KR101075155B1 - Apparatus and method for confocal laser scanning microscopy system using line scanning way - Google Patents

Abstract

According to the present invention, a beam of a focal point type reflected by a sample is incident and separated into a plurality of beams spatially or temporally. The present invention relates to an image processing apparatus and method for applying to a confocal laser scanning microscope system using a method. It can be effective.

Confocal Laser Scanning Microscope, Laser Beam

Description

Image processing apparatus and method for application to confocal laser scanning microscope system using the line scanning method {APPARATUS AND METHOD FOR CONFOCAL LASER SCANNING MICROSCOPY SYSTEM USING LINE SCANNING WAY}

The present invention relates to a confocal laser scanning microscope system, and more particularly, to incident beams in the form of a line focus reflected from a sample, to separate them into a plurality of beams spatially or temporally, and to separate each beam in a different spatial region. It relates to a method of obtaining an image by having a transmission area and then synthesizing them.

In general, since its introduction in 1961 by Minsky, Confocal Laser Scanning Microscopy (CLSM) has been widely used as an essential test equipment in various industries and bio fields.

Such a confocal laser scanning microscope is known to have an excellent resolution of about 40% compared to a general microscope, and particularly, it has excellent utility in that it is possible to acquire three-dimensional images without having to make physical fragments.

In addition, the confocal laser scanning microscope measures the structure by using reflected light to the excitation beam, or generates light such as fluorescence, Raman scattering, and second harmonic generation. Images of physical and chemical phenomena in the focal plane can be imaged.

In addition, it can be applied to Fluorescence Resonance Energy Transfer (FRET), Fluorescence Lifetime Imaging Microscopy (FLIM) and the like, and its application range is gradually increasing.

Confocal laser scanning microscopy (CLSM) is capable of imaging high-resolution micronized sections of biological tissue up to several hundred microns deep. In particular, biosamples are available for both reflection and transmission imaging, and in vivo In the condition, skin can be imaged up to about 50 to 100 microns.

Such a confocal laser scanning microscope method is known as a point scanning method and a line scanning method. Since the point scan method is a TV scan line method, a frame image acquisition speed is slow.

On the other hand, the scanning operation can be performed in a faster time as the pre-scanning method instead of the scanning method of the point scan method, but the resolution is somewhat lower than that of the point scanning method, so there is a continuous demand for improvement.

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide a confocal laser scanning microscope system using a line scanning method which is inexpensive in a relatively simple manner.

It is another object of the present invention to provide a confocal laser scanning microscope system with improved resolution in a prescan method.

In order to achieve the above object, the first aspect of the present invention is an image processing apparatus for applying to a confocal laser scanning microscope system using a line scanning method, the beam of the line focus form reflected from the sample is incident to a plurality of directions Beam splitter for separating and outputting; A plurality of slits arranged to have each of the separated beams have transmissive regions in different spatial regions; And an image processor for synthesizing each beam output through the plurality of slits with each other.

According to a second aspect of the present invention, there is provided an apparatus for applying to a confocal laser scanning microscope system using a line scanning method, in which a beam having a line focus type reflected by a sample is incident to separate the beam into a plurality of beams in time. A plurality of slits arranged to have transmission regions in different spatial regions, respectively; And an image processor for synthesizing each beam output through the plurality of slits with each other.

According to a third aspect of the present invention, there is provided a method for applying to a confocal laser scanning microscope system using a line scanning method, the method comprising: injecting a beam in the form of a line focus reflected from a sample and outputting the beam in a plurality of directions; Processing the separated beams to have transmissive regions in different spatial regions; It is to provide an image processing method comprising the step of synthesizing the processed beams with each other.

A fourth aspect of the present invention provides a method for applying to a confocal laser scanning microscope system using a line scanning method, the method comprising: incident a beam in the form of a line focus reflected by a sample and separating the beam into a plurality of beams in time; Processing the separated beams to have transmissive regions in different spatial regions; The present invention provides an image processing method comprising synthesizing the processed beams with each other.

Preferably, the method may further include separating and processing a beam having a line focus form reflected by the sample for each wavelength.

The image processing apparatus may be applied to a linear or nonlinear confocal microscope of two-photon microscopy, multi-photon microscopy, second-harmonic generation, and multi-harmonic generation.

According to the confocal microscope system using the line scanning method of the present invention as described above, there is an effect that does not require the previously unstable and complicated image processing.

According to the present invention, by acquiring a two-dimensional image using a pre-scanning method, there is an advantage of having the advantages of a high speed image acquisition speed and a high resolution at the same time.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. The embodiments of the present invention are provided to enable those skilled in the art to more fully understand the present invention.

