WO2016021745A1

WO2016021745A1 - Work station including sample holder for cryogenic electron microscope for correlative imaging detection of light microscope and electron microscope, correlative imaging detection apparatus including same, and imaging detection method and imaging system using same

Info

Publication number: WO2016021745A1
Application number: PCT/KR2014/007230
Authority: WO
Inventors: 정현석; 전상미
Original assignee: 한국기초과학지원연구원
Priority date: 2014-08-05
Filing date: 2014-08-05
Publication date: 2016-02-11

Abstract

The present invention relates to: a work station for correlative measurement of a light microscope and an electron microscope, more specifically, a work station that includes a sample holder for a cryogenic electron microscope for correlative imaging detection of a light microscope and an electron microscope; a correlative imaging detection apparatus including the same; and an imaging detection method and an imaging system using the same, wherein the work station comprises: a grid accommodating part (150) that is formed on the upper surface of the work station (10) for locating a sample grid in a light microscope and has a coolant accommodating portion (310), a grid accommodating recess (340), and a through-hole (156) that are formed therein, wherein liquid nitrogen for cooling the sample grid is supplied into the coolant accommodating portion (310), the grid accommodating recess (340) is used to locate the sample grid in a space that is separated from the coolant accommodating portion (310) by an accommodating-portion partition wall (323), and the through-hole (156) is formed on a side of the grid accommodating recess (340) to communicate with the outside; and a sample holder (14) that is attached/detached to locate a mounting head (380) in the grid accommodating part (150) through the through-hole (156), wherein the mounting head (380) on which the sample gird is seated is formed at a tip end of an extension tube (180) through which liquid nitrogen is supplied from a cooler (170) outside the grid accommodating part (150).

Description

Workstation including cryogenic electron microscope specimen holder for linked imaging detection of optical microscope and electron microscope, linked imaging detection apparatus including the same, imaging detection method and imaging system using the same

The present invention relates to a workstation for a cooperative measurement of a light microscope and an electron microscope, a cooperative measuring apparatus including the same, a measuring method and a measuring system using the same.

In general, as a device for observing biological samples in the medical and biotechnology fields, light microscopes and electron microscopes are widely used.

An optical microscope is a microscope made to investigate the position and characteristics of a particular component or element by injecting a fluorescent dye that fluoresces under ultraviolet light close to visible rays into a sample. In this case, the fluorescent dye used is characterized by absorbing ultraviolet light of short wavelength and emitting energy of long wavelength. For example, optical microscopy is performed by immunofluorescence staining of antibodies capable of reacting with a specific antigen when the immunoassay is performed in a hospital, followed by observing it, and is classified as a fluorescence microscope by this feature. Such an optical microscope can be usefully used for a sample which has a fluorescent substance itself or can be adsorbed on a fluorescent material. Therefore, the optical microscope can be used to identify the path of infection of bacteria or viruses, to locate functional proteins in the cell, and to change the environment. It is widely used for identifying biological phenomena such as specific protein expression and intracellular detection / testing of biological samples.

Meanwhile, an electron microscope uses an electron beam instead of visible rays used in an optical microscope and an electron lens instead of a glass lens to create an enlarged image of an object. Say. The electron microscope scans the surface of a sample placed in a vacuum with a fine electron beam to accelerate the electrons from the filament and scanning electron microscopy (SEM) to display an enlarged image, and transmits the electron beam exiting the hole of the anode to the specimen. Transmission electron microscopy (TEM), which obtains an image and magnifies the image with an electron lens, can be classified.

Recently, due to the industrialization of ultra-fine technology in the industrial field, technologies for accurately observing the surface and internal shape of fine materials have been gradually developed, and the technology of the medical biotechnology field has been rapidly developed by the introduction of the electron microscope. . Since electron microscopes have high resolution, they can not only observe objects with higher magnification than optical microscopes, but also can correlate the physical structure of the objects with physical properties such as hardness and reflectivity. It has the advantage of being able to analyze the size and type and relative amounts of elements and compounds.

However, the electron microscope can observe the state within the ultra-fine area with high magnification, but has a disadvantage in that it is not suitable for observing the characteristics of the large area. Therefore, recently, in observing living cells, by linking the optical microscope and the electron microscope, a method of intensively observing the extracted region through the electron microscope after extracting the necessary signal through the optical microscope has been used.

In the related art, in order to observe a biological sample by linking an optical microscope and an electron microscope, for example, after a biological sample is clouded in a liquid, the pipette is placed on a support membrane treated with a predetermined pipette, and water is removed with a filter paper, and then liquid nitrogen is removed. It is necessary to carry out a pretreatment step of rapidly cooling to prevent ice crystals from being supplied, and then to carry and observe the rapidly cooled biological sample grid by an optical microscope, and then again by electron microscope.

