KR20170016773A - Confocal microscopy and method of processing image using the same - Google Patents

Confocal microscopy and method of processing image using the same Download PDF

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KR20170016773A
KR20170016773A KR1020150179848A KR20150179848A KR20170016773A KR 20170016773 A KR20170016773 A KR 20170016773A KR 1020150179848 A KR1020150179848 A KR 1020150179848A KR 20150179848 A KR20150179848 A KR 20150179848A KR 20170016773 A KR20170016773 A KR 20170016773A
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lens
unit
photographing
optical probe
image
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KR1020150179848A
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KR101898220B1 (en
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김필한
안진효
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한국과학기술원
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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0052—Optical details of the image generation
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/0036—Scanning details, e.g. scanning stages
    • G02B21/0048—Scanning details, e.g. scanning stages scanning mirrors, e.g. rotating or galvanomirrors, MEMS mirrors
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/0004—Microscopes specially adapted for specific applications
    • G02B21/002—Scanning microscopes
    • G02B21/0024—Confocal scanning microscopes (CSOMs) or confocal "macroscopes"; Accessories which are not restricted to use with CSOMs, e.g. sample holders
    • G02B21/008—Details of detection or image processing, including general computer control

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  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Optics & Photonics (AREA)
  • Microscoopes, Condenser (AREA)
  • Engineering & Computer Science (AREA)
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Abstract

The present invention relates to a confocal microscope and an image processing method using the confocal microscope. The confocal microscope according to the embodiment of the present invention outputs at least two laser light sources having different wavelengths to a photographed region, A light source for emitting light reflected from the photographing part to the photographing part; a photographing part for acquiring a two-dimensional image by scanning light in a confocal manner; And a coupling unit coupling the lens of the photographing unit and the optical probe and controlling the movement of the optical probe.

Description

TECHNICAL FIELD The present invention relates to a confocal microscope and an 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 with a resolution in the depth direction, which can obtain three-dimensional information of a specimen and is widely used in various industrial and biotechnological fields. The confocal microscope is known to have excellent resolution of about 40% compared with a general microscope. Especially, it is superior in that it is possible to acquire three-dimensional image without making a physical intercept.

However, there is a limitation in that it is difficult to capture the internal structure of the living animal model and to image it because the limit size of the invisible objective lens and the penetration depth of the laser light source are not sufficient.

Because of these limitations, most cell imaging studies using confocal microscopy are performed by observing only the epidermal status of the skin or by histologic analysis of in vitro samples.

In order to solve this problem, an endoscopic microscope method is used to transmit the light source passing through the objective lens of the confocal microscope to the inside of the tissue through a small lens. However, the approach to the skin and the internal organ tissue There is a problem that a light source and a detection signal may be lost in the process of transmitting a light source from an objective lens to a small lens.

Disclosure of Invention Technical Problem [8] The present invention provides a confocal microscope capable of microscopically approaching a living tissue, photographing the same, and imaging the same, and an image processing method using the same.

A confocal microscope according to an embodiment of the present invention includes at least two laser light sources having different wavelengths to a photographing site and a two-dimensional image obtained by scanning the light reflected from the photographing site in a confocal manner, An optical probe which transmits light output from the photographing unit to the photographing unit and transmits light reflected from the photographing unit to the photographing unit by invasion of the photographing unit; And a coupling part for controlling the movement of the optical probe.

The optical probe may include a needle tip to be infiltrated into the living body, and a lens portion located 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 unit 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 changing the traveling direction of the light by reflecting the light.

The coupling unit may include an x, y axis movement unit that controls movement of the optical probe in the x and y axis directions, and a z axis movement unit that controls movement of the optical probe in the z axis direction.

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

The front end of the main body may have a slope in its cross section.

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

And a protective layer surrounding the needle tip.

The protective layer may be formed 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 for controlling the laser light source to focus on the photographing site, A scanning unit for generating a scanning image, and an image obtaining unit for generating an image excited by the laser light source by passing the scanning image through a slit portion provided on a confocal plane.

An image processing method using a confocal microscope according to an embodiment of the present invention includes a step of infiltrating an optical probe into an in-vivo imaging region through the skin, a step of irradiating a laser light source to the imaging region, And acquiring the image.

