KR101845438B1 - Device for structured illumination microscopy - Google Patents

Abstract

The present invention provides an optical system for illuminating a specimen with structured illumination of an interference fringe pattern incident from the outside and restoring a modulated frequency by the structured illumination to obtain an image of the specimen in the structured illumination microscope apparatus; And a lens unit for converting the structured illumination path such that the structured illumination corresponds to a projection area of an apparatus provided with the optical system, wherein the lens unit is inserted 100 mm to 150 mm in the inner direction of the apparatus in which the optical system is provided .
The present invention provides an additional configuration of an optical system and a lens unit compatible with an SLM module for generating structured illumination from an external light source, thereby reducing the cost of a structural illumination microscope system using a 0.45 "DMD module .

Description

{DEVICE FOR STRUCTURED ILLUMINATION MICROSCOPY}

The present invention relates to a structural illumination microscope, and more particularly to a structural illumination microscope compatible with a DMD module.

High spatial resolution of the microscope is required to precisely measure and inspect the industrial microstructures mass produced by highly integrated / high-precision equipment. In particular, in the bio / medical research field, high spatial resolution of the microscope is required to accurately observe and judge the cell-based image.

However, the resolution of a general optical microscope has a problem that the diffraction limit resolution is limited due to the property that light is dispersed while passing through the lens. In addition, despite the development of materials and processing techniques, these limitations have not been exceeded.

In recent years, there has been a growing awareness of the usefulness and development potential of high-resolution optical imaging techniques that can overcome these optical limitations mathematically and physically. Fluorescence excitation detection methods such as stimulated emission depletion (STED), photoactivation localization microscopy (PALM), and fPALM (fluorescence PALM) are used as methods for realizing ultra high resolution. In addition, structured illumination microscopy (SIM) techniques can provide high resolution using patterned illumination.

Structured illumination microscopy (SIM) is a high-resolution microscope that dramatically improves the lateral spatial resolution of a microscope by illuminating a specimen with bright and dark interference fringe patterns in the form of sine waves. Structural illumination microscopes can be applied to industrial and biomedical / medical applications because they can be applied irrespective of reflected light / fluorescence, without requiring special properties of the fluorescence factor. Structural illumination microscopes have been evaluated as an optical image acquisition technology that improves the spatial resolution of optical microscopes and exceeds diffraction limited resolution.

Structural illumination microscopes can be classified according to how the illumination pattern is generated, either by using a diffraction grating, by using a spatial light modulator (SLM) or by using an LED array. The SLM must be precisely displaced and rotated the diffraction grating to obtain a phase-shifted structured illumination image with an active element capable of controlling the pupil function of the light. In this regard, attempts have been made to use a DMD (Digital Micro Mirror Device) device as a pattern generator.

DMD is a device that emits a 16 micron fine mirror on a silicon wafer at 1 micron intervals to control the reflection of light through the mirror. When an object shines on the surface of an aluminum mirror, a mirror that collects the light through the lens scans the moving image.

Korean Patent No. 10-1541610 (hereinafter referred to as "prior art") as a related art related to a structural illumination microscope modulates a grid pattern generated through a spatial filter using an electrical signal to acquire high resolution image information Lt; / RTI > microscope based on a spatial filter. Here, the prior art suggests that a DMD device can be used to generate a pattern of a structured illumination microscope. However, the above-mentioned prior art does not disclose how to construct a structured illumination microscope for compatibility of DMD elements. Currently, products using DMD modules for structural illumination microscopes are being offered at considerably higher prices. A commercially available structural illumination microscope commercially available on the market is provided to be compatible with 0.75 "or 0.9" DMD modules with a 1 "optic system. However, since 0.75" or 0.9 "DMD modules are too expensive, The present applicant believes that the present invention can be applied to a structure illumination microscope capable of being mounted on a conversion illumination microscope, To provide a structured illumination microscope that is compatible with a significantly lower 0.45 "DMD module.

Korean Patent No. 10-1541610

It is an object of the present invention to provide a structured illumination microscope capable of supplying a structured illumination microscope with a remarkably reduced unit price so as to be compatible with a 0.45 "DMD device.