Therefore, in the related art, a rapid cooling process of a biological sample through a separate device may be carried by an optical microscope, and a manual operation may be accompanied by a tool such as tweezers in the process of transporting a biological sample observed by the optical microscope to an electron microscope. There is no choice but to. Therefore, in the prior art, breakage or damage of the sample grid occurred in each process of carrying the sample grid from which signals were extracted by a pretreatment process and an optical microscope. In addition, the sample grid pretreated by rapid cooling has to maintain a constant temperature during the final delivery to the electron microscope, there is a problem that the sample transport operation is very difficult and cumbersome, and takes a lot of time.

As a technique for solving the above problems, Korean Patent No. 10-1396420, which is pre-registered by the applicant of the present invention, is pre-processed by dipping the sample grid in liquid nitrogen and transported the pre-treated sample grid and mounted on the optical microscope. A description is given of a coupled cryogenic specimen preparation device of an electron microscope, which is configured such that a series of processes to observe and observe can be performed continuously in a single specimen preparation device. To this end, the domestic registration No. 10-1396420, as shown in Figure 7, injects liquid nitrogen through the injection port 2 formed in the main body 1 of the specimen preparation device (A), formed in the main body (1) After removing the sample grid from the sample grid transport container mounted on the sample storage part 5 of the sample mounting part, attaching the sample grid to the sample mounting part 4, and mounting the specimen preparation device A equipped with the sample grid on an optical microscope. A technique is described which makes it possible.

However, although the domestic registration No. 10-1396420 can be mounted immediately with the optical microscope after the pre-treatment, the input to the electron microscope can still be made by manually transporting the grid. Therefore, damage may occur in the process of introducing the sample grid into the electron microscope. In particular, such an electron microscope has a complicated measurement environment and a mounting environment of the sample grid, so that the transport of the sample grid is very difficult and many hours. There is a problem that takes.

Accordingly, it is a time to urgently need to introduce a technology for solving the above-described problems of the prior art so that the pretreatment of the sample grid and the injection into the electron microscope as well as the optical microscope can be continuously performed.

Thus, the present invention in view of the above point, the pre-treatment process of the specimen grid, the measurement through the optical microscope and the electron microscope can be carried out through a single device, the specimen grid in the optical microscope It is an object of the present invention to provide a linked measuring device and a measuring method using the same for a cooperative measurement of an optical microscope and an electron microscope that do not need to be moved in order to maintain the cooling state.

In a workstation in which a pretreatment process of cooling the specimen grid according to the present invention is performed, and the specimen grid is inserted into the specimen mounting holder, the image observation in the optical microscope and the electron microscope is continuously performed without carrying the specimen grid.

An inlet portion through which liquid nitrogen is supplied from the outside of the work station, an outlet hole through which the supplied liquid nitrogen is discharged, a coolant accommodating portion accommodating the liquid nitrogen, and a coolant accommodating portion, and the specimen grid The grid receiving groove may include a grid receiving portion formed in the grid receiving groove and including a temperature sensor for measuring the temperature of the specimen grid, and a through hole formed through the outside.

In addition, the linked imaging detection apparatus of the optical microscope and the electron microscope according to the present invention,

The workstation and a cooler for storing the liquid nitrogen for performing a pretreatment process for cooling the specimen grid, and an extension tube formed extending to one side of the cooler, connected to the workstation during the pretreatment process, the pretreatment process is And a specimen mounting holder which, after being performed, is detached from the workstation and inserted into the measuring section of the electron microscope for linked measurements.

In addition, the linked imaging system for the coordinate measurement of the optical microscope and the electron microscope according to the present invention,

And a work station for performing a pretreatment process of cooling a specimen grid, a cooler in which liquid nitrogen is stored, and an extension tube extending to one side of the cooler, and connected to the work station during a pretreatment process. A specimen mounting holder which is detached from the workstation after being performed and inserted into the measuring section of the electron microscope for linkage measurement; an optical microscope for measuring a signal for identifying an analyte on a specimen grid preprocessed by the workstation; and An electron microscope that analyzes the signal measured by an optical microscope.

Finally, the imaging detection method in conjunction with the optical microscope and the electron microscope according to the present invention,

At a spaced position below the optical microscope, the specimen grid may be positioned and the workstation is formed with a grid receiving portion including a through hole formed to penetrate to the outside on one side, inserting the specimen mounting holder through the through hole Positioning a specimen grid inside the grid receptacle, then cooling, placing the cooled specimen grid in the specimen mounting holder, and forming a signal on the cooled specimen grid using an optical microscope And after forming the signal, separating the specimen mounting holder from the work station.

According to such a linked measuring device and a measuring method according to the present invention, the pre-treatment process for cooling the specimen grid, and the measurement by the optical microscope is performed in a single linked measuring device, and after the measurement by the optical microscope to the electron microscope It is possible to reduce the time required to move the specimen grid and to prevent damage to the specimen grid by allowing the specimen to be fed as it is without having to move the specimen grid.

According to the present invention, the frozen state of the specimen grid can be continuously maintained during the movement of the specimen grid after the measurement through the optical microscope, thereby improving the accuracy of the measurement through the electron microscope.

1 is a perspective view of a workstation and an associated measuring device including the same according to a first embodiment of the present invention.

