KR20220052256A - Method for stochastic optical reconstruction microscopy imaging and stochastic optical reconstruction microscopy imaging apparatus - Google Patents

Abstract

Disclosed are an imaging method of an ultra-high-resolution fluorescence microscope, and an imaging device of an ultra-high-resolution fluorescence microscope. The present invention comprises: a step of culturing cells on cover glass; a step of fixating the cultured cells; a step of marking the fixated cells with fluorescent substances; a step of immersing the fluorescently marked cells in an imaging buffer solution; a step of measuring fluorescence of the cells by using an ultra-high-resolution fluorescence microscope to perform imaging of the fluorescence of the cells and analyzing a fluorescence signal; and a step of filtering the fluorescence signal. Laser power of the ultra-high-resolution fluorescence microscope is 6 mW to 50 mW.

Description

Ultra-high-resolution fluorescence microscopy imaging method and ultra-high-resolution fluorescence microscopy imaging apparatus

The present invention relates to an ultra-high-resolution fluorescence microscope imaging method and an ultra-high-resolution fluorescence microscope imaging device, and more particularly, to an ultra-high-resolution fluorescence microscope imaging method and an ultra-high-resolution fluorescence microscope imaging device capable of imaging even with a low-intensity laser.

Recently, various ultra-high-resolution fluorescence microscopy techniques have been developed that exceed the theoretical resolution of optical microscopy, which is also called the diffraction limit of light. Among them, STORM (Stochastic Optical Reconstruction Microscopy) technology causes a photoswitching phenomenon at high laser power to separate overlapping fluorescent molecules (corresponding to phosphors or fluorescent dyes) by time to achieve ultra-high resolution. It is an imaging technique with

Existing STORM technology has a problem in that ultra-high-resolution imaging is impossible because fluorescence intensity is low and optical switching is impossible at low laser power.

More specifically, the conventional STORM technology has a difficulty in that ultra-high-resolution imaging is impossible because optical switching does not occur well at low laser power and the intensity of fluorescence that determines image resolution also drops sharply.

On the other hand, in the case of a high-intensity laser power, the phosphor can be rapidly photobleached, and in particular, in the case of a bio sample, the structure can be destroyed by the high-intensity laser power. need.

Republic of Korea Patent Publication No. 2020-0024794, "Super-resolution imaging" Republic of Korea Patent Publication No. 2018-0130567, "Generalized Stochastic Super-Resolution Sequencing"

In an embodiment of the present invention, by adjusting the chemical composition (concentration and/or pH) of the imaging buffer solution, it is possible to increase the fluorescence intensity at low laser power, activate optical switching, and to improve resolution. An object of the present invention is to provide a fluorescence microscope imaging method and an ultra-high-resolution fluorescence microscope imaging apparatus.

In addition, the imaging buffer solution is to provide an ultra-high-resolution fluorescence microscope imaging method and an ultra-high-resolution fluorescence microscope imaging apparatus capable of rapidly and strongly causing optical switching in organic phosphors even under low-intensity laser power conditions.

An embodiment of the present invention provides an ultra-high-resolution fluorescence microscopy imaging method and ultra-high-resolution fluorescence microscopy imaging method capable of imaging even at low laser power by filtering signal errors caused by poor optical switching at low intensity laser power through image analysis. to provide the device.

Ultra-high-resolution fluorescence microscopy imaging method according to an embodiment of the present invention comprises the steps of culturing cells on a cover glass (cover glass); fixing the cultured cells; labeling the fixed cells with a fluorescent substance; immersing the fluorescently-labeled cells in an imaging buffer solution; measuring and imaging the fluorescence of the cells using an ultra-high-resolution fluorescence microscope, and analyzing the fluorescence signal; and filtering the fluorescence signal, wherein a laser power of the ultra-high-resolution fluorescence microscope is 6mW to 50mW.

In the ultra-high-resolution fluorescence microscope, the full width at half maximum (FWHM) and the fluorescence intensity of the distribution of the imaging centroid of a single phosphor may be adjusted according to the laser power.

The ultra-high-resolution fluorescence microscope may be Stochastic Optical Reconstruction Microscopy (STORM).

The pH of the imaging buffer solution may be 7.0 to 9.0.

The imaging buffer solution may include a Tris (2-amino-2-hydroxymethyl-1,3-propanediol) solution, a thiol compound, and an oxygen scavenger system.

The concentration of the thiol compound in the imaging buffer solution may be 5 mM to 200 mM.

The oxygen scavenger system may include glucose oxidase, glucose, and catalase.

The fluorescent label is at least one of a cyanine series dye, atto series, fluorescein seires, rhodamine series, BODIPY dye, and phorphyrin series. It may be labeled with either dye.

The filtering may remove a background signal of 800 (A.U.) to 2000 (A.U.) detected by the EM-CCD camera sensor.

An ultra-high-resolution fluorescence microscope apparatus according to an embodiment of the present invention comprises: a plate on which fluorescently-labeled cells supported in an imaging buffer solution are disposed; a light source unit irradiating a laser to the fluorescently labeled cells; and a detector measuring and imaging the fluorescence of the cells and analyzing the fluorescence signal, wherein the laser power is 6mW to 50mW.