According to an aspect of the present invention, there is provided a structured illumination microscope, comprising: a structure illuminator for irradiating a specimen with structured illumination of an interference fringe pattern incident from outside, restoring a modulated frequency by the structured illumination, Optical system; And a lens unit for converting the structured illumination path such that the structured illumination corresponds to a projection area of an apparatus provided with the optical system, wherein the lens unit is inserted 100 mm to 150 mm in the inner direction of the apparatus in which the optical system is provided .

Preferably, the optical system includes a connection tube having one side inserted into the apparatus and the other side exposed to the outside of the apparatus, and the other side receives the structural illumination, and the lens unit is provided in the connection tube .

Preferably, the connection tube is compatible with a Spatial Light Modulator (SLM) module including a DMD (Digital Micro Mirror Device) element that generates a pattern of structural illumination.

Preferably, the lens portion includes: a first lens for condensing the structured light; And a second lens for converting transmitted light of the first lens into parallel light corresponding to a projection area of the optical system.

Preferably, the first lens is provided in the other direction of the connection tube, and the second lens is provided in the one direction of the connection tube.

Preferably, the first lens is located at 90 to 110 mm from the instrument in the other direction of the connection tube, and the second lens is located at 100 to 150 mm from the instrument in the one direction of the connection tube .

Preferably, the first lens has a focal length of 10 mm to 30 mm, and the second lens has a focal length of 100 mm to 150 mm.

Preferably, the first lens may be designed to have a magnification of 1.0 to 2.5 times.

According to the present invention, there is provided a structural illumination microscope equipped with a lens part which is compatible with a DMD module of 0.45 ". In particular, the structural illumination microscope according to the present invention is characterized in that the lens part is not inserted too deeply or too shallowly, "It is provided in optimized design conditions that are coupled to the hardware of the structural illumination microscope, so it does not interfere with other parts inside the microscope or lose the ability of the photoconversion. As a result, it is possible to interchange the 0.45 "DMD module, which is remarkably inexpensive to the structural illumination microscope, to lower the unit price to about 1/10 of the conventional expensive equipment.

1 shows a structural illumination microscope system according to an embodiment of the present invention.
Fig. 2 shows a lens section and an SLM module of a structural illumination microscope constituting the structured illumination microscope system of Fig. 1; Fig.
Fig. 3 is a conceptual diagram for explaining that the projection area of the lens portion is compatible with the structure illuminating microscope of Fig. 2 to calculate magnification. Fig.
4 is a conceptual diagram for explaining the calculation of the focus of the lens unit according to the embodiment of the present invention.
FIG. 5 shows a connection tube and an SLM module manufactured by simulating the structural illumination microscope system of FIG. 1;

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. However, the present invention is not limited to or limited by the exemplary embodiments. Like reference numerals in the drawings denote members performing substantially the same function.

The objects and effects of the present invention can be understood or clarified naturally by the following description, and the purpose and effect of the present invention are not limited by the following description. In the following description, well-known functions or constructions are not described in detail since they would obscure the invention in unnecessary detail.

Fig. 1 shows a structural illumination microscope system 1 according to an embodiment of the present invention. 1, the structural illumination microscope system 1 may include a structural illumination microscope 10, a specimen 3, a CCD camera 5, a light source 7 and an SLM module 30. [

The structural illumination microscope 10 is an optical instrument for SIM (Structured Illumination Microscopy) imaging. The structural illumination microscope (10) is a high-resolution microscope that dramatically improves the lateral spatial resolution of a microscope by illuminating a specimen with a bright and dark interference fringe pattern in the form of a sine wave. The structural illumination microscope 10 can be applied regardless of the reflected light / fluorescence without requiring special properties of the fluorescence factor. The structural illumination microscope 10 is mainly used for cell-based imaging in bio / medical research because it enhances the spatial resolution of optical microscopes and exceeds diffraction limited resolution.