2 is a side view of the workstation shown in FIG. 1 and the associated measurement device including the same;

3 is an enlarged perspective view of the grid receiving portion of the workstation shown in FIG.

4 is a plan view and a side view of the grid receptacle shown in FIG. 3;

5 is a perspective view of a workstation and an associated measuring device including the same according to a second embodiment of the present invention;

FIG. 6 is a side view of the workstation shown in FIG. 5 and the associated measurement device including the same; FIG.

FIG. 7 is an enlarged perspective view of the grid receiving portion of the workstation shown in FIG. 5; FIG.

8 is a plan view and a side view of the grid receptacle shown in FIG. 7;

9 is an enlarged perspective view of the measuring lens holder according to the first and second embodiments of the present invention;

10 is a side view of a specimen mounting holder according to the first and second embodiments of the present invention.

11 is a perspective view of a workstation and a cooperative measurement apparatus including the same according to the first embodiment of the present invention.

12 is a perspective view showing the prior art.

DETAILED DESCRIPTION Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. Since the exemplary embodiments of the present invention may be embodied in various different forms, one of ordinary skill in the art to which the present invention pertains may be described herein. It is not limited to the Example to make.

1 is a perspective view of a workstation and a cooperative measuring apparatus including the same according to an embodiment of the present invention, and FIG. 2 is a side view of the workstation and the cooperative measuring apparatus including the same shown in FIG. 1.

1 and 2 together, the associated measuring device according to the present embodiment is a workstation 10 for performing a pretreatment process for cooling the specimen grid, and connected to the workstation 10 during the pretreatment process and pretreatment. A specimen mounting holder 14 is included that is detached from the workstation 10 after the procedure is performed so that it can be inserted into the measurement section of the electron microscope for linked measurements.

The workstation 10 is provided with a box part 120 in which the grid receiving part 150 is formed, and an external terminal part which is provided at the lower part of the box part 120 and is an external device connecting terminal for connecting to an external measuring device or control device. An upper plate 110 having an opening for covering the upper portion of the box portion 120 and exposing the grid accommodating portion 150 to an upper portion thereof, and an upper plate 110 vertically supporting the upper plate 110. A support 123, a base plate 140 supporting the lower end of the vertical support 123, and a horizontal support 142 formed on one side of the base plate 140 to horizontally support the workstation 10. It may be formed to include.

Here, the first handle 121 and the second handle 141 are provided on the upper and base plates 140 of the box part 120, respectively. The second handle 141 is formed to be rotatable in a vertical direction and a horizontal direction. For example, the second handle 141 is hinged to the handle is rotatable in the vertical and horizontal directions. Through this, when detached from the workstation 10 to insert the specimen mounting holder in the electron microscope, it can be easily separated.

In addition, the box portion 120 is formed of a thermally conductive material containing copper on the inside, and the outside is formed of the hardest acetal material among the resin types. In particular, a heat insulating layer formed of a heat insulating material containing styrofoam is formed in a multilayer structure between the inner side and the outer side.

The grid receiving portion 150 is formed to accommodate the specimen grid to be measured.

The grid accommodating part 150 is an inlet hole into which an inlet pipe 124 for supplying liquid nitrogen for cooling the specimen grid from the liquid nitrogen tank (not shown) provided outside of the workstation 10 is inserted ( 324 and a discharge hole 325 into which the discharge pipe 125 for discharging the liquid nitrogen supplied therein to the outside is inserted.

Here, the inlet pipe 124 and the discharge pipe 125 may be provided with a valve (not shown), respectively, to control the inflow and discharge of liquid nitrogen. This may facilitate the workstation 10 to move with the electron microscope.

The grid receiving part 150 includes a receiving part cover 152 that covers or opens the upper part, and a handle 154 is formed on the upper part of the receiving part cover 152 so that the grid holding part 150 can be easily covered and opened by a hand. do.

For reference, the accommodating part cover 152 may be formed of a transparent material such as acrylic, glass, or synthetic resin, but is not limited thereto and may be formed of various materials. Preferably, the accommodating part cover 152 may be formed of a material containing acrylic to facilitate observation.

In addition, the receiving part cover 152 may be formed with an opening 155 having a long hole shape to allow the work tool to be inserted into the grid receiving part 150 while covering the upper part of the grid receiving part 150. have.

In addition, according to the present invention, the workstation 10 for performing the pre-treatment process for cooling the specimen grid, the cooler 170 in which the liquid nitrogen is stored, and the extension tube extending to one side of the cooler 170 ( A specimen mounting holder which is connected to the workstation 10 during the pretreatment process, and which is detached from the workstation 10 after the pretreatment process is carried out so that it is inserted into the measuring part of the electron microscope for the associated measurement. A linkage measuring apparatus for linkage measurement between an optical microscope and an electron microscope including 14 is provided.

In the present embodiment, since the workstation may be applied to the workstation, a description thereof will be omitted below.