The full width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor measured with the ultra-high-resolution fluorescence microscope may be in the range of 21.55 nm to 50.00 nm.

The ultra-high-resolution fluorescence microscope may be Stochastic Optical Reconstruction Microscopy (STORM).

The concentration of the thiol compound in the imaging buffer solution may be 5 mM to 200 mM.

The pH of the imaging buffer solution may be 7.0 to 9.0.

The detector filters the fluorescence signal, and the filtering may remove a background signal of 800 (A.U.) to 2000 (A.U.) detected by the EM-CCD camera sensor.

According to an embodiment of the present invention, by adjusting the chemical composition (concentration and/or pH) of the imaging buffer solution, it is possible to increase the fluorescence intensity at low laser power, activate optical switching, and improve resolution. A high-resolution fluorescence microscope imaging method and an ultra-high-resolution fluorescence microscope imaging apparatus can be provided.

In addition, the imaging buffer solution can quickly and strongly cause optical switching in the organic phosphor even under a low laser power condition.

According to an embodiment of the present invention, an ultra-high-resolution fluorescence microscope imaging method and an ultra-high-resolution fluorescence microscope capable of imaging even at low laser power by filtering signal errors caused by poor optical switching at low intensity laser power through image analysis An imaging device may be provided.

1 is a flowchart illustrating an ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention.
2A to 2F are ultra-high-resolution STORM images and fluorescence intensity graphs of intracellular microtubules taken with different laser power, and FIG. 3 is a fluorescence intensity (Brightness; mean photon number) according to FIGS. 2A to 2F. ), and FIG. 4 is a graph showing the full width at half maximum (FWHM) for the distribution of the imaging position (centroid) of a single phosphor according to FIGS. 2A to 2F .
FIG. 5 shows super-resolution STORM images of intracellular microtubules taken by varying the concentration of a thiol compound (corresponding to the concentration of MEA), and FIG. 6 is a fluorescence intensity (Brightness; mean) according to FIG. photon number), and FIG. 7 is a graph showing the full width at half maximum (FWHM) for the distribution of the imaging position (centroid) of the single phosphor according to FIG. 5 .
8 is a diagram showing an ultra-high-resolution STORM image of intracellular microtubules taken by varying the pH of the imaging buffer solution, and FIG. 9 is a graph showing the fluorescence intensity (Brightness; mean photon number) according to FIG. 8, FIG. 10 is a graph illustrating a full width at half maximum (FWHM) for a distribution of an imaging centroid of a single phosphor according to FIG. 8 .
11 shows an ultra-high-resolution STORM image of intracellular microtubules photographed using the imaging buffer solution according to Comparative Example 1 and an ultra-high-resolution STORM image of intracellular microtubules photographed using the imaging buffer solution according to Example 1. 12 is a graph showing the fluorescence intensity (mean photon number) according to FIG. 11 , and FIG. 13 is a full width at half maximum for the distribution of the imaging position (centroid) of the single phosphor according to FIG. 11 . , FWHM) is a graph showing.
14 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 15 is a second of intracellular microtubules taken using the imaging buffer solution according to Comparative Example 1 at high laser power (100 mW). This is a high-resolution STORM image.
FIG. 16 is a graph showing the full width at half maximum with respect to the distribution of imaging centroids of a single phosphor according to FIG. 15 , and FIG. 17 is a graph showing fluorescence intensity according to FIG. 15 .
18 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 19 is an ultra-high-resolution STORM of intracellular microtubules taken using the imaging buffer solution according to Comparative Example 1 at a weak laser power (12 mW). image, and FIG. 20 is a graph showing the full width at half maximum for the distribution of imaging centroids of a single phosphor according to FIG. 19, and FIG. 21 is a graph showing the fluorescence intensity according to FIG.
22 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 23 is an ultra-high-resolution STORM of intracellular microtubules taken using the imaging buffer solution according to Example 2 at a weak laser power (12 mW). image, and FIG. 24 is a graph showing the full width at half maximum for the imaging centroid distribution of a single phosphor according to FIG. 23 , and FIG. 25 is a graph showing the fluorescence intensity according to FIG. 23 .
26 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 27 is an ultra-high-resolution STORM of intracellular microtubules taken using the imaging buffer solution according to Example 3 at a weak laser power (12 mW). image, and FIG. 28 is a graph showing the full width at half maximum for the imaging centroid distribution of a single phosphor according to FIG. 27 , and FIG. 29 is a graph showing the fluorescence intensity according to FIG. 27 .
30 is an ultra-high-resolution STORM image and graph illustrating the effect of ultra-high-resolution fluorescence imaging according to signal error filtering image analysis in the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings and contents described in the accompanying drawings, but the present invention is not limited or limited by the embodiments.

The terminology used herein is for the purpose of describing the embodiment and is not intended to limit the present invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, "comprises" and/or "comprising" refers to the presence of one or more other components, steps, operations and/or elements mentioned. or addition is not excluded.

As used herein, “embodiment”, “example”, “aspect”, “exemplary”, etc. are to be construed as advantageous in any aspect or design described as being preferred or advantageous over other aspects or designs. is not doing

Also, the term 'or' means 'inclusive or' rather than 'exclusive or'. That is, unless stated otherwise or clear from context, the expression 'x employs a or b' means any of natural inclusive permutations.