According to the embodiment of the present invention, the inside of a living tissue can be finely approached and photographed and imaged. According to the 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 diagram of a confocal microscope according to an embodiment of the present invention.
2 is a view showing a detailed configuration of a confocal microscope according to an embodiment of the present invention.
3 is a view showing a structure of a confocal microscope optical probe according to an embodiment of the present invention.
4 is an exemplary illustration of a confocal microscope optical probe according to one embodiment of the present invention.
5 is a cross-sectional view illustrating a structure of a confocal microscope optical probe according to an embodiment of the present invention.
6 is a view showing a structure of a confocal microscope coupling unit according to an embodiment of the present invention.
7 is a flowchart illustrating a method of image processing using a confocal microscope according to an embodiment of the present invention.
FIGS. 8 and 9 are views showing photographing results obtained using a confocal microscope according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In order to clearly illustrate the present invention, parts not related to the description are omitted, and similar parts are denoted by like reference characters throughout the specification.

Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

Now, a confocal microscope according to an embodiment of the present invention will be described in detail with reference to the drawings.

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

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

2, the photographing unit 10 includes a light source 110, beam splitters 120a, 120b, 120c, and 120d, a scan unit 130, an objective lens 140, and an image acquisition unit 150, . ≪ / RTI >

The light source unit 110 irradiates the excitation light source of the photographing unit 10 to excite the photographed region. The light source unit 110 includes two or more light sources having different wavelengths in order to capture an object to be observed marked with a different fluorescent sample. In this embodiment, the light source unit 110 is composed of 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 number and the wavelength of the light source may be variously changed.

The light irradiated from the light source unit 110 passes through the scan unit 130 through at least one beam splitter 120a, 120b, 120c and 120d, passes through the objective lens 140 and the optical probe 20, Investigate the inside shooting area.

The optical probe 20 penetrates through the skin of the living tissue to transmit the light irradiated from the light source unit 110 to the imaging site inside the living tissue and reflects the light reflected from the inside of the living tissue back to the imaging unit 10 ). The shape of the optical probe 20 may vary, but in the present invention, an optical probe in the form of a needle (needle) that can be easily inflated will be described as an example.

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

The light reflected from the inside of the living tissue returns to the photographing unit 10 via 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 through which the light of the fluorescent material excited by the light source unit 110 enters. The objective lens 140 outputs a video signal having an image of the imaging region labeled with the fluorescent material to the scanning unit 130. In the present embodiment, the objective lens 140 is a 250 x 250

Figure pat00001
, Or using a 20x objective lens to have a field of view of 500x500
Figure pat00002
However, the present invention is not limited to this, and it may be set to have various fields of view by setting a lens having an appropriate magnification.

The scan unit 130 reads the image signal input through the objective lens 140 and constructs a two-dimensional array of pixels. At this time, 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 acquiring unit 150 passes the scanning image signals generated from the scanning unit 130 to at least one or more beam splitters 151a, 151b, and 151c, and separates the scanning image signals for each wavelength band. The separated scanning image signal passes through a band pass filter (BPF) 152 and a condenser lens 153 and then passes through a slit 154 provided on a confocal plane surface of the photomultiplier tube (PMT) 155, respectively.

The beam splitters 151a, 151b, and 151c separate and align the scanning image generated from the scanning unit 130, and may be formed of a Dichroic Beam Split (DBS).

The band-pass filter unit 152 is located in the path of the light split from the beam splitters 151a, 151b, and 151c, and obtains the separated light to pass the light in the spectral range of the designated visible light region.

The photomultiplier 155 is located in the path of the light passed from the bandpass filter 152 and detects the fluorescence signal that has passed through the bandpass filter 152 and generates an electrical signal and transmits it to the computing unit 40 .

The computing unit 40 acquires a video signal photographed by the photographing unit 10. At this time, the computing unit 40 may correct the photographing result value to obtain a physically meaningful result.