Specimen mounting holder 14 is provided on one side of the workstation 10 is detachably coupled, the front end of the extension tube 180 extending to one side is inserted into the side of the workstation 10 grid receiving portion 150 It is coupled to be located in the grid receiving groove 340 of the).

At this time, the specimen mounting holder 14 of various lengths seat the cooler 170 to the base plate 140, while supporting the lower portion of the cooler 170 to slide in a direction to be separated from the grid receiving portion 150 In the direction of insertion into the grid accommodating part 150, it may be supported by a sliding guide 190 having a stopper for limiting insertion. In addition, the sliding guide 190 is slid back and forth so that the specimen mounting holder 14 can be easily moved.

FIG. 3 is an enlarged perspective view of the grid accommodating part of the linked measurement device shown in FIG. 1, and FIG. 4 is a plan view and a side view of the linked measurement device shown in FIG. 3.

3 and 4 together, the grid receiving portion 150 forms an inner space that is open toward the upper portion of the workstation 10, and is formed in the box portion 120 to form the upper plate 110. Exposed upward through the opening.

In the inner space of the grid accommodating part 150, a coolant accommodating part 310 accommodating liquid nitrogen supplied therein, and a grid accommodating groove 340 which is separated from the coolant accommodating part 310 to form a space in which the specimen grid is located. ) Is formed, and a through hole 156 penetrating to the outside of the workstation 10 is formed at one side of the inside.

The grid accommodating part 150 further forms an inlet hole 324 and an outlet hole 325 at the other side of the inside. Liquid nitrogen is introduced into the inlet hole 324, and liquid nitrogen is discharged from the outlet hole 325. In particular, the discharge hole 325 is formed at a position higher than the inlet hole 324 so that the liquid nitrogen can be discharged while maintaining a constant water level.

In addition, an inflow partition 312 having a '-' shape is formed inside the grid accommodating part 150 to prevent splashing of the liquid nitrogen flowing from the inflow hole 324. The coolant accommodating part 310 and the grid accommodating groove 340 are separated from each other by the accommodating partition wall 323 so that the liquid nitrogen contained in the coolant accommodating part 310 does not flow into the grid accommodating groove 340.

Here, the receiving portion partition wall 323 is made of copper, and is designed to be slightly lower than the height of the entire box portion. This design allows the specimen grid 152 to be closed during image observation and the cold air of liquid nitrogen flows toward the grid receiving groove 340 where the specimen grid is frozen in the grid receiver 150, whereby the specimen grid is liquid nitrogen. Do not melt without.

The grid accommodating groove 340 is formed to accommodate the specimen grid therein, and a lens groove into which the measuring lens is inserted into the lower portion of the grid accommodating groove 340 to measure the specimen grid through an optical microscope. 342 is formed at the bottom.

The workstation according to the present invention is to measure the specimen grid by connecting the specimen grid with an optical microscope and an electron microscope. For example, when observing a living cell specimen, the position of the signal observed through the optical microscope is prevented from changing. It is preferred to use liquid nitrogen to rapidly freeze the specimen grid, but not limited thereto.

In addition, the grid accommodating part 150 is a cooling part groove 320 for inserting a grid holder (not shown) for storing a plurality of the specimen grid into the lower side of the coolant accommodating part 310 to maintain the state locked in liquid nitrogen. Is formed.

Then, the holder holder groove 330 is further formed to deliver the grid holder from the cooling unit groove 320 to be seated on one side of the grid receiving groove 340.

For reference, the grid holder may be formed in a circular shape, and the cooling unit groove 320 and the holder seating groove 330 may be formed in a circular shape so as to insert the grid holder.

In this case, the cooling unit groove 320 and the holder seating groove 330 may be formed to prevent protrusions of the grid holder by forming

protrusions

321 and 331 respectively having a concave-convex structure with the grid holder on one side. In particular, the holder seating groove 330 may enable the specimen grid to be stably taken out of the grid holder.

In addition, a temperature sensor 343 may be formed inside the grid accommodating hole so that the user can adjust the temperature by sensing the internal temperature cooled by the coolant.

In detail, the temperature measured by the temperature sensor 343 serves as a basis for controlling an external control device connected through the external terminal unit 122, and the external control device controls the inflow and discharge of liquid nitrogen to adjust the temperature of the sample grid. Keep it.

5 is a perspective view of a workstation and a cooperative measuring apparatus including the same according to the second embodiment of the present invention, and FIG. 6 is a side view of the workstation and the cooperative measuring apparatus including the same shown in FIG. 5.

5 and 6 together, the workstation according to the second embodiment has the same basic configuration and operation as the workstation according to the first embodiment, but the workstation according to the second embodiment is more portable.

The work station 10 removes the inlet pipe 124 and the inlet hole 324 from which the liquid nitrogen is supplied from the outside to facilitate movement with an electron microscope, but the outlet 125 and the outlet hole 325 have overflows of the liquid nitrogen. It is maintained to prevent overflow.