Also, as used herein and in the claims, the singular expression "a" or "an" generally means "one or more," unless stated otherwise or clear from the context that it relates to the singular form. should be interpreted as

The terms used in the description below have been selected as general and universal in the related technical field, but there may be other terms depending on the development and/or change of technology, customs, preferences of technicians, and the like. Therefore, the terms used in the description below should not be construed as limiting the technical idea, but as illustrative terms for describing the embodiment.

In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the corresponding description. Therefore, the terms used in the description below should be understood based on the meaning of the term and the content throughout the specification, rather than the simple name of the term.

Meanwhile, terms such as first and second may be used to describe various components, but the components are not limited by the terms. The terms are used only for the purpose of distinguishing one component from another.

In addition, when a part such as a film, layer, region, configuration request, etc. is said to be “on” or “on” another part, it is not only in the case that it is directly on the other part, but also another film, layer, region, or component in the middle. Including cases where etc. are interposed.

Unless otherwise defined, all terms (including technical and scientific terms) used herein may be used with the meaning commonly understood by those of ordinary skill in the art to which the present invention belongs. In addition, terms defined in a commonly used dictionary are not to be interpreted ideally or excessively unless clearly defined in particular.

Meanwhile, in describing the present invention, if it is determined that a detailed description of a related well-known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted. In addition, the terms used in this specification are terms used to properly express the embodiment of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Accordingly, definitions of these terms should be made based on the content throughout this specification.

1 is a flowchart illustrating an ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention.

The ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention comprises the steps of culturing cells on a cover glass, fixing the cultured cells, and labeling the fixed cells with a fluorescent substance. , immersing the fluorescently-labeled cells in an imaging buffer solution, measuring and imaging the fluorescence of the cells using an ultra-high-resolution fluorescence microscope, analyzing the fluorescence signal, and filtering the fluorescence signal.

The ultra-high-resolution fluorescence microscopy imaging method according to an embodiment of the present invention can realize high resolution by using a laser power of a low intensity corresponding to 12% of the laser intensity mainly used in the prior art.

At this time, the laser power of the ultra-high-resolution fluorescence microscope can be 6mW to 100mW, and when the laser power is 6mW or less, optical switching is not good, and when it exceeds 100mW, photobleaching occurs quickly and it is possible to image living cells. When there is a problem to damage the sample. Preferably, the laser power of the ultra-high-resolution fluorescence microscope may be 6mW to 50mW.

In addition, in the ultra-high-resolution fluorescence microscope, the full width at half maximum (FWHM) and the fluorescence intensity for the distribution of the imaging centroid of a single phosphor can be adjusted according to laser power.

The full with at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor is the position when one phosphor is photoswitched for each frame and its emission is photographed. means distribution. That is, in the case of a phosphor whose emission is photographed with high precision, it has a narrow distribution and has a small FWHM value, but in the case of a phosphor with low precision, it has a narrow area distribution. By being spread out and imaged, the distribution area can be widened to have a large full width at half maximum (FWHM).

Accordingly, in the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention, the resolution may be improved as the full width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor is reduced. That is, the ultra-high-resolution fluorescence microscope imaging apparatus may determine the resolution criterion as the full width at half maximum (FWHM) with respect to the distribution of the imaging centroid of a single phosphor.

In addition, the fluorescence intensity (Brightness, meaning photon number) when a single phosphor is imaged by a camera in an ultra-high-resolution fluorescence microscope imaging device (eg, STORM) is the precision of the location of the phosphor. can be decided

That is, in the case of a bright phosphor (the higher the brightness), the centroid can be determined with very high precision by making a sharp point spread function (PSF) with high intensity, whereas the dark In the case of a phosphor (the lower the brightness), the point diffusion function is made not clear, and the centroid may be uncertainly determined with low precision.

에 반비례하기 때문에, 형광 세기(brightness)가 높을수록 높은 정확도(small error)로 위치를 잡을 수 있어 해상도가 증가될 수 있다.Theoretically, the degree of this localization error is

Since it is inversely proportional to , the higher the fluorescence intensity (brightness), the higher the position can be positioned with high accuracy (small error), the resolution can be increased.

First, the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention proceeds with the steps of culturing cells on a cover glass and fixing the cultured cells.

The cell may include at least one of a cell, a cell type, a polynucleotide, a protein, a polysaccharide, a mucopolysaccharide, a proteoglycan, a lipid, a microorganism, a virus, and a molecule, and preferably, a protein.

Various methods used in the art may be used without limitation as a method for culturing and fixing cells.

Thereafter, in the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention, a step of labeling the fixed cells with a fluorescent substance is performed.

The immobilized cells are labeled with a fluorescent substance on a desired target, and various antibody staining methods known in the art can be used for the method of labeling the fluorescent substance. may include steps.

The fluorescent label is at least one of cyanine series, atto series, fluorescein seires, rhodamine series, BODIPY dyes, and phorphyrin series. It can be labeled with one dye.