(First embodiment)

1 is a schematic diagram showing the configuration of a confocal laser scanning microscope system using a line scanning method according to a first embodiment of the present invention.

1, the confocal laser scanning microscope system using the line scanning method according to the first embodiment of the present invention, the light source 100, the first and second beam splitter (Beam Splitter, BS) (200a and 200b) ), Lens 300, scanner 400, objective lens (OL) 500, beam splitter (BS) 600, first and second image acquirers 700a And 700b), and the like.

In the present system, the image processing apparatus 700 constitutes a main feature of the present invention, and includes a beam splitter 600 for injecting a beam of a line focus form reflected from a sample and separating and outputting the beam in a plurality of directions; Synthesizing each beam output through the plurality of image acquirers 700a and 700b and the plurality of image acquirers 700a and 700b arranged so that each separated beam has a transmission region in different spatial regions An image processor 800 for processing.

Hereinafter, an example of a series of processes in which a beam in the form of a line focal point reflected by the sample and incident on the image processing apparatus 700 is generated will be described. However, it is a matter of course that other methods that are not limited to this process can be employed.

The light source 100 may use a laser as spot light (focus light) that scans the surface of the sample 10.

The first beam splitter 200a is a means for reflecting or passing the beam so that the traveling direction of the beam (light) is changed. The first beam splitter 200a passes through the laser beam irradiated from the light source 100 to travel toward the scanner 400. The reflection beam reflected from the sample 10 and returned along the first incident light path travels in the direction of the image acquisition apparatuses 700a and 700b.

The second beam splitter 200b is a means for reflecting or passing the beam so that the traveling direction of the beam (light) is changed. The second beam splitter 200b changes the path so that the beam scanned from the scanner 400 travels in the direction of the sample 10. In this case, the path of the reflected beam reflected from the sample 10 and returned along the first incident light path is directed toward the scanner 400.

The lens 300 performs a function of outputting the beam passing through the first beam splitter 200a as a beam of line focus type. The lens 300 may be formed of, for example, a cylindrical lens, which causes the linear lens to form a linear focus, which is used when the beam is irradiated onto the sample surface of the sample 10. In other words, it plays a role in making the line shape. The position of the lens 300 is not limited to the position shown in FIG. 1, and may be located anywhere on the path of the beam between the light source and the sample.

In addition, since the cylindrical lens has a cylindrical shape, the focus is focused on only one surface. According to this phenomenon, the linear lens forms a linear focus on the sample surface.

The scanner 400 performs a function of scanning a beam of a line focus type output from the lens 300 by changing a path in a vertical direction. That is, the scanner 400 is a Y-direction scanner and scans a beam in the X-direction ray focal form passing through the lens 300 in the Y-direction perpendicular to the longitudinal direction of the focal point.

The objective lens 500 is disposed between the second beam splitter 200b and the sample 10, and focuses an incident beam to form an image of an object to irradiate the sample 10. .

The beam splitter 600 is disposed between the first beam splitter 200a and the first and second image acquisition apparatuses 700a and 700b.

Additionally, at least one lens (L1 and L2) is disposed between the second beam splitter 200b and the objective lens 500 to make the beam converted from the second beam splitter 200b into a parallel beam. May be

Next, a detailed configuration of the image processing apparatus 700 will be described.

The first image acquisition apparatus 700a filters at least one first condenser lens 710a for condensing the beam transmitted from the beam splitter 600 and a beam condensed by the first condenser lens 710a. A first slit portion 720a having a slit S and a first collimating lens 730a for converting a beam passing through the slit S of the first slit 720a into a parallel beam And a first photodetector 740a that photoelectrically converts the parallel beam converted from the first collimating lens 730a and outputs the converted electrical signal to the image processing apparatus 800. The second image acquisition apparatus 700b is also similar in structure to the first image acquisition apparatus 700a.

However, the first image acquisition apparatus 700a and the second image acquisition apparatus 700b have each beam having a transmission region in different spatial regions. FIG. 2A illustrates the structure of the first slit part 720a and the second slit part 720b as an example.

The first slit part 720a and the second slit part 720b are arranged so that each separated beam has a transmission area in a different spatial area. Referring to FIG. 2A, the odd-numbered (A) space becomes a transmissive region and the even-numbered (B) space becomes an opaque region. With this configuration, the first slit portion 720a and the second slit portion 720b obtain images of different spatial regions, respectively, and these images are combined with each other by the image processor 800 to form a complete image.