In addition, the receiving cover 152 is formed with a first inlet hole 156 and a second inlet hole 157, the first inlet hole 156 is inserted into the first inlet pipe 360, the second inlet hole 157 is a second inlet pipe 370 is inserted. Detailed description of the first inlet pipe 360 and the second inlet pipe 370 will be described later.

Configurations other than those described above are the same as the workstation according to the first embodiment.

7 is an enlarged perspective view of the grid receiving portion of the workstation shown in FIG. 5, and FIG. 8 is a plan view and a side view of the grid receiving portion shown in FIG. 7.

7 and 8 together, the grid receiving portion 150 according to the second embodiment of the inlet hole 324 is removed, only the discharge hole 325 is maintained.

The grid accommodating part 150 flows in liquid nitrogen contained in the portable cooling part 350 through the first inflow pipe 360 and the second inflow pipe 370. The portable cooling unit 350 may be formed in a funnel or container shape. In addition, the first inflow pipe 360 introduces liquid nitrogen into the grid receiving groove 340, and the second inflow pipe 370 introduces liquid nitrogen into the coolant accommodation portion 310.

Here, the first inlet pipe 360 may be inserted into the first inlet hole 156 to introduce liquid nitrogen into the grid receiving groove 340, and the second inlet pipe 370 may be the second inlet hole 157. The liquid nitrogen may be introduced into the coolant accommodating part 310.

Alternatively, the first inflow pipe 360 and the second inflow pipe 370 may enter the liquid nitrogen through the opening 155 of the accommodation cover 152 shown in the first embodiment.

At this time, the first inlet pipe 360 connected to the portable cooling unit 350 is basically formed at a position higher than the second inlet pipe 370. As a result, the first inlet pipe 360 may stop the inflow of liquid nitrogen faster than the second inlet pipe 370, which is the liquid nitrogen flow into the coolant accommodating part 310 and maintains the temperature at the same time as the grid receiving groove ( 340 may stop the introduction of liquid nitrogen to facilitate image measurement.

In addition, the first inlet pipe 360 and the second inlet pipe 370 may further include a valve to adjust the amount of liquid nitrogen inlet.

Configurations other than those described above are the same as those of the grid receiving portion 150 according to the first embodiment.

9 is an enlarged perspective view of the measuring lens holder according to the first and second embodiments of the present invention.

Referring to FIG. 9, the measuring lens holder 400 is provided to prevent freezing of the measuring lens. The measuring lens holder 400 includes a holder body 410, a gas injection unit 420, and a buffer 430.

The holder body 410 is inserted into the measuring lens, the gas injection unit 420 is formed on one side of the holder body 410 in order to prevent the freezing or frost of the measuring lens by injecting an inert gas such as nitrogen gas. . In addition, the buffer 430 is installed to prevent shock absorption and vibration of the measurement lens inserted into the holder body 410. That is, the buffer 430 provides a measurement lens to protect from external shock.

Therefore, the measuring lens holder 400 secures a problem that when the sample cooled by liquid nitrogen and the measuring lens are close to each other, the measuring lens is frozen or frost is generated due to a temperature difference to obtain an accurate image.

10 shows a side view of the specimen mounting holder shown in the first and second embodiments of the present invention.

Referring to FIG. 5, the specimen mounting holder 14 includes a cooler 170 capable of storing liquid nitrogen therein, an extension pipe 180 extending from one side of the cooler 170 to supply liquid nitrogen, and an extension pipe 180. A seating head 380 is formed at an extended tip of the head and provided with a grid seating groove 382 to seat the specimen grid.

The cooler 170 is preferably formed in a small size that can be easily carried and moved by the hand.

The liquid nitrogen stored in the cooler 170 is supplied to the seating head 380 through the extension pipe 180, thereby supplying the liquid nitrogen to the specimen grid seated on the seating head 380.

The specimen mounting holder 14 is manufactured so that the seating head 380 formed at the tip of the extension tube 180 may be inserted into a measurement unit of a conventional general electron microscope to position the specimen grid.

At this time, the extension tube 180 of the specimen mounting holder 14 forms a step in which the diameter becomes smaller at the front end side in a plurality of places, so that the extension tube 180 can be stopped exactly at the inserted position to the end, an electron microscope or a workpiece It can be formed to accurately position the specimen grid inside the station 10.

Here, the extension pipe 180 is formed to extend to one side of the box portion 120 in which the coolant is stored, the inner side is formed of a heat insulating material such as styrofoam, the outer side is formed of the hardest acetal material of the resin type.

In addition, the extension tube 180 is provided with a cog wheel therein to adjust the thickness and length according to the specimen mounting holder 14 having various thicknesses and lengths, or the extension tube 180 according to the size of the specimen mounting holder 14; Equipped with a removable structure to fit the size.

Hereinafter, the operation of the workstation of the present invention and the associated measurement device including the same according to the above configuration will be described in detail.

According to the present invention, in linking the measurement of the optical microscope and the electron microscope, the specimen mounting holder detachable to the grid receiving portion 150 and the measuring portion of the electron microscope for measuring the optical microscope, respectively Mount the cryogenic specimen grid in the grid seating groove (382) at the end of (14) and allow measurements to be made.