Thereafter, in the ultra-high-resolution fluorescence microscopy imaging method according to an embodiment of the present invention, the step of loading the fluorescently-labeled cells in an imaging buffer solution is performed.

The imaging buffer solution may include a Tris(2-amino-2-hydroxymethyl-1,3-propanediol) solution, a thiol compound, and an oxygen scavenger system.

ME(beta-mercaptoethanol) 중 적어도 어느 하나를 포함할 수 있다.Preferably, the thiol compound is MEA (2-mercaptoethylamine) and

It may include at least one of beta-mercaptoethanol (ME).

In the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention, at 12 mW laser power, the optical switching speed reaching equilibrium is adjusted according to the concentration of the imaging buffer solution, the half maximum width is adjusted, and the degree of deprotonation is changed according to the pH. The resolution may be adjusted by adjusting the fluorescence intensity and the full width at half maximum.

The optical switching phenomenon refers to a phenomenon in which a fluorescent dye can reversibly switch between an on-state in which fluorescence can be emitted and an off-state in which it cannot emit fluorescence. it means.

At this time, since the concentration and pH of the imaging buffer solution can affect the process of changing the fluorescent dye from on-state to off-state, the population between the two states can be controlled. and the optical switching speed may be adjusted accordingly. In addition, in general, since fluorescent dyes can sensitively change their fluorescence intensity depending on the surrounding buffer solution conditions, the full width at half maximum (FWHM) for the imaging centroid distribution of a single phosphor under various imaging buffer solution conditions can be adjusted. can

The concentration of the thiol compound in the imaging buffer solution may be 5 mM to 200 mM, and if the concentration of the thiol compound is less than 5 mM, there is a problem in that the optical switching speed is too low, and exceeds 200 mM If the fluorescence intensity is reduced, there is a problem.

Preferably, the ultra-high-resolution fluorescence microscopy imaging method according to an embodiment of the present invention may include 20 mM MEA in the imaging buffer solution, whereby the fluorescence intensity may be increased by 1.8 times and the resolution by 1.26 times.

The pH of the imaging buffer solution may be 7.0 to 9.0, and if the pH of the imaging buffer solution is less than 7.0, there is a problem in that the optical switching speed decreases and it is difficult to apply to living cells. There is a problem that cannot be applied.

Preferably, the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention may include MEA of pH 9.0 in the imaging buffer solution to have a pH of 9.0, and the concentration of MEA at this time may be 100 mM, Due to this, the fluorescence intensity can be increased by 1.7 times and the resolution by 1.05 times.

Therefore, in the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention, by adjusting the concentration or pH of the imaging buffer solution, light switching occurs quickly and strongly in the organic phosphor even under a laser power condition of a low intensity of 6 mW to 50 mW, It is possible to shoot high-resolution images.

Tris(2-amino-2-hydroxymethyl-1,3-propanediol) solution may be used as a pH buffer in the imaging buffer solution, and preferably, Tris-HCl 8.0 buffer solution may be used under high laser intensity conditions. and Tris-HCl 7.0 or Tris-HCl 9.0 buffer solution may be used under low laser intensity conditions.

The oxygen scavenger system has an effect of suppressing a decrease in the fluorescence intensity of a fluorescence sample due to oxygen, and may include glucose oxidase, glucose and catalase.

Thereafter, the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention performs imaging by measuring fluorescence of cells using an ultra-high-resolution fluorescence microscope, and analyzing the fluorescence signal.

Preferably, the fluorescence emitted from the fluorescent molecule is detected through an EM-CCD or sCMOS camera sensor and an image can be obtained based on the fluorescence intensity read for each pixel.

At this time, if a limit value is set for the background fluorescence intensity, a Point Spread Function (PSF) is recognized for a signal exceeding the limit value in each frame, and a two-dimensional By fitting with a Gaussian curve, the centroid of the point spread constant can be determined as the position of a single molecule.

The final STORM image can be reconstructed by collecting all centroids obtained for each frame and correcting for drift over time.

The ultra-high-resolution fluorescence microscope may be any one of Stochastic Optical Reconstruction Microscopy (STORM), Stimulated Emission Depletion Microscopy (STED), PhotoActivation Localization Microscopy (PALM), and Structured Illumination Microscopy (SIM), but preferably, Stochastic Optical Reconstruction (STORM) microscopy).

Finally, the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention proceeds with the step of filtering the fluorescence signal.

In the ultra-high-resolution fluorescence microscopy imaging method, when imaging is performed at low laser power, optical switching does not occur well and the brightness of the phosphor is low, which may cause false fluorescence signals (localization). This may occur because the point spread function (PSF) is not well fitted with a two-dimensional Gaussian function and is classified as a background signal.

The background signal is an average value of pixel signal intensity between close frames in a state where there is no fluorescence emission of the phosphor, and the background ( background) value can be given.

Therefore, the ultra-high-resolution fluorescence microscopy imaging method according to an embodiment of the present invention removes a background signal of a certain intensity or more, thereby removing an erroneous background signal indicating a slow light switching phenomenon and improving localization precision.