The structure illumination microscope 10 may include an optical system 101 and a lens unit 103. [

The optical system 101 can acquire the image of the specimen 3 by irradiating the specimen 3 with the structure illumination of the interference fringe pattern incident from the outside and restoring the frequency modulated by the structure illumination. The optical system 101 may be compatible with the SLM module 30 to receive structural illumination. The optical system 101 may be composed of optical elements for acquiring images of the specimen inside the microscope. The optical system 101 includes a CCD camera 5 and a CCD camera 5. The optical system 101 may be a 1 "optic system. &Quot; May be provided on the microscope apparatus together with the optical system 101. In this specification, the hardware to which the optical system 101 is provided is referred to as an " apparatus ".

In general, the optical system 101 of the 1 "optic system can adjust the size of the optic to some extent, but its range is extremely limited. In this embodiment, the 1" . However, when the size of the optic exceeds 1.2 ", interference occurs in the hardware inside the microscope. That is, the optical system 101 of the microscope changes hardware design according to the optic system, 10 are not compatible with the various SLM modules 30. The structured illumination microscope 10 according to the present embodiment is designed to be compatible with a low cost SLM module 30 so that a separate lens can be assembled in hardware The configuration of the present invention capable of interfacing a low-cost SLM module 30 with a limited optic size in consideration of the hardware characteristics of the structural illumination microscope 10 will be described later with reference to FIG.

The optical system 101 may include a connection tube 1011. The connection tube 1011 can be coupled and fixed to an apparatus in which the optical system 101 is provided. The connection tube 1011 can be understood as a tube which is exposed to light by opening one side of the microscope on a hardware device on which the optical system 101 is provided.

One side of the connection tube 1011 is inserted into the inside of the apparatus, the other side is exposed to the outside of the apparatus, and the structure lighting can be received from the other side. Note that, in this embodiment, the length of one side of the inserted tube 1011 inserted is limited by the characteristics of the structural illumination microscope 10. The connecting tube 1011 has a limited size of the microscope, and if the length inserted into the microscope is too long when the apparatus is manufactured, it may interfere with another optical element in front. Conversely, if the inserted length of one side of the connecting tube 1011 is too short, light can not be reflected or reflected in the epi-fluorescence port, and uniform light can not be irradiated. Therefore, the connecting tube 1011 must be properly inserted to the vicinity of the objective lens of the microscope or the filter cube turret.

The connecting tube 1011 according to the present embodiment accommodates the lens portion 103 to be described later with the limited hardware characteristics as described above. Thus, the lens section 103 can be compatible with the structured illumination microscope 10 and the low-cost SLM module 30. [

The lens unit 103 can convert the structural illumination path such that the structural illumination corresponds to the projection area of the apparatus provided with the optical system 101. [ The lens portion 103 may be provided in the connection tube 1011. The lens unit 103 may include a first lens 1031 and a second lens 1033. Accordingly, the length of the lens unit 103 can be changed according to the characteristics of the first lens 1031 and the second lens 1033. [ 2 shows the lens section 103, the connecting tube 1011 and the SLM module 30 of the structural illumination microscope 10.

Referring to FIG. 2, the lens unit 103 may be inserted 100 mm to 150 mm in the inner direction of the apparatus in which the optical system 101 is provided. The connection tube 1011 follows a hardware suggestion as described above. The restriction condition of the connection tube 1011 is that the length A of one side inserted into the inside of the apparatus is in the range of 100 to 150 mm. As described above, when the length (A) of the portion of the connecting tube 1011 inserted into the inside of the apparatus is less than 100 mm, the light is interfered with and reflected by the epi-fluorescence port, This is because there is a problem that is lost.

If the length A of the side where the connection tube 1011 is inserted is 150 mm or more, interference with other components located in front of the connection tube 1011 occurs inside the device, and the structure illumination microscope 10 may not be utilized. Therefore, the lens section 103 should also be designed to be installed in the range of 100 to 150 mm inward of the structural illumination microscope 10. Particularly, in Fig. 2, a configuration provided in the range of 100 mm to 150 mm in the inward direction of the structural illumination microscope 10 is shown as a second lens 1033 that converts the structured illumination into parallel light that is compatible with the optical system 101 .

