KR20070023354A - Method for Single-Molecule Detection in Cell Using a Combination of Differential Interference Contrast Microscopy and Total Reflection Fluorescencee Microscopy and the Apparatus therefor - Google Patents

Abstract

The present invention is to measure the accurate image of the living cell by the transmission light differential interference microscopy, and apply a total reflection optical microscope technique using a light source of one or two or more different wavelengths immediately, without moving the sample. The present invention uses a prism that allows light to pass through both the left and right sides and all the top and bottom without moving the sample, using a method of combining the total reflection fluorescence microscope technology of the prism shape and the transmitted light differential interference microscope technology to induce total reflection The two methods can be applied together to a sample to determine the precise distribution of one or more single biomolecules attributable to light sources of different wavelengths from living cells as well as from one or more single biomolecules within the cell, and Analyze the actions and properties of single biomolecules in real time.

Differential effect microscope, total reflection fluorescence microscope, single biomolecule

Description

Method for Single-Molecule Detection in Cell Using a Combination of Differential Interference Contrast Microscopy and Total Reflection Fluorescencee Microscopy and the Apparatus therefor }

1 shows a single molecule detection device in which a differential interference microscope and a triangular prism-shaped total reflection fluorescence microscope are combined.

FIG. 2A is an image of living cells in solution using a total reflection fluorescence microscope technique, FIG. 2B is an image of identical cells measured using a differential interference microscope technique, FIG. 2C is an integrated image of FIG. 2A and FIG. to be.

FIG. 3 shows a single molecule detection device in which a differential interference microscope and a total reflection fluorescence microscope of a dove prism having a transparent and flat top and bottom surfaces are combined.

Figures 4a to 4c is a microscopic image of the differential interference effect of the measured cells after incubating the living cells on the glass cover glass of various kinds of prisms and general glass materials.

5A to 5C are images measured using a 100 objective lens with cells cultured on a cover glass on a dove prism having a flat top and bottom surfaces.

6A is a superimposition of the images of FIGS. 5A and 5C, and FIG. 6B is a superimposition of the images of FIGS. 5B and 5C.

7A to 7C are images obtained by mixing the DNA-nanoparticles with the cells and incubating for 16 hours using the apparatus shown in FIG. 3 for the cultured cells.

FIG. 8A overlaps the images of FIGS. 7A and 7C, and FIG. 8B overlaps the images of FIGS. 7B and 7C.

9 to 11 are devices combining a differential interference microscope technique and a dove prism type multi-wavelength total reflection fluorescence microscope technique, and FIG. 9 is a case of using two wavelengths derived from two light sources in opposite directions. FIG. 10 shows light of two or more different wavelengths in one direction, and FIG. 11 shows light of two or more different wavelengths in both directions.

Figure 12 illustrates various prisms that can be used in the present invention.

The present invention is to determine the precise detection and characteristics of a single molecule in living cells, characterized by a combination of the measurement technology of the differential interference microscope and the total reflection fluorescence microscope.

Single-molecule detection (SMD) technology enables research at the atomic or molecular level in a variety of technical fields, and in particular in the life sciences or related fields, it can be used to identify life phenomena, diagnose diseases, and Research into the principles of biological systems, including reactions and mechanisms of interaction between molecules, is being actively conducted.

Fluorescence detection using fluorescent dyes has been widely used as a method of detecting single biomolecules in solution and measuring interaction and behavior between molecules. However, in recent years, single biomolecule detection and manipulation experiments have attracted attention as in vitro experiments in vitro.

Optical techniques, such as interference reflection microscopy (IRM) and total reflection fluorescence microscopy (TIRFM), have been used to detect single biomolecules at solid / liquid interfaces, such as cell wall and solution contact areas. In addition, the theory that the total reflection fluorescence microscopy technique showed more stable and reproducible and stable results than the interference reflection microscopy at the cell membrane and substrate interface, and experimentally proved that the noise in the solution can be minimized.

The principle of total reflection fluorescence microscope technology is as follows. When light passes through two different media with different indices of refraction, such as glass and water, Snell's formula causes refraction and scattering, with total internal reflection (TIF) in the direction of the more dense medium at a particular angle of incidence. ) Occurs. At this time, an EFL (evanescent field layer) of about 150 nm occurs in a solution on the opposite surface of a solid such as glass or a polymer material. In this region, the incident light intensity is minimized by minimizing the disturbance effect (noise) that can come from the incident angle of light. Properly increasing can effectively maximize the signal-to-noise ratio, increasing the detection sensitivity to the level where single molecules can be detected.

Despite these advantages, however, the single-molecule detection method using total reflection fluorescence microscopy can only measure single biomolecules at EFL within about 150 nm at the high-book / liquid interface, so that single biomolecules such as genes or proteins It is difficult to measure in which area it is distributed.