Preferably, the filtering is performed by removing the background signal of 800 (A.U.) to 2000 (A.U.) detected by the EM-CCD camera sensor and filtering, thereby removing the signal error that was misplaced by overlapping light emission to remove the super high resolution Image quality can be improved. For example, the value may be a range for a green channel in a background histogram (BG histogram).

When the distribution of the background value for each localization is drawn, it is largely divided into two curves, a high background and a low background. At this time, the localization corresponding to the high background is judged as a signal error and these can be removed

According to the embodiment, the ultra-high-resolution fluorescence microscopy imaging method according to the embodiment of the present invention is applied to various fluorescence imaging or analysis as well as ultra-high-resolution fluorescence microscopy technology (STORM). It is applicable to all fluorescence analysis that has been used.

When imaging a biosample using a high-intensity laser (eg, 100mW), the structure of the bio-sample may be destroyed by the high-intensity laser, but the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention is low Since imaging is performed using a laser of high intensity, high-resolution imaging is possible without destroying the structure of the bio sample, and furthermore, live cell imaging is possible.

In addition, in the case of a fluorescence microscope device using a high-intensity laser (eg, 100mW), an expensive ultra-high-resolution fluorescence microscope setup cost is required, but the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention uses a low-intensity laser can be used to reduce manufacturing costs.

In addition, in the case of a fluorescence microscope device using a high-intensity laser (eg, 100 mW), photobleaching occurs quickly and imaging time is short. can be used to increase the imaging time that can be imaged because photobleaching occurs slowly.

The imaging time may be 10 to 15 minutes at 60 Hz when imaging a structure with a high density such as microtubules, and if the imaging time is less than 10 minutes, it is difficult to obtain a high-quality image (or the shape of the target is unclear due to the low localization number) There is a problem that is observed frequently), and there is a problem that light fading occurs if it exceeds 30 minutes.

The ultra-high-resolution fluorescence microscope apparatus according to an embodiment of the present invention includes a plate on which fluorescently-labeled cells supported in an imaging buffer solution are disposed, a light source unit that irradiates a laser to the fluorescently-labeled cells, and the fluorescence of the cells by measuring the fluorescence and fluorescence signal It includes a detector that analyzes the

Since the ultra-high-resolution fluorescence microscope apparatus according to the embodiment of the present invention includes the same components as the ultra-high-resolution fluorescence microscope imaging method according to the embodiment of the present invention, the same components will be omitted.

Preferably, the ultra-high resolution fluorescence microscope may be STORM (Stochastic Optical Reconstruction Microscopy).

The ultra-high-resolution fluorescence microscope apparatus according to an embodiment of the present invention includes a plate on which fluorescently-labeled cells supported in an imaging buffer solution are disposed.

Fluorescently-labeled cells supported in an imaging buffer solution may be placed on the plate.

The concentration of the thiol compound in the imaging buffer solution may be 5 mM to 200 mM, and the pH of the imaging buffer solution may be 7.0 to 9.0.

The ultra-high-resolution fluorescence microscope device according to an embodiment of the present invention includes a light source for irradiating a laser to the fluorescently labeled cells.

The light emitted from the light source from the light source reaches the fluorescently-labeled cells to excite the fluorescent chromosome, and then is condensed by the lens of the ultra-high-resolution fluorescence microscope to enlarge and filter the image.

In this case, the laser power of the light source is 6mW to 50mW.

The ultra-high-resolution fluorescence microscope apparatus according to an embodiment of the present invention includes a detector for measuring and imaging the fluorescence of cells, and analyzing the fluorescence signal.

The detector may analyze a signal of the phosphor emitted by the incident laser, and analyze the phosphor signal emitted by the incident laser using a Gaussian point intensity distribution function to obtain position information of the phosphor.

The detector filters the fluorescence signal, and the filtering can remove a background signal of 800 (A.U.) to 2000 (A.U.) detected by the EM-CCD camera sensor.

A full width at half maximum (FWHM) for a centroid distribution of a single phosphor of an ultra-high-resolution fluorescence microscope may be 24 nm to 50.00 nm.

Experimental Example 1: Preparation of imaging buffer solution

[Comparative Example 1]: 100 mM + pH 8.0

In the case of a pH 8.0 MEA 100 mM-based imaging buffer solution, first, 50 mM Tris 8.0 buffer solution was mixed with 7.81 mM glucose oxidase, 5% glucose (w/v), and 0.16 mM catalase as an oxygen scavenger system. Then, 100 mM MEA was added to room temperature (21 ℃) and mix well.

[Example 1]: 20 mM + pH 9.0

For imaging buffer solution based on pH 9.0 MEA 20mM, first, 50 mM Tris 8.0 buffer solution was mixed with 7.81 mM glucose oxidase as an oxygen scavenger system, 5% glucose (w/v), and 0.16 mM catalase 0.16 mM. Then, 20 mM MEA was added, followed by pH with NaOH. After adjusting to 9.0, mix well at room temperature (21℃).

[Example 2]: 20 mM

[Example] In the case of an imaging buffer solution based on pH 8.0 MEA 20 mM, first, 50 mM Tris 8.0 buffer solution was mixed with 7.81 mM glucose oxidase, 5% glucose (w/v), and 0.16 mM catalase as an oxygen scavenger system, and then 20 mM MEA was added. Mix well at room temperature (21℃).