In FIG. 2A, the first slit part 720a and the second slit part 720b of FIG. 1 are shown as an example of each configuration, but the present invention is not limited thereto. FIG. 2B is a diagram illustrating a case where the shapes of the first slit portion 720a and the second slit portion 720b are different from those of FIG. 2A. Of course, the first slit 720a and the second slit 720b may have various shapes other than those of FIGS. 2A and 2B.

On the other hand, the first image acquisition apparatus 700a and the second image acquisition apparatus 700b are not limited to obtaining an image by dividing into two, as shown in FIGS. 1, 2A, and 2B. Therefore, it is also possible to set it as three or more. In FIG. 2C, when three image acquisition apparatuses exist, three slit portions are formed and an example of the configuration thereof is illustrated. To this end, for example, the split optical power of the beam splitter 600 is 2: 1, and the light corresponding to 2/3 is separated again using a 1: 1 optical splitter to split the beam with three equal powers. It may be possible.

Meanwhile, the first and second condensing lenses 710a and 710b and the first and second collimating lenses 730a and 730b are cylindrical lenses that form a linear focus like the lens 300 described above. Can be done.

Meanwhile, the first and second slits 720a and 720b play a role as a filter for removing noise. Only light focused in a line shape at the focal planes of the first and second condensing lenses 710a and 710b is disposed to be extracted by passing through the slit S. That is, the center of the slit mask is located on the optical axes of the first and second condensing lenses 710a and 710b, respectively.

The first and second photodetectors 740a and 740b may be formed of photodetectors for converting light obtained through the slits S of the first and second slits 720a and 720b into an electric signal corresponding to the amount of light thereof. have.

The first and second photodetectors 740a and 740b may be a linear array detector or a linear array that forms an image of information of the sample 10 from the linear light passing through the slit S described above. It may be implemented as a line-scan camera, for example, it is preferably made of a charge coupled device (CCD) camera. For example, when using a linear scanning camera with a line rate of about 60 kHz (readout speed ~ 60,000 lines / second), a frame image with 512 x 512 pixels is significantly faster at about 120 frames per second. You can get it.

The image processor 800 may be embodied by a conventional computer. The first and second photodetectors 740a and 740b may be configured to start, end, or transmit an image by an instruction from the computer. Controlled. The computer receives the image data photographed from the first and second photodetectors 740a and 740b and performs a processing to display the image data on a monitor.

In particular, the image processor 800 applies the image detected from the first and second image acquisition apparatuses 700a and 700b to, for example, any one of a Richardson-Lucy (RL) algorithm, a Tikhonov Regularization (TR) or a local linear filter. Can be corrected. In addition, the image processor 800 may use a conventional image processing method.

The operation of the confocal laser scanning microscope system using the line scanning method according to the first embodiment of the present invention configured as described above will be described in detail as follows.

First, the laser beam irradiated from the light source 100 passes through the first beam splitter 200a, and then the beam passing through the first beam splitter 200a passes through the lens 300 and is output as a beam of line focus type. do.

Thereafter, the beam of the line focus type output from the lens 300 proceeds to the scanner 400, and then the path is changed in the vertical direction by the scanner 400 and scanned in the direction of the second beam splitter 200b. .

Then, the beam scanned from the scanner 400 is reflected back by the second beam splitter 200b and irradiated to the sample 10 through the lenses L1 and L2 and the objective lens 500.

Then, the light reflected from the sample 10 is returned back along the light path that originally came. In this process, the first beam splitter 200a reflects the reflected beam that is reflected by the sample 10 and returns along the first incident light path and travels toward the first and second image acquisition apparatuses 700a and 700b. Convert

In this case, the reflected beam path-converted by the first beam splitter 200a passes through the beam splitter 600. In this case, the beam splitter 600 separates the reflected beam path-converted through the first beam splitter 200a. . That is, the beam is divided into two parts, and some beams are transmitted to travel in the direction of the first image acquisition apparatus 700a, and some beams are reflected and travel in the direction of the second image acquisition apparatus 700b.

The two images obtained as described above may be corrected by the image processor 800 and the two images may be synthesized to form a two-dimensional image having a greatly improved resolution.

(2nd Example)

3 is a schematic diagram showing the configuration of a confocal laser scanning microscope system using the line scanning method according to the second embodiment of the present invention. The description of the second embodiment will be described mainly for differences that are distinguished from the first embodiment for the convenience of description.

Referring to FIG. 3, in the second embodiment, the image acquisition apparatus 900 is configured as one. The image acquisition apparatus 900 includes at least one first condensing lens 910 for condensing the beams, and a slit S to filter the beams condensed by the first condensing lens 910. Photoelectric conversion of the collimated lens 930 and the collimated beam converted from the first collimated lens 930 to convert the beam passing through the slit 920, the slit S of the slit S 920 into a parallel beam And a photodetector 940 for outputting the converted electric signal to the image processing apparatus 800.

