 transmits the light irradiated from the light source unit 110 to the image capturing site inside the biological tissue, and transmits the light reflected from the image capturing site located inside the biological tissue to the image capturing unit 10.

The lens unit 220 may have three lens structures having the same diameter as shown in FIG. 5, and the lens unit 220 according to the present exemplary embodiment is a coupling lens positioned near the photographing unit 10. And a relay lens 223 positioned between the coupling lens 221 and the image lens 222.

Each lens may be formed of a gradient-index (GRIN) lens, and the NA (Numerical Aperture) of the coupling lens 221 and the image lens 222 is 0.45 to 0.55, and the NA of the relay lens 223 is 0.15 to 0.25.

As shown in FIG. 5, the optical probe 20 may further include a mirror 230 attached to the distal end of the image lens 222. The mirror unit 230 reflects light to change a path of light. The mirror unit 230 may be coated with aluminum, and the laser light source passing through the lens unit 220 is irradiated to the photographing site.

The mirror unit 230 is disposed at an angle of 45 degrees to the longitudinal direction of the lens unit 220 to change the traveling direction of the laser light source in a second direction perpendicular to the first direction through which the laser light source passes through the lens unit 220. Can be. However, the present invention is not limited thereto, and the mirror unit 230 may be disposed in various directions to change a traveling direction of the laser light source. Accordingly, the confocal microscope 1 according to the present embodiment irradiates a laser light source to a photographing site in various directions as well as a depth direction in which the needle tip 210 invades living tissue, and transmits light reflected therefrom. Receive the image.

Hereinafter, the coupling unit 30 will be described in detail with reference to FIG. 6. 6 is a view showing the structure of the coupling portion of the confocal microscope according to the present embodiment.

Referring to FIG. 6, the coupling part 30 according to the present exemplary embodiment may include a main body part, an x and y axis moving part 320, including a plate 311, a rod part 312, and an adapter 313. It includes a z-axis moving unit 330, and the probe holder 340.

The x, y-axis moving part 320 controls the movement of the planar direction (x, y-axis) of the optical probe 20, and the z-axis moving part 330 is a depth (height) direction (of the optical probe 20) ( z-axis) control. Accordingly, the confocal microscope 1 according to the present embodiment finely and accurately controls the movement in the three-axis direction of the optical probe 20, so that the body tissue of the desired site can be photographed.

The probe holder 340 fixes the position of the optical probe 20 to the photographing unit 10, and may prevent the movement of the optical probe 20 while imaging the photographed portion.

Referring to FIG. 7, the confocal microscope according to the embodiment of the present invention invades the optical probe 20 to the in vivo photographing site through the skin (S100). The optical probe 20 may include a needle tip 210 having an inclined tip portion 212 to facilitate penetration into the living body.

The needle tip 210 may include an opening 213 formed by removing one side of the side part such that its cross section has a semi-circle shape in order to obtain an image in close contact with a photographed part of the living body.

In addition, the needle tip 210 may further include a protective layer surrounding the outer surface of the needle tip to prevent foreign substances from infiltrating into the biological tissue.

Next, the laser light source is irradiated to the imaging area inside the biological tissue (S200). The laser light source is irradiated from the light source unit 110 of the imaging unit 10, and may have a wavelength band of 405nm, 488nm, 561nm, and 640nm, respectively. The laser light source may be transmitted to the imaging area inside the biological tissue through the lens unit 200 of the optical probe 20.

Then, the light is received from the photographed portion receives the image (S300). The light reflected from inside the biological tissue is returned to the imaging unit 10 through the optical probe 20. The reflected light passes through the objective lens 140 and the scan unit 130 in order to be transferred to the image acquisition unit 150, whereby the confocal microscope can acquire an image of the photographed portion.

In the image processing method using confocal according to an embodiment of the present invention, by directly invading through the skin of the living body and imaging it, it is possible to obtain an image of directly photographing internal organ tissues, which may occur due to the use of optical fibers. The loss of the light source and the detection signal can be reduced.