[Example 3]: pH 9.0 + 100 mM

[Example] In the case of an imaging buffer solution based on pH 9.0 MEA 100 mM, first, 50 mM Tris 8.0 buffer solution was mixed with 7.81 mM glucose oxidase, 5% glucose (w/v), and 0.16 mM catalase as an oxygen scavenger system, then 100 mM MEA was added. After adjusting the pH to 9.0 with NaOH, mix well at room temperature (21°C).

Experimental Example 2: Ultra-high-resolution STORM shooting

The cell line is COS-7 cell line, and the culture solution of the cell lines is Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and 1% penicillin/streptomycin (v/v) added. and cells were cultured at a temperature of 36.5 °C in an incubator under humid conditions of 95% air/5% CO2. The culture solution was changed once every 3 to 4 days, and the cells were aliquoted every week.

Cells are fixed with 3% paraformaldehyde (PFA) + 0.1% glutaraldehyde (GA), and the target to be imaged is fluorescently labeled through general immunofluorescence.

Before STORM imaging, put the imaging buffer into the cells labeled with fluorescence. And the fluorescence is emitted using a laser of 6mW to 100mW.

Finally, the fluorescence emanating from the target is imaged through an Electron multiplying Charge-Coupled Device (EMCD).

2A to 2F are ultra-high-resolution STORM images and fluorescence intensity graphs of intracellular microtubules taken with different laser power, and FIG. 3 is a fluorescence intensity (Brightness; mean photon number) according to FIGS. 2A to 2F. ), and FIG. 4 is a graph showing the full width at half maximum (FWHM) for the distribution of the imaging position (centroid) of a single phosphor according to FIGS. 2A to 2F .

Table 1 is a table showing the result values for FIGS. 2A to 2F and FIG. 3 .

[Table 1]

Referring to FIGS. 2A to 2F, 4 and Table 1, as the laser power is decreased, the half width at half maximum is increased compared to the ultra-high-resolution STORM photographed using an imaging buffer solution containing 100 mW of MEA. It can be seen that the resolution is improved.

However, since the fluorescence intensity is reduced as the laser power is reduced, the resolution may be controlled by adjusting the fluorescence intensity and the half width according to the laser power value.

Preferably, the ultra-high resolution fluorescence microscope imaging method according to an embodiment of the present invention can apply a laser power of 12 mW, which is through time trace vs. intensity at various laser intensities. This is because the quality of resolution can be improved by changing the composition of the imaging buffer solution.

If the laser power is 9mW or 6mW, as can be seen from the time trace vs. intensity graph ( FIGS. 2 to 4 ), the photoswitching property may be slightly reduced than 12mW.

However, in the ultra-high-resolution fluorescence microscopy imaging method according to an embodiment of the present invention, resolution and optical switching characteristics can be improved by adjusting the concentration and pH of the imaging buffer solution to be optimized for a laser power of 9 mW or 6 mW.

FIG. 5 shows super-resolution STORM images of intracellular microtubules taken by varying the concentration of a thiol compound (corresponding to the concentration of MEA), and FIG. 6 is a fluorescence intensity (Brightness; mean) according to FIG. photon number), and FIG. 7 is a graph showing the full width at half maximum (FWHM) for the distribution of the imaging position (centroid) of the single phosphor according to FIG. 5 .

Table 2 is a table showing the result values for FIGS. 6 and 7 .

[Table 2]

5 to 7 and Table 2, as the concentration of MEA included in the imaging buffer solution is decreased, the fluorescence intensity is increased up to 20 mM, and the full width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor is decreased. It can be seen that the optical switching characteristics and resolution are improved.

In particular, in the case of the present invention using 20 mM compared to 100 mM, which is the MEA concentration used in the prior art, it can be seen that the fluorescence intensity is increased and the half width is reduced, so that the optical switching characteristics and resolution are improved even when a low intensity laser is used. there is.

FIG. 8 is a super-resolution STORM image of intracellular microtubules taken by varying the pH of the imaging buffer solution, and FIG. 9 is a graph showing the fluorescence intensity (Brightness; mean photon number) according to FIG. 8, FIG. 10 is a graph illustrating a full width at half maximum (FWHM) for a distribution of an imaging centroid of a single phosphor according to FIG. 8 .

Table 3 is a table showing the result values for FIGS. 9 and 10 .

[Table 3]

Referring to FIGS. 8 to 10 and Table 3, the fluorescence intensity increases as the pH of the imaging buffer solution increases or decreases based on the previously used pH 8.0, and the half width for the distribution of the imaging centroid of a single phosphor It can be seen that (FWHM) is decreased to improve optical switching characteristics and resolution.

In particular, when the imaging buffer solutions of pH 7 and pH 9 are used, it can be seen that the fluorescence intensity is remarkably improved, and the half width at half maximum for the distribution of the imaging centroid of a single phosphor is reduced, so that the optical switching characteristics and resolution are greatly improved. .