According to the invention, prior to measuring the specimen grid on an optical microscope, the seating head 380 of the specimen mounting holder 14 is placed in the grid receiving groove 340 of the grid receiving portion 150 at the workstation 10. . After filling the grid receiving portion 150 with liquid nitrogen, the grid holder storing the cryogenic specimen is first placed in the receiving portion 320, and when the temperature of the grid receiving groove 340 is stabilized to −185 degrees or less, the grid holder is cooled with water. The mounting head 380 of the specimen mounting holder 14 which is lifted up from the receiving portion 320 and seated in the holder seating groove 330 of the grid receiving portion 150, and the specimen grid is positioned adjacently by using a tweezer or the like. ) Is mounted on the grid seating groove 382, which is a sample insertion part of the head, and the measurement is performed with a lens (not shown) located under the optical microscope.

When the signal is measured through the optical microscope, the specimen mounting holder 14 is separated from the workstation 10 to separate the seating head 380 on which the specimen grid is seated from the grid receiving portion 150 and then into the inside of the electron microscope. Insertion is carried out for imaging measurements based on the signal of the optical microscope.

At this time, the liquid nitrogen by the cooler 170 of the specimen mounting holder 14 is continuously supplied from the time point at which the specimen grid measured by the optical microscope is separated from the workstation 10 to the electron microscope until the measurement is completed. By supplying, the state of the specimen grid according to the signal measured by the optical microscope can be accurately maintained in the cryogenic phase.

Therefore, according to the linked measuring device according to the present invention, the pretreatment process for cooling the specimen grid and the measurement through the optical microscope is performed in a single linked measurement device, it is necessary to move the specimen grid after the measurement through the optical microscope It can be put into the measuring section of the electron microscope as it is.

Therefore, the inconvenience of manually moving the specimen grid for the electron microscope measurement can be eliminated, and the freezing of the specimen grid is continuously maintained during the transfer to the electron microscope, thereby improving the accuracy of the measurement through the electron microscope. .

In addition, damage and breakage can be prevented by manually moving the specimen grid.

Referring to FIG. 11, a height adjusting device 510 is further provided to support a lower surface of the base plate 140 of the linked measuring device according to the previous embodiment to adjust the vertical height of the linked measuring device.

The height adjusting device 510 is a ground plate 520 supporting the bottom of the ground, a folding member 540 for forming a vertical driving stroke by a plurality of links between the ground plate 520 and the base plate 140 and Insertion of the operating shaft 530 is a screw thread formed in the folding member 540, it may be formed including a drive lever 550 for driving the folding member 540 up and down.

According to this configuration, it can be moved up and down integrally in the state in which the specimen mounting holder 14 is inserted into the workstation 10 located in the optical microscope, it is possible to increase the accuracy of the measurement through the optical microscope, the electron microscope This makes the measurement in conjunction with the system more efficient and easier.

Hereinafter, a measuring method in which an optical microscope and an electron microscope in accordance with another aspect of the present invention will be described.

For reference, the measuring method according to the present embodiment may be executed using the workstation and the associated measuring device including the same.

In addition, in the following when referring to the configuration of the coordinate measuring device, a configuration having the same function as the configuration of the above-described reference numerals will be described with the same reference numerals, and descriptions that overlap with the description of the above-described coordinate measuring device A brief description will be made to avoid.

According to this embodiment, the specimen mounting holder 14 is inserted into one side of the grid receiving portion 150 for positioning the specimen grid on the optical microscope, so that the specimen is inserted into the grid receiving groove 340 of the grid receiving portion 150. The first step of positioning the seating head 380 of the specimen mounting holder 14, the grid is mounted, and after filling the liquid nitrogen in the grid receiving portion 150, the grid holder storing the cryogenic specimen to the receiving portion 320 Firstly, after waiting for the temperature of the grid accommodating groove 340 to stabilize below −185 degrees, the grid holder is lifted from the coolant accommodating part 320 and seated on the holder seating groove 330 of the grid accommodating part 150. In the second step, the cryogenic grid stored in the grid holder is mounted in the grid seating groove 382, which is a sample inserting portion of the seating head 380 of the grid receiving portion 150 to measure the signal through the optical microscope Step 3 and above By separating the mounting holder 14 from the grid receiving portion it may be made of a fourth step of measuring the input and the mounting head 380 by an electron microscope.

The third step uses a method of picking up the specimen grid cooled by the grid holder with tweezers and transferring it to the seating head 380.

At this time, the grid holder is lifted up from the coolant accommodating part 310 and can be easily moved to the grid accommodating groove 340 positioned adjacent to the holder seating groove 330 of the grid accommodating part 150.

And, even after the grid holder is lifted up from the coolant accommodating part 310, since the liquid nitrogen is continuously affected by the inside of the grid accommodating part 150, the frozen state can be continuously maintained.