Accordingly, the image acquisition apparatus 900 of the second embodiment obtains a complete image by obtaining images transmitted in different spatial regions from the beams transmitted continuously in time and synthesizing them in the image processor 800. To this end, an example of a possible slit portion 920 is shown in FIGS. 4A to 4D.

Referring to FIG. 4A, the slit portion divides one line into two different spatial regions, and configures the first slit to have a transmissive region A at an odd number in the first time (1-1), and the second time (1). In the case of -2), the even-numbered transmission region B is configured. Then, the image having the transmission region is synthesized in different spaces acquired in 1-1 time and 1-2 hours to obtain the completed image information of the first line. To this end, the slit portion 920 of the image acquisition apparatus 900 is preferably configured to perform a vertical linear motion to obtain the above-described image information. That is, the slit portion has a two-dimensional shape in the form of an M x N matrix, and the unit sets 1-1 and 1-2 arranged to have transmissive regions in different spatial regions are repeatedly arranged in a predetermined direction.

Another way is shown in FIGS. 4B-4D. 4B shows that the slit portion is configured in the form of a rotatable plate, and a set of units arranged to have a transmission region in different spatial regions is repeatedly arranged in the circumferential direction. FIG. 4A shows that information having different transmission regions is obtained by linear movement of the slit portion, while FIG. 4B shows related information by rotational movement of the slit portion.

According to another method, as shown in FIG. 4C, the slit is configured to periodically vibrate.

According to another method, as shown in FIG. 4D, a slit is secured in a temporally different spatial region using a spatial light modulator (SLM). The spatial light modulator can secure different spatial transmission regions by adjusting the transparency / opacity of the unit pixels with matrix pixels. Examples of the spatial light modulator include an electrically driven SLM method and an optically driven SLM method, and the electrically driven SLM method includes LCOS (Liquid Crystal On Silicon), DMD (Digital Micromirror Device), and p-Si TFT (poly-). Si Thin Film Transistor). The method using the spatial light modulator has an advantage of obtaining a complete image by acquiring a plurality of images having different spatial transmission regions in various and complicated shapes and synthesizing them with each other.

On the other hand, the spatial light modulator can be a one-dimensional or two-dimensional form, and either a transmissive or reflective type. Therefore, by using the spatial light modulator, the slit pattern can be made in a one-dimensional or two-dimensional array form, and the slit method can be made a transmissive or reflective type. By making the above-described slit pattern change with time using the spatial light modulator, it is possible to make various slit patterns change with time even in a fixed state without mechanical movement (translation, rotation, periodic repetition).

(Third Embodiment)

5 is a schematic diagram showing the configuration of a confocal laser scanning microscope system using the line scanning method according to the third embodiment of the present invention. The description of the third embodiment will be mainly focused on differences that are distinguished from the first embodiment for the convenience of description. Referring to FIG. 5, in the third embodiment, the difference is that the filter 1100 is added in front of the beam separator 600.

Using the third embodiment, it is possible to implement a confocal laser fluorescence microscope of the line scanning method. Briefly describing the confocal laser fluorescence microscope, the fluorescent material is first administered to the cells to be observed. Only the light that is in focus is separated from the light that is lasered into the cell. Analyze this to make an image. Because the laser can penetrate deep into the cell, it can be viewed in three-dimensional stereoscopic images, which makes it an essential device for biological research.

In order to implement a confocal laser fluorescence microscope, it is necessary to obtain a different image for each wavelength. To this end, in the third embodiment, it is possible to add a filter 1100 that can be selected and filtered for each wavelength. However, the additional position of the filter 1100 is not limited to the beam separator 600 and may be added at various positions. In more detail, when the filter 1100 is added in front of the beam separator 600, the filter 1100 is separated by wavelength using the filter 1100, and then passed through the beam separator 1100, as in the first embodiment. It is also a method of performing image processing, and it is also possible to implement each beam passing through the beam splitter 600 to pass through the filter. In this case, the filter 1100 may be disposed at appropriate positions of the image processing apparatuses 1000a and 1000b, respectively.

In addition, although the addition of the filter 1100 was demonstrated in the form added to 1st Embodiment, it can also be added to a predetermined part of 2nd Embodiment. In this case, of course, in order to implement a confocal laser fluorescence microscope, different images can be obtained for each wavelength.

In addition, the present invention is not limited to the above-described first, second, and third embodiments, and can be modified and applied in various ways.