11 shows an ultra-high-resolution STORM image of intracellular microtubules photographed using the imaging buffer solution according to Comparative Example 1 and an ultra-high-resolution STORM image of intracellular microtubules photographed using the imaging buffer solution according to Example 1. 12 is a graph showing the fluorescence intensity (mean photon number) according to FIG. 11 , and FIG. 13 is a full width at half maximum for the distribution of the imaging position (centroid) of the single phosphor according to FIG. 11 . , FWHM) is a graph showing.

Table 4 is a table showing the result values for FIGS. 12 and 13 .

[Table 4]

In the MEA concentration range and pH range of the present invention (Example 1: pH 9.0 + 20mM MEA), when ultra-high-resolution STORM is photographed using low-intensity laser power, fluorescence intensity is improved, but high-intensity laser power (100 mW ), it can be seen that the fluorescence intensity is reduced as shown in FIGS. 11 to 13 and Table 4 when super high-resolution STORM is photographed.

That is, since the full width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor can be adjusted according to the laser power, the optimal conditions (concentration and pH) to obtain bright fluorescence intensity are a weak laser (12 mW) and a high It can be different in laser (100 mW).

14 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 15 is a second of intracellular microtubules taken using the imaging buffer solution according to Comparative Example 1 at high laser power (100 mW). This is a high-resolution STORM image.

Referring to FIGS. 14 and 15 , it can be seen that the resolution is improved when the image is taken with the super high-resolution STORM imaging method rather than the general fluorescence imaging method.

FIG. 16 is a graph showing the full width at half maximum (FWHM) for the distribution of imaging centroids of a single phosphor according to FIG. 15 , and FIG. 17 is a graph showing fluorescence intensity according to FIG. 15 .

The full width at half maximum (FWHM) profile for the centroid distribution of a single phosphor shown in FIG. 16 is the position when one phosphor is photoswitched for each frame and its emission is photographed. The distribution is shown. In the case of a phosphor whose emission is photographed with high precision, it has a narrow distribution and has a small FWHM value, but in the case of a phosphor with low precision Since the image is spread over a wide area, the distribution area can be widened to have a large full width at half maximum (FWHM).

Therefore, in the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention, the resolution is improved as the full width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor is reduced. The criterion can be determined as the width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor. That is, the full width at half maximum (FWHM) for the distribution of the imaging centroid of a single phosphor may correspond to the resolution.

The intensity profile (time trace) shown in FIG. 17 is data for showing the optical switching phenomenon, and represents the change in signal intensity per frame in the space of pixels where each phosphor is located. It is data.

Through this, it is possible to distinguish between a fluorescence on-state and a fluorescence off-state, and it is possible to determine how quickly a photoswitching phenomenon, which is a conversion between the two states, occurs. can be checked.

In addition, the photoswitching phenomenon occurs quickly, and the shorter the on-state time is, the less the probability of overlapping with the light emission of other phosphors is reduced. can get

14 to 17 , it can be seen that the imaging buffer solution of Comparative Example 1 exhibits fast optical switching characteristics at high laser power.

18 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 19 is an ultra-high-resolution STORM of intracellular microtubules photographed using the imaging buffer solution according to Comparative Example 1 at a weak laser power (12 mW). image, and FIG. 20 is a graph showing the full width at half maximum for the distribution of imaging centroids of a single phosphor according to FIG. 19, and FIG. 21 is a graph showing the fluorescence intensity according to FIG.

18 to 21 , the imaging buffer solution according to Comparative Example 1 shows slow light switching in the graph of fluorescence intensity over time, and in the STORM image, it overlaps and is incorrectly assigned and the resolution is lowered by localization. It can be seen that it is difficult to distinguish each microtubule.

22 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 23 is an ultra-high-resolution STORM of intracellular microtubules taken using the imaging buffer solution according to Example 2 at a weak laser power (12 mW). image, and FIG. 24 is a graph showing the full width at half maximum for the imaging centroid distribution of a single phosphor according to FIG. 23 , and FIG. 25 is a graph showing the fluorescence intensity according to FIG. 23 .

Referring to FIGS. 22 to 25 , it can be seen that, unlike FIGS. 18 to 21 , it exhibits a fast optical switching characteristic, and microtubules are well distinguished in the STORM image, so that an ultra-high-resolution structure is observed.

Table 5 is a table showing the result values according to FIGS. 20 to 24 .

[Table 5]

14 to 25 and Table 5, at low intensity laser power (12 mW), compared to Comparative Example 1, which is an imaging buffer solution containing 100 mM MEA, which is conventionally used, Example 2 an imaging buffer solution containing 20 mM MEA It can be seen that the fluorescence intensity is increased, so that the light switching occurs quickly, and the half-maximum width for the distribution of the imaging centroid of a single phosphor is reduced, and thus the resolution is improved.

26 is a fluorescence image of intracellular microtubules taken by a general fluorescence imaging method, and FIG. 27 is an ultra-high-resolution STORM of intracellular microtubules taken using the imaging buffer solution according to Example 3 at a weak laser power (12 mW). image, and FIG. 28 is a graph showing the full width at half maximum for the imaging centroid distribution of a single phosphor according to FIG. 27 , and FIG. 29 is a graph showing the fluorescence intensity according to FIG. 27 .