As described above, the confocal microscope 1 including the optical probe 20 is capable of approaching the internal organs tissue directly through the skin of the living body and controlling the position of the optical probe 20, The photographing can be freely controlled and the inside of the photographed tissue can be imaged. Further, the confocal microscope 1 including the optical probe 20 can invade the precise position, and can reduce the loss of the light source and the detection signal that may 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. FIG.

FIG. 3 is a cross-sectional view of a confocal microscope optical probe according to an embodiment of the present invention, and FIG. 4 is a view exemplarily showing a confocal microscope optical probe according to an embodiment of the present invention. And FIG. 5 is a view illustrating 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 portion 220.

The needle tip 210 has a tubular body 211 having a circular or elliptical cross-sectional configuration for infiltrating the imaging site, that is, the deep part of the living tissue, and the inside thereof may be hollow and hollow. The lens unit 220 is positioned inside the main body 211.

In this embodiment, the distal end portion 212 of the needle tip 210 body 211 may be formed to have a predetermined inclination, as shown in FIG. 3, to facilitate the 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 that exposes at least a part of the lens portion 220. In this embodiment, the opening 213 may be formed by removing one side of the side surface of the main body 211 so that the opening 213 has a semicircular shape in cross section so that the opening 213 can be more closely attached to the imaging region inside the biotissue.

On the other hand, the diameter of the needle tip 210 may be 0.5 to 1.0 mm, and the needle tip 210 may be low-invaded in the living body, and the length may be 10 mm to 30 mm.

Further, it may further include a protective layer surrounding the surface of the needle tip 210 to prevent foreign substances from entering the living tissue when infiltrated. 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 imaging unit within the living tissue and transmits the light reflected from the imaging unit located inside the living tissue to the imaging unit 10 again.

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 embodiment may include a coupling lens And a relay lens 223 positioned between the image lens 222 and the coupling lens 221 and the image lens 222,

The numerical aperture (NA) of the coupling lens 221 and the image lens 222 may be 0.45 to 0.55, and the NA of the relay lens 223 may be a numerical aperture 0.15 to 0.25.

The optical probe 20 may further include a mirror part 230 attached to the distal end of the image lens 222, as shown in FIG. The mirror 230 reflects light to change the path of light. The mirror unit 230 may be coated with aluminum, and the laser light source that has passed through the lens unit 220 is turned on to illuminate the photographed region.

The mirror unit 230 is disposed at an angle of 45 degrees with respect 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 the lens unit 220 . However, the present invention is not limited thereto, and the mirror 230 may be arranged in various directions to change the traveling direction of the laser light source. Accordingly, the confocal microscope 1 according to the present embodiment irradiates the laser light source to not only the depth direction in which the needle tip 210 is infiltrated by the living tissue but also the imaging region in various directions, and transmits the reflected light therefrom Acquired images can be obtained.

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

6, the engaging portion 30 according to the present embodiment includes a main body including a plate 311, a bar portion 312, and an adapter 313, an x, y axis moving portion 320, a z-axis moving part 330, and a probe holder 340.

The x-axis moving unit 320 controls the movement of the optical probe 20 in the plane direction (x, y axis) and the z axis moving unit 330 controls the moving direction of the optical probe 20 in the depth z axis). Accordingly, the confocal microscope 1 according to the present embodiment can photograph a body tissue at a desired site by finely and accurately controlling the movement of the optical probe 20 in three axial directions.

The probe holder 340 fixes the position of the optical probe 20 to the imaging section 10, thereby preventing movement of the optical probe 20 during imaging of the imaging site.

7 is a flowchart illustrating a method of image processing using a confocal microscope according to an embodiment of the present invention.

Referring to FIG. 7, the confocal microscope according to an embodiment of the present invention infiltrates the optical probe 20 through the skin into an in-vivo imaging region (S100). The optical probe 20 may include a needle tip 210 having an inclined tip 212 to facilitate entry into the living body.

 The needle tip 210 may include an opening 213 formed by removing one side of a side portion so as to have a semicircular shape in cross section in order to closely adhere to an imaging region inside the living body and acquire an image.

In addition, the needle tip 210 may further include a protective layer surrounding the outer surface of the needle tip 210 to prevent foreign matter from entering the biotissue.