In addition, the fourth step is a liquid by the cooler 170 provided in the specimen mounting holder 14 until the specimen mounting holder 14 is separated from the grid receiving portion 150 and the measurement by the electron microscope is completed. Supplying nitrogen to the specimen grid to maintain cooling.

As described above, according to the measuring method in conjunction with the optical microscope and the electron microscope according to the present invention, in the pre-treatment process before the measurement of the specimen grid through the optical microscope to enable the rapid input to the optical microscope after cooling, the specimen grid In addition to preventing breakage, the specimen grid can be kept frozen.

In addition, it is possible to easily and easily move the specimen grid by one device without having to move it to a separate stage to input the specimen grid to the electron microscope after measurement through the optical microscope, so that the specimen grid may be generated during the movement of the specimen grid. Possible breakage can be prevented and the continuous supply of liquid nitrogen by the cooler is possible.

As described above, although the present invention has been described by way of limited embodiments and drawings, the present invention is not limited thereto and is intended by those skilled in the art to which the present invention pertains. Of course, various modifications and variations are possible within the equivalent scope of the claims to be described below.

[Description of the code]

10: workstation 14: specimen mounting holder

110: upper plate 120: box portion

121: first handle 122: external terminal portion

123: vertical support 124: inlet pipe

125: discharge pipe 140: base plate

141: second handle 142: horizontal support

150: grid receiving portion 152: receiving portion cover

154: cover handle 155: opening

156: first inlet hole 157: second inlet hole

158: through hole 170: cooler

180: extension tube 190: sliding guide

310: coolant accommodating part 312: inflow partition

320: cooling portion groove 323: receiving portion partition wall

324: inlet hole 325: outlet hole

330: holder seating groove 340: grid receiving groove

342: lens groove 343: temperature sensor

350: portable cooling unit 360: first inlet pipe

370: second inlet pipe 380: seating head

382: grid seating groove 400: measuring lens holder

410: holder body 420: gas injection portion

430: buffer 510: height adjusting device

520: ground plate 530: working shaft

540: folding member 550: drive lever

Claims

In a workstation in which a pretreatment process of cooling a specimen grid is performed, and a specimen grid is inserted into a specimen mounting holder, image observation in an optical microscope and an electron microscope is continuously performed without carrying the specimen grid.

An inlet portion through which liquid nitrogen is supplied from the outside of the work station and a discharge hole through which the supplied liquid nitrogen is discharged;

A coolant accommodating part in which the liquid nitrogen is accommodated;

A grid accommodating groove formed separately from the coolant accommodating part and in which the specimen grid is located;

A temperature sensor formed in the grid receiving groove to measure a temperature of the specimen grid; And

The workstation for penetrating the optical microscope and the electron microscope including a; including a; grid receiving portion including a through-hole formed through the outside on one side.
The method of claim 1,

And a receptacle cover for opening or closing the upper portion of the grid receptacle.
The method of claim 2,

The receiving portion cover,

And a long hole-type opening configured to allow a work tool to be inserted into the grid receiving portion while covering the upper portion of the grid receiving portion.
The method of claim 1,

The inlet portion,

And an inlet hole formed at a lower level than the outlet hole on one surface of the outlet hole, in which the liquid nitrogen is introduced from the outside.
The method of claim 4, wherein

The workstation for measuring the connection between the optical microscope and the electron microscope, characterized in that the 'b' shaped inlet partition is formed to prevent the splashing of the liquid nitrogen flowing from the inlet hole inside the grid receiving portion.
The method of claim 2,

The receiving portion cover is formed with two inlet holes,

The inlet portion,

The two inlet holes;

A first inlet pipe inserted into one of the two inlet holes to introduce liquid nitrogen into the grid receiving groove; And

And a second inlet pipe inserted into the other one to introduce the liquid nitrogen into the coolant accommodating part.
The method of claim 3,

The inlet portion,

A first inlet pipe for introducing liquid nitrogen into the grid receiving groove; And

And a second inlet pipe for introducing liquid nitrogen into the coolant accommodating part.

The first inlet pipe and the second inlet pipe,

And a work station for measuring the linkage between the optical microscope and the electron microscope, characterized by passing through the opening.
The method of claim 7, wherein

The first inlet pipe and the second inlet pipe,

Is connected to the portable cooling unit for supplying while carrying the liquid nitrogen,

The first inlet pipe is formed at a position higher than the second inlet pipe, the workstation for measuring the coupling between the optical microscope and the electron microscope.
The method of claim 1,

The grid receiving portion,

Cooling groove for inserting the grid holder for storing a plurality of the specimen grid to maintain the state immersed in liquid nitrogen; And

A holder seating groove which picks up the grid holder from the cooling unit groove and seats on one side of the grid receiving groove; Workstation for measuring the optical microscope and the electron microscope linked to the, characterized in that it comprises a.
The method of claim 9,

The cooling unit groove and the holder seating groove,

And a protrusion having a concave-convex structure to remove the specimen grid from the grid holder.
The method of claim 1,

And a lens groove into which a measurement lens for measuring the received specimen grid is formed below the grid receiving groove.
The method of claim 1,