Referring to FIGS. 26 to 29 , it can be seen that fast light switching characteristics are shown in the graph of measuring fluorescence intensity according to time, and microtubules are well distinguished in the STORM image, so that an ultra-high-resolution structure is observed.

Table 6 is a table showing the fluorescence intensity of Comparative Example 1 (pH 8.0) and Example 3 (pH 9.0) and the half maximum width for the imaging position (centroid) distribution of a single phosphor.

[Table 6]

Referring to FIGS. 26 to 29 and Table 6, the fluorescence intensity of Example 2 compared to Comparative Example 1 is increased at a weak laser power (12 mW) to improve optical switching characteristics, and the imaging centroid distribution of a single phosphor It can be seen that the half width is reduced and the resolution is improved.

30 is an ultra-high-resolution STORM image and graph illustrating the effect of ultra-high-resolution fluorescence imaging according to signal error filtering image analysis in the ultra-high-resolution fluorescence microscope imaging method according to an embodiment of the present invention.

Referring to FIG. 30 , it can be seen that the ultra-high-resolution fluorescence microscopy imaging method according to the embodiment of the present invention enables ultra-high-resolution STORM imaging even at a laser power of low intensity of 12 mW by filtering the background signal.

On the other hand, the embodiments of the present invention disclosed in the present specification and drawings are merely presented as specific examples to aid understanding, and are not intended to limit the scope of the present invention. It will be apparent to those of ordinary skill in the art to which the present invention pertains that other modifications based on the technical spirit of the present invention can be implemented in addition to the embodiments disclosed herein.

Claims (15)

culturing the cells on a cover glass;
fixing the cultured cells;
labeling the fixed cells with a fluorescent substance;
immersing the fluorescently labeled cells in an imaging buffer solution;
measuring and imaging the fluorescence of the cells using an ultra-high-resolution fluorescence microscope, and analyzing the fluorescence signal; and
filtering the fluorescence signal;
including,
The imaging method of the ultra-high-resolution fluorescence microscope, characterized in that the laser power of the ultra-high-resolution fluorescence microscope is 6mW to 50mW.
According to claim 1,
In the ultra-high-resolution fluorescence microscope, the full width at half maximum (FWHM) and the fluorescence intensity of the distribution of the imaging centroid of the single phosphor are adjusted according to the laser power.
According to claim 1,
The ultra-high-resolution fluorescence microscope is an imaging method of an ultra-high-resolution fluorescence microscope, characterized in that STORM (Stochastic Optical Reconstruction Microscopy).
According to claim 1,
The imaging method of the ultra-high-resolution fluorescence microscope, characterized in that the pH of the imaging buffer solution is 7.0 to 9.0.
According to claim 1,
The imaging buffer solution is a Tris (2-amino-2-hydroxymethyl-1,3-propanediol) solution, a thiol (thiol) compound, and an oxygen scavenger (oxygen scavenger) system, characterized in that it comprises an ultra-high-resolution fluorescence microscope imaging Way.
6. The method of claim 5,
The imaging method of the ultra-high-resolution fluorescence microscope, characterized in that the concentration of the thiol compound in the imaging buffer solution is 5 mM to 200 mM.
7. The method of claim 6,
The oxygen scavenger system is an imaging method of an ultra-high-resolution fluorescence microscope, characterized in that it comprises a glucose oxidase (glucose oxidase), glucose (glucose) and hydrogen peroxide lyase (catalase).
According to claim 1,
The fluorescent label is at least one of cyanine series, atto series, fluorescein seires, rhodamine series, BODIPY dyes, and phorphyrin series. An imaging method of an ultra-high-resolution fluorescence microscope, characterized in that it is labeled with any one dye.
According to claim 1,
The filtering is an imaging method of an ultra-high-resolution fluorescence microscope, characterized in that removing the background signal of 800 (AU) to 2000 (AU) detected by the EM-CCD camera sensor.
a plate on which fluorescently-labeled cells supported in an imaging buffer solution are placed;
a light source unit irradiating a laser to the fluorescently labeled cells; and
a detector measuring and imaging the fluorescence of the cells and analyzing the fluorescence signal;
including,
The laser power is an ultra-high-resolution fluorescence microscope imaging apparatus, characterized in that 6mW to 50mW.
11. The method of claim 10,
The ultra-high-resolution fluorescence microscope imaging apparatus, characterized in that the full width at half maximum (FWHM) for the distribution of the centroid of a single phosphor of the ultra-high-resolution fluorescence microscope is 24.00 nm to 50.00 nm.
11. The method of claim 10,
The ultra-high-resolution fluorescence microscope is an ultra-high-resolution fluorescence microscope imaging apparatus, characterized in that the STORM (Stochastic Optical Reconstruction Microscopy).
11. The method of claim 10,
The ultra-high-resolution fluorescence microscope imaging apparatus, characterized in that the concentration of the thiol compound in the imaging buffer solution is 5 mM to 200 mM.
11. The method of claim 10,
The imaging buffer solution has a pH of 7.0 to 9.0.
11. The method of claim 10,
the detector filters the fluorescence signal,
The filtering is an ultra-high-resolution fluorescence microscope imaging apparatus, characterized in that removing the background signal of 800 (AU) to 2000 (AU) detected by the EM-CCD camera sensor.

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