Next, the laser light source is irradiated to the imaging region inside the biotissue (S200). The laser light source is irradiated from the light source unit 110 of the photographing unit 10 and may have wavelength bands of 405 nm, 488 nm, 561 nm, and 640 nm, respectively. The laser light source can be transmitted to the imaging site inside the biotissue through the lens unit 200 of the optical probe 20. [

Then, light reflected from the photographing site is received and an image is acquired (S300). The light reflected from the inside of the living tissue returns to the photographing unit 10 via 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, whereby the confocal microscope can acquire images of the photographed region.

The image processing method using confocal according to the embodiment of the present invention can directly acquire an image of the internal organs tissue by directly invading and imaging the inside through the skin of the living body, It is possible to reduce the loss of the light source and the detection signal.

FIGS. 8 and 9 are views showing photographing results obtained using a confocal microscope according to an embodiment of the present invention.

FIG. 8 is a view showing an image obtained by successively photographing and imaging 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 fluorescence signal and blood vessels were labeled with a red fluorescence signal.

The skin is composed of the epidermis of the outermost layer, the epidermis, the dermis, and the subcutaneous tissue. Referring to FIG. 8, the distribution of blood vessels and cells in the dermal layer located at the base of the skin, which could not be imaged by a general optical microscope, As shown in Fig.

Fig. 9 is a view showing a photographing result obtained by photographing and imaging the inside of a cancer tissue located in the tissue deep part of a living body. In order to perform effective imaging inside the tissue, blood cells and low oxygen regions in cancer cells and cancer tissues are labeled with different fluorescence signals in this embodiment.

Referring to FIG. 9, images of cancerous cells labeled with green and red, oxygenated blood vessels inside the cancerous tissue, and low oxygen regions labeled with blue were taken. The confocal microscope according to the present example is a simple However, it can be confirmed that the image signal can be acquired by penetrating into the tissue deep part of the living body.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (13)

A photographing unit for outputting at least two laser light sources having different wavelengths to a photographing site and scanning the light reflected from the photographing site in a confocal manner to obtain a two-
An optical probe that transmits light output from the photographing unit to the photographing site and transmits light reflected from the photographing site to the photographing unit by invasion of the photographing site;
And a coupling unit coupling the lens of the photographing unit and the optical probe and controlling the movement of the optical probe,
Wherein the optical probe is a needle-shaped confocal microscope. The method of claim 1,
Wherein the optical probe comprises:
A needle tip that invades into the body, and
And a lens portion located inside the needle tip. 3. The method of claim 2,
The lens unit includes:
A coupling lens positioned on the photographing unit side,
An image lens positioned on the photographing site side, and
And a relay lens positioned between the coupling lens and the imaging lens. 4. The method of claim 3,
Wherein the coupling lens, the image lens, and the relay lens are made of a gradient-index (GRIN) lens. 3. The method of claim 2,
Wherein the optical probe comprises:
And a mirror unit which is located at an end of the lens unit and changes the traveling direction of the light by reflecting the light. The method of claim 1,
The coupling unit may include an x, y axis moving unit for controlling movement of the optical probe in the x and y axis directions,
And a z-axis moving part for controlling movement of the optical probe in the z-axis direction. 3. The method of claim 2,
Wherein the needle tip includes a tubular body, and one end of the body includes an opening exposing at least a portion of the lens portion. 8. The method of claim 7,
Wherein a front end portion of the main body has a slope in its cross section. 9. The method of claim 8,
Wherein the inclination angle of the tip portion is 1 to 20 degrees. 3. The method of claim 2,
And a protective layer surrounding the needle tip. 11. The method of claim 10,
Wherein the protective layer is made of an optical adhesive film. The method of claim 1,
Wherein,
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 on the photographing portion,
A scanning unit for reading an image observed from the photographing site to generate a two-dimensional array scanning image, and
And an image acquiring section for passing the scanning image through a slit section provided on a confocal plane to generate an excited image by the laser light source. As a method of image processing using a confocal microscope,
Introducing an optical probe into the in vivo imaging site through the skin,
Irradiating at least two laser light sources having different wavelengths to the imaging region, and
And a step of acquiring an image by receiving the light reflected from the photographing part.
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