The grid receiving portion,

And a measuring lens holder to prevent freezing or frost of the measuring lens when the sample cooled by the liquid nitrogen and the measuring lens are close to each other.
The method of claim 12,

The measuring lens holder,

A holder main body into which the measuring lens is inserted;

A gas injection unit for injecting an inert gas to one side of the holder body; And

And a buffer for protecting the measurement lens inserted into the holder body from an external shock, wherein the measuring lens is connected to the optical microscope and the electron microscope.
The method of claim 1,

A box portion in which the grid receiving portion is formed;

An upper plate covering an upper portion of the box portion and having an opening for exposing at least a portion of the grid receiving portion;

A support for supporting the upper plate in a vertical direction; And

A base plate supporting a lower end of the support part; Workstation for measuring the optical microscope and the electron microscope linked to the further comprising.
The method of claim 14,

The box unit,

The inside is formed of a thermally conductive material, the outside is formed of an acetal material,

Between the inner side and the outer side, the insulating layer formed of a heat insulating material is formed in a multi-layered structure, the workstation for measuring the connection between the optical microscope and the electron microscope.
The method of claim 1,

And a temperature sensor configured to measure a temperature inside the grid accommodating hole.
The workstation of any one of claims 1 to 16 for performing a pretreatment procedure to cool the specimen grid; And

And an extension tube formed extending to one side of the cooler, connected to the workstation during the pretreatment process, and separated from the workstation after the pretreatment process is performed, for an associated microscope Specimen mounting holder to be inserted into the measuring portion of the; Linked imaging detection device of the optical microscope and the electron microscope comprising a.
The method of claim 17,

The extension tube,

The inside is formed of a heat insulating material, the outside is formed of acetal material,

Gears inside to adjust thickness and length,

Linked imaging detection device of the optical microscope and the electron microscope, characterized in that to form a removable structure according to the size according to the size of the specimen mounting holder.
The method of claim 17,

The specimen mounting holder,

And a seating head formed at the tip of the extension tube, the seating head having a grid seating groove for seating a specimen grid.
The method of claim 17,

At the tip of the extension tube,

At least one step of gradually decreasing diameter is formed of the associated imaging detection device of the optical microscope and the electron microscope.
The method of claim 17,

The workstation,

A base plate for supporting the specimen mounting holder,

And a sliding guide having a stopper for sliding the specimen mounting holder in a direction to be separated from the grid accommodating portion and for limiting a depth at which the specimen mounting holder is inserted into the grid accommodating portion. Linked Imaging Detection Device.
The method of claim 17,

The workstation,

And a base plate for supporting the specimen mounting holder.

Linked imaging detection device for a coordinated measurement of the optical microscope and the electron microscope, characterized in that it further comprises; height adjustment device for adjusting the vertical height of the workstation by supporting the lower surface of the base plate.
The method of claim 22,

The height adjusting device includes a ground plate supporting the bottom of the ground;

A folding member driven up and down by a plurality of links between the ground plate and the base plate; And

And a driving lever for inserting an operating shaft horizontally screwed into the folding member to drive the folding member up and down. 2.
17. A work station according to any one of claims 1 to 16 for performing a pretreatment procedure to cool the specimen grid;

And an extension tube formed extending to one side of the cooler, connected to the workstation during the pretreatment process, and separated from the workstation after the pretreatment process is performed, for an associated microscope Specimen mounting holder to be inserted into the measuring portion of the;

An optical microscope for measuring a signal for identifying an analyte on a specimen grid preprocessed by the workstation; And

An electron microscope for analyzing the signal measured by the optical microscope; Linked imaging system for measurement of the linkage between the optical microscope and the electron microscope comprising a.
The method of claim 24,

And the cooler continuously supplies liquid nitrogen while the specimen mounting holder is separated from the workstation and inserted into the interior of the electron microscope.
Positioning a workstation on which a grid receptacle is formed at a spaced position below the optical microscope, in which a specimen grid may be positioned and a through hole formed through one side;

Inserting a specimen mounting holder through the through hole;

Placing a specimen grid inside the grid receptacle and then cooling;

Positioning the cooled specimen grid in the specimen mounting holder;

Forming a signal on the cooled specimen grid using an optical microscope; And

After the signal is formed, separating the specimen mounting holder from the workstation; and an optical microscope and an electron microscope in association with each other.
The method of claim 26,

And separating the specimen mounting holder from the work station, and then measuring the specimen mounting holder by using an electron microscope to measure the optical microscope and the electron microscope.
The method of claim 26,

The positioning of the cooled specimen grid on the specimen mounting holder comprises: picking the specimen grid cooled from the grid holder with tweezers and transferring the specimen to a seating head.
The method of claim 26,

After separating the specimen mounting holder from the grid receiving portion, the liquid nitrogen by the cooler provided in the specimen mounting holder is supplied to the specimen grid until the measurement by the electron microscope is completed, to maintain cooling An imaging detection method in which an optical microscope and an electron microscope are used.