For example, the slits 720a, 720b, 720c, 920, and 930 are illustrated and described in a transmissive slit method, but may be similarly applied by fabricating in a reflective slit method. In addition, the contents of the first, second, and third embodiments are various linear or nonlinear, including reflection, transmission, polarization, fluorescence, two-photon microscopy, multi-photon microscopy, second-harmonic generation, and multi-harmonic generation. The same applies to the confocal microscope of the method.

By using the confocal laser scanning microscope system using the line scanning method according to an embodiment of the present invention configured as described above, it can be effectively used for the study of the coupling (Coupling) with Ca ++ and Astrocyte, and blood vessels in brain research. .

In addition, studies have shown that the scattering coefficient changes very rapidly and dynamically during the transmission of neurons, and changes in the light transmission signal in one region have been detected in one dimension, but there are no studies in which two to three dimensions are imaged. . Therefore, it can be expected that the application of the present invention can be very helpful in identifying the signaling system in the brain in the future.

In addition, according to the present invention, it is possible to implement an ultrafast three-dimensional image of a sample having a three-dimensional structure such as cells. Therefore, it can be of great help in identifying the three-dimensional structure and inter-mechanical dynamics of constituents in cells, and it can be applied to general biological fields such as rapid cilia movement of microorganisms to enable more research. Non-destructive inspection can be performed at high speed for inspection, etc., which can contribute to productivity improvement.

Although a preferred embodiment of the image processing apparatus and method for applying to the confocal laser scanning microscope system using the line scanning method according to the present invention described above, the present invention is not limited to this, but the claims and the details of the invention It is possible to carry out various modifications within the scope of the description and the accompanying drawings, which also belong to the present invention.

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1 is a schematic diagram showing the configuration of a confocal laser scanning microscope system using a line scanning method according to a first embodiment of the present invention.
2A to 2D are diagrams illustrating examples of respective configurations of the first slit part 720a and the second slit part 720b of FIG. 1.

3 is a schematic diagram showing the configuration of a confocal laser scanning microscope system using a line scanning method according to a second embodiment of the present invention.

4A through 4D are diagrams illustrating examples of the slit 920 of FIG. 3.

5 is a schematic diagram showing the configuration of a confocal laser scanning microscope system using the line scanning method according to the third embodiment of the present invention.

Claims (14)

delete delete delete delete Apparatus for applying to a confocal laser scanning microscope system using a line scanning method, A plurality of slits arranged to have a transmissive region in different spatial regions by injecting beams in a line focus form reflected from a sample to separate the beams into a plurality of beams in time; And Including an image processor for synthesizing each beam output through the plurality of slits with each other, The plurality of slits are formed in a two-dimensional form of an M x N matrix form, the unit set arranged to have a transmission region in the different spatial region is repeatedly arranged in a predetermined direction. Apparatus for applying to a confocal laser scanning microscope system using a line scanning method, A plurality of slits arranged to have a transmissive region in different spatial regions by injecting beams in a line focus form reflected from a sample to separate the beams into a plurality of beams in time; And Including an image processor for synthesizing each beam output through the plurality of slits with each other, The plurality of slits are configured in the form of a rotatable plate, wherein the unit set arranged to have a transmissive region in the different spatial region is repeatedly arranged in the circumferential direction. Apparatus for applying to a confocal laser scanning microscope system using a line scanning method, A plurality of slits arranged to have a transmissive region in different spatial regions by injecting beams in a line focus form reflected from a sample to separate the beams into a plurality of beams in time; And Including an image processor for synthesizing each beam output through the plurality of slits with each other, The plurality of slits are formed in a line shape having a spatially selective transmission region, and by vibrating periodically to obtain an image reflected in different spatial regions of the sample. Apparatus for applying to a confocal laser scanning microscope system using a line scanning method, A plurality of slits arranged to have a transmissive region in different spatial regions by injecting beams in a line focus form reflected from a sample to separate the beams into a plurality of beams in time; And Including an image processor for synthesizing each beam output through the plurality of slits with each other, And the plurality of slits to secure a transmissive region in the different spatial regions in time using a spatial light modulator. The method according to any one of claims 5 to 8, And a filter for separating and processing a beam having a line focus form reflected by the sample for each wavelength. The method according to any one of claims 5 to 8, The plurality of slits are transmissive or reflective image processing apparatus. The method according to any one of claims 5 to 8, The image processing apparatus is applied to a linear or nonlinear confocal microscope of reflection, transmission, polarization, fluorescence, two-photon microscopy, multi-photon microscopy, second-harmonic generation, and multi-harmonic generation. delete delete delete

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