The principle of differential interference microscope technique is as follows. Although similar to conventional interferometers in that the measurement and comparison beam systems interfere with each other, the difference is that the comparison beam also passes through the object. The distance between the two beams is very close in units of about 1 μm. Sometimes it is carefully chosen to be below the minimum resolution distance defined by the diffraction limit. The principle of differential interference in transmitted light is more complicated than the differential interference of reflected light because of the use of two Wollaston prisms. In the differential interference microscope, the image of the sample can be viewed by the difference in the optical path passing through. Although the observed height and depth only show local differences in the gradient of the optical path through the sample, the flexibility of the image obtained by the differential interference effect is excellent. Recently, measurement of living cells in microchips, detection and manipulation of single gene molecules, and kinetic measurements using transmitted light differential interference microscopes have been reported.

Differential effect microscopes are only used to obtain images of fixed samples using conventional coverslips and slide glasses. In general, the measurement of a sample using a transmission differential interference microscope is measured by placing the sample on a flat surface, such as glass biochips, slide glass and cover slip. However, even if a single biomolecule labeled with a fluorescent substance is detected simultaneously on the outer wall, the inner wall, above or below the cell, it is not easy to determine where the biomolecule is present in the living cell sample, and the intensity of the irradiated light source While measuring single biomolecules bound with fluorescent materials, it is difficult to obtain accurate molecular images by the photobleaching effect.

The present invention is a method and apparatus for combining the conventional total reflection microscopy technique and the differential interference effect microscope technique as described above to measure the accurate distribution of single biomolecules in living cells and to analyze the action and properties of the biomolecules in real time. Its purpose is to provide.

The present invention is characterized by measuring the accurate image of the living cell by the transmitted light differential interference effect microscopy method, and applying a total reflection optical microscope technique using a light source of one or two or more different wavelengths immediately without moving the sample. .

In order to apply the total reflection fluorescence microscopy technique immediately without moving the sample in the present invention, it is essential to use a prism through which light passes through both the left and right sides and the top and the bottom. Using a method that combines total reflection fluorescence microscopy technique with transmitted light differential interference microscopy technique that induces total reflection, two methods are applied together to a sample, and not only living cells but also one or two single biomolecules within the cells. From this, the precise distribution of one or two or more single biomolecules caused by light sources of different wavelengths and the action and characteristics of single biomolecules are analyzed in real time.

According to the present invention, the living cells are first placed on the cover glass and then placed on the cover glass on the prism of the prism type total reflection fluorescence microscope. The prism may use a regular triangular right-angle prism, but when using a prism with a flat bottom and top, such as a rhombus dove prism that transmits light well on both left and right sides, and on both sides of the top and bottom. More pronounced cell images can be obtained.

The detection of single biomolecules in cells consists of the detection of fluorescence generated in single biomolecules in the approximately 50-400 nm EFL layer of a total reflection fluorescence microscopy technique induced by a laser irradiated on one or both prisms. Overlapping the exact cell image obtained by using the microdispersive microscopy technique on the fixed cells and the image of a single biomolecule in the cell, the measurement of the distribution and position of the single biomolecule in living cells, and the action and characteristics of the biomolecule interactions It is possible.

If each molecule is stained with a compound such as quantum particles, fluorescent dyes, or nanoparticles, which can induce fluorescence, by using a unique fluorescent light generated by a single biomolecule itself or having no natural fluorescence, Or light of one or two or more different wavelengths derived from two or more light sources. Select the desired wavelength from mercury lamps, arc lamps, deuterium lamps, fluorescent lamps, white light, etc., or use one or two or more various lasers having unique wavelengths such as various kinds of gas lasers, semiconductor lasers, laser diodes, etc. Use as a light source of wavelength.

By adjusting the angle of incidence of the irradiated light source, the total reflection fluorescence is induced on the surface of glass or prism, or in the total reflection fluorescence caused by using the objective lens for total reflection fluorescence microscope. Single-molecule individual images, molecular-molecule interactions, or single molecules in vivo are detected in real time.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a method and apparatus for detecting single molecules in living cells using microdispersive effect microscopy and total reflection fluorescence microscopy techniques according to the present invention will be described in detail.

<Example 1>

Example 1 detects a single gene molecule bound to nanoparticles in a living cell using a single molecule detection device in which a differential interference effect microscope technique and a triangular prism-shaped total reflection fluorescence microscope technique are combined.

1 shows a single molecule detection device in which a differential interference microscope and a triangular prism-shaped total reflection fluorescence microscope are combined. In Example 1, Olympus upright microscopes (Upright Olympus BX51, Olympus Optical Co., Ltd., Shinjuku-ku, Tokyo, Japan) were used as standard, and the objective lens was an oil type 100 × (Olympus UPLFL 100 × / 1.3 NA). , WD 0.1). Single cells were incubated in a commercially available cover glass (No. 1 Corning, 22 mm square) to observe living cell molecules, and 5 μl of the culture solution was fixed to the prism in consideration of the thickness of the cells. CCD (Cascade 512B, Photometrics, Tucson, AZ, USA) cameras mount directly on top of microscopes with halogen lamps. The exposure time of the CCD is 10 to 300.

A halogen lamp and a DIC slider (U-DICT, Olympus) are mounted to detect the differential interference image in the apparatus shown in FIG. A mercury lamp and a filter cube (U-MWB2, 450-480 nm / 50 nm, Olympus) were installed to measure fluorescence images, and an argon laser with a wavelength of 488 nm was used to detect the sample using a total reflection fluorescence microscope technique. mount model 35-LAP-431-220, Melles Griot, Irvin, CA, USA) on the left side of the microscope.