The first lens 1031 for transmitting light in accordance with the design conditions of the second lens 1033 may be provided in the outer direction of the apparatus. In this specification, the expression of the lens portion 103 inserted in the range of 100 mm to 150 mm in the inward direction of the device can be understood to mean the second lens 1033. Under the above conditions, the design of the first lens 1031 and the second lens 1033 according to the present embodiment is performed as follows.

The first lens 1031 can condense the structured light. The first lens 1031 can condense the structured light to produce an image on the focal length of the second lens 1033. [ The first lens 1031 may be provided on the other side of the connection tube 1011. In this embodiment, the first lens 1031 may be positioned at 90 to 110 mm from the device in the other direction of the connection tube 103. More preferably, the first lens 1031 may be located at a distance of 50 mm from the device in the other direction of the connection tube 103. The range in which the first lens 1031 can be installed in FIG. In the present specification, the expression "the other direction of the connection tube 1011" means a direction from the apparatus interface to the apparatus outer side.

Fig. 3 shows a conceptual diagram for explaining that the magnification is calculated by completing the projection area of the lens unit 103 in the structured illumination microscope 10. Fig.

Referring to FIG. 3, the lens unit 103 may combine the first lens 1031 and the second lens 1033 to have a specific magnification (m) in order to use the DMD device efficiently. The aspect ratio of the optic region of the optical system 101 is generally 1: 1. On the other hand, the ratio of the optic region in which the DMD device emits is about 16:10, and both are different. Therefore, in order to optimize the DMD element to the structure illumination microscope 101, it is appropriate to limit the length of each edge of the reflection surface to a square shape equal to the height of the DMD element. Since the height of the 0.45 "DMD is about 6 mm, it is desirable that the magnification (m) is designed to be four times as large as possible in order to project the optical system to a 1" (25.4 mm) maximum.

In this embodiment, however, since the pupil diameter PD for maintaining and installing the structure of the objective lens of the structural illumination microscope 101 differs depending on the model and magnification of the objective lens, the first lens 1031 and the second lens 1033 May be 1.0 to 2.5 times, which can be obtained by adjusting the distance between the DMD element and the first lens 1031.

Fig. 4 shows a conceptual diagram for explaining the calculation of the focus of the lens unit 103 according to the embodiment of the present invention.

를 만족시킬 수 있다.Referring to FIG. 4, the first lens 1031 may be implemented as a single lens. The SLM module 30 including the DMD element may exist between the focal length f of the first lens 1031 and the focal length 2 times 2f. In this embodiment, Object represents the structure illumination generated in the DMD element, and Image represents the image in which the structure illumination passes through the first lens 1031 and is made inside the connection tube 1011. a represents the distance between the first lens 1031 and the DMD, and b represents the distance between the first lens 1031 and the image. The magnification m of the first lens 1031 is given by the following equation

Can be satisfied.

와 렌즈의 초점거리(f)를 산출하기 위한 수식

와 연립하여 식

를 만족 시킬 수 있다. 특히, 본 실시예에서는 사용하기에 가장 적합한 대물렌즈를 고려하여 제1 렌즈(1031)의 배율(m)을 2로 산출하였다. Further, the first lens 1031 is a lens

And the focal distance f of the lens,

And

Can be satisfied. Particularly, in this embodiment, the magnification m of the first lens 1031 is calculated to be 2 in consideration of the most suitable objective lens to be used.

In this embodiment, the first lens 1031 may be designed to have a focal length of 10 mm to 30 mm. More preferably, the first lens 1031 may be designed to have a focus of 25 mm.

In this embodiment, the focal length f of the first lens 1031 is designated as the minimum focal length that can be calculated using a high quality single 1 "convex lens that can be obtained inexpensively on the market, and is calculated as 25 mm. The distance a between the first lens 1031 and the DMD to be calculated is 37.5 mm and the distance b between the first lens 1031 and the image is 75 mm. When the distance f increases, a + b increases exponentially and the use of the structural illumination microscope 10 becomes impossible. However, the focal length f of the first lens 1031 may be used up to 30 mm Further, the first lens 1031 may be composed of a composite lens, so that a shorter focal length can be realized, leading to a variety of design of the structural illumination microscope 10. [

The second lens 1033 can convert the transmitted light of the first lens 1031 into parallel light corresponding to the projection area of the optical system 101. [ The second lens 1033 can reach the objective lens of the apparatus through the connecting tube 103 with parallel light. The second lens 1033 may be provided in one direction of the connection tube 1011. [ The second lens 1033 may be positioned at a distance of 100 mm to 150 mm from one side of the connecting tube 1011 in the apparatus. More preferably, the second lens 1033 can be positioned at 125 mm from the instrument in one direction of the connecting tube 1011.