FIG. 8 is a view illustrating imaging results obtained by continuously photographing somatic cells and blood vessels existing in the epidermis and dermis of the skin while inserting the needle tip 210 in the depth direction from the surface of the skin. At this time, somatic cells were labeled with a green fluorescent signal, and blood vessels were labeled with a red fluorescent signal.

The skin is composed of the epidermis, the basement, the dermis, and the subcutaneous tissue, which are epithelial cells of the outermost layer. Referring to FIG. You can see that the image.

FIG. 9 is a diagram illustrating a photographing result obtained by photographing and imaging an inside of a cancer tissue located in a deep tissue part of a living body. In order to perform effective imaging inside the tissue, in this embodiment, cancer cells, blood vessels inside the cancer tissue, and low oxygen regions were labeled with different fluorescent signals.

Referring to FIG. 9, images of cancer cells labeled with green and blood vessels inside with cancer tissues labeled with red and low oxygen regions labeled with blue were photographed. The confocal microscope according to the present embodiment is a simple transmission image of skin only. In addition, it can be seen that the image signal can be obtained by penetrating the deep tissue of the living body.

FIG. 10 is a diagram showing the results of continuous imaging while penetrating the inside of cancer tissue located in the deep tissue part of the living body in the depth (z-axis) direction. Referring to FIG. 10, it can be seen that an image up to 6.8 mm deep of a living tissue is obtained. The confocal microscope according to the present embodiment can confirm that imaging of a transmission depth that cannot be taken by a general confocal microscope is possible. have.

Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to the scope of rights.

Claims

A photographing unit which outputs at least two laser light sources having different wavelengths to a photographing part, and acquires a 2D image by scanning light reflected from the photographing part by a confocal method;

An optical probe which invades the photographing part, transmits the light output from the photographing part to the photographing part, and transmits the light reflected from the photographing part to the photographing part;

A coupling unit for coupling the lens of the photographing unit and the optical probe and controlling the movement of the optical probe,

The optical probe has a needle shape confocal microscope.
In claim 1,

The optical probe,

A needle tip that invades the living body, and

Confocal microscope including a lens portion located inside the needle tip.
In claim 2,

The lens unit,

A coupling lens positioned on the photographing unit side;

An image lens positioned at the side of the photographing portion, and

A confocal microscope comprising a relay lens positioned between the coupling lens and the image lens.
In claim 3,

The coupling lens, the image lens and the relay lens is a confocal microscope consisting of a gradient (index-index, GRIN) lens.
In claim 2,

The optical probe,

Located at the end of the lens portion, by reflecting the light, the confocal microscope further comprises a mirror for changing the direction of travel of the light.
In claim 1,

The coupling unit, x, y-axis moving unit for controlling the movement in the x, y axis direction of the optical probe, and

A confocal microscope including a z-axis moving unit for controlling the movement in the z-axis direction of the optical probe.
In claim 2,

The needle tip includes a tubular body and one end of the body includes an opening that exposes at least a portion of the lens portion.
In claim 7,

A confocal microscope having a tip of the main body having an inclined cross section.
In claim 8,

The inclination angle of the tip portion is a confocal microscope of 1 to 20 degrees.
In claim 2,

A confocal microscope further comprising a protective layer surrounding the needle tip.
In claim 10,

The protective layer is a confocal microscope composed of an optical adhesive film.
In claim 1,

The photographing unit,

A light source unit for outputting at least two laser light sources having different wavelengths,

An objective lens in which the laser light source is adjusted to focus at the photographing site;

A scanning unit which reads the image observed from the photographing site and generates a scanning image in a 2D array form, and

And an image acquisition unit for passing the scanning image to a slit unit installed in a confocal plane to generate an image excited by the laser light source.
As an image processing method using a confocal microscope,

Invading the optical probe through the skin to the in vivo imaging area,

Irradiating at least two laser light sources having different wavelengths to the imaging area, and

The image processing method using a confocal microscope comprising the step of obtaining an image by receiving the light reflected from the photographed portion.