The range in which the second lens 1033 can be installed in FIG. In this specification, the direction of one side of the connection tube 1011 from the device means the direction from the device interface to the connection tube 1011 inserted inside the device. The second lens 1033 may be designed to have a focus of 100 mm to 150 mm. More preferably, the second lens 1033 can be designed to have a focal length of 125 mm.

When the focal length of the second lens 1033 is more than 150 mm, there is a problem that the connection tube 1011 for installing the second lens 1033 becomes too long inside the apparatus. Therefore, when the focal length of the second lens 1033 exceeds 150 mm, it is impossible to use the structural illumination microscope 10 due to interference with other components of the structural illumination microscope 10. When the focal length of the second lens 1033 is smaller than 100 mm, the light is interfered with and reflected in the epi-fluorescence port, and uniform light can not be irradiated, making it impossible to utilize the structured illumination microscope 10. In this embodiment, the focus of the second lens 1033 is calculated to be 125 mm in consideration of the above-described range.

The SLM module 30 may change the light emitted from the light source 7 into a structured light. SLM module 30 may include a DMD device as an apparatus for generating a pattern of structural illumination. In particular, in this specification, a SLM module 30 including a 0.45 "DMD device can be used. The conventional structure illumination microscope 10 can only connect 0.75 " and 0.9" DMD devices expensive with a 1 " A 0.45 "DMD device that is compatible with the structured illumination microscope 10 disclosed herein can reduce the fabrication cost of the structured illumination microscope system 1 to a level of 1/10. Through this embodiment, a structured illumination microscope system 1) can be improved.

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. will be. Therefore, the scope of the present invention should not be limited to the above-described embodiments, but should be determined by all changes or modifications derived from the scope of the appended claims and equivalents of the claims.

1: Structural Lighting Microscope System
3: The Psalms
5: CCD camera
7: Light source
10: Structure illumination microscope
101: Optical system 103:
1011: connecting tube 1031: first lens
1033: Second lens
30: SLM module

Claims (8)

A structure illuminating microscope comprising an optical system for illuminating a specimen with structured illumination of an interference fringe pattern incident from outside and restoring a frequency modulated by the structured illumination to obtain an image of the specimen,
A lens unit for interfacing a DMD (Digital Micro Mirror Device) element for generating an interference fringe pattern of the structured illumination microscope with the structural illumination microscope is provided between the structured illumination microscope and the DMD element,
The lens unit includes a first lens for condensing the structured light; And
And a second lens for converting transmitted light of the first lens into parallel light corresponding to a projection area of the optical system,
Wherein the second lens is inserted and positioned in a range of 100 mm to 150 mm inward of an instrument provided with the optical system.
The method according to claim 1,
The structure illumination microscope includes:
And a connection tube for receiving the structural illumination from the other side of the apparatus, wherein one side of the connection tube is inserted into the inside of the apparatus, the other side of the connection tube is exposed to the outside of the apparatus,
The lens unit includes:
And a structural illumination microscope provided in the connection tube.
The method according to claim 1,
The optical system is a 1 "optic system, and the DMD device is a 0.45" DMD device.
delete The method according to claim 2, wherein
The first lens is provided in the other direction of the connection tube,
And the second lens is provided in the one direction of the connection tube.
The method according to claim 2, wherein
And the first lens is located at a distance of 50 mm from the instrument toward the other side of the connection tube.
3. The method of claim 2,
The first lens is designed to have a focal length of 10 mm to 30 mm,
And the second lens is designed to have a focus of 100 mm to 150 mm.
The method according to claim 1,
Wherein the first lens is designed to have a magnification of 1.0 to 2.5 times.

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