Light from the laser passes through a triangular prism (RP) (fused-silica prism, Melles Griot, Irvine, CA, USA; Prism UVGSFS, A = B = C = 25.4 mm). The light from the laser is adjusted using a prism (RP) and an oil (IO) with a refractive index of 1.518 to adjust the refractive index and adjust the angle of incidence of the light so that total reflection occurs at the interface between the cells and the cover slip (CS). At this time, the intracellular single gene molecule is observed in the formed EFL region. To reduce photobleaching of the sample, laser light is only applied to the sample during fluorescence measurements of single gene molecules using a mechanical shutter (MS) in front of the prism (RP) (VMM-D1 Shutter Driver, Vincent Associates, Rochester, NY, USA). To be investigated. For image measurement and data processing of single biomolecules, a software called MetaMorph 6.3 (Universal Imaging Co., PA, USA) is used.

Sample preparation

Penicillin-streptomycin used as antibiotics such as Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's Buffered Saline (dPBS), Fetal Bovine Serum (FBS), Trypsin-EDTA, and antibiotics was purchased from Gibco (GIBCO, Invitrogen, Grand Island, NY, USA) do. DMEM is prepared by melting one package of DMEM purchased in 1 liter of pure tertiary distilled water. The frozen FBS is dissolved in boiling water of 55.7 ° C for 30 minutes before experimenting, and then mixed with 10% of DMEM and 1% of antibiotics to prepare a culture solution, and dPBS is used to wash the cells. All experimental instruments that may come into contact with cells should be sterilized.

-Nano beads

The nanobead polymer used in the present invention has a molecular weight of 14,000 and has about 64 primary amines on the surface, and PAMAM-Dendrimer (Sigma-Aldrich, Yongin, Korea) dissolved in methanol is used. Take an appropriate amount with a micro-pipette and dry the methanol using nitrogen gas. Be careful not to blow nitrogen gas too hard as this may damage the sample. When all the methanol is evaporated, it is left to freeze-drying for one day, and the fluorescent dye FITC is prepared to bind.

-Cell culture

Store the fresh culture in refrigerated water for about 10 minutes in a water bath at 37 ° C, which is optimal for cell culture. Separately prepare dPBS and Trypsin-EDTA, which are necessary reagents for culturing cells in the clean room. In order to cleanly remove the previous culture of the cells in culture, the pipette was washed three times using dPBS. Put the appropriate amount of Trysin-EDTA using a micro-pipette so that the cells fall from the cell culture dish (35 × 10 mm, Sewon Plastic Labware, Korea), wet the cells, and then completely remove Trysin-EDTA and incubator ( Store at 37 ° C., 5% CO ₂ ) for about 5 minutes. Meanwhile, a 22 mm square cover glass purchased from Corning is placed in a new cell culture dish to measure the image of the cells, and the new culture is dropped onto the cover glass. In order to measure the image shape of the cells with time, several cell culture dishes with cover glass are prepared. After 5 minutes of incubation, the plate containing the cells was struck several times by hand to allow the cells to fall from the plate. If you pour a new culture solution and pipette it several times, the cells that you have cultured fall off individually, and then spray the appropriate amount onto the cover glass in a new cell culture dish prepared in advance. Cells prepared by the above procedure are stored in an incubator and mixed with nanoparticles to observe the degree of insertion of nanoparticles into the cells before measurement. The cell culture dish was taken out one by one at regular time intervals and the image of the cells was measured.

- Experiment result

FIG. 2A is an image of living cells in solution using a total reflection fluorescence microscope technique. FIG. Figure 2b is an image of the same cell measured using a differential interference microscope technique. FIG. 2C is an integrated image of FIG. 2A and FIG. 2B, which shows precisely where DNA-nanoparticles having fluorescence are distributed in cells.

<Example 2>

Example 2 detects a single gene molecule bound to nanoparticles in a living cell using a single molecule detection device that combines a differential interference microscopy technique with a total reflection fluorescence microscopy technique using a walking prism in a transparent rhombus shape. It is.

In the single-molecule detection device shown in FIG. 3, the prism is not using a triangular prism (RP), and the rhombic dove prism (DP) (BK7, 15 mm x 63 mm x 15 mm) is transparent on all sides. -Optics Co., LTD., Korea) is the same as the apparatus of FIG.

Sample preparation

Same as Example 1 above

-DNA and nanobead binding

The nanobeads used in this example are PAMAM-Dendrimers having a molecular weight of 14,000, and are prepared to have about 64 primary amines on the surface and to combine a fluorescent dye called FITC. DNA is a plasmid expressing luciferase and has a size of 8-kb. The nanoparticles and the DNA are mixed in a 4: 1 ratio to prepare the DNA to which the nanoparticles are bound.

-Cell culture

In the same manner as in Example 1, in Example 2, instead of introducing the fluorescent dye-bound nanoparticles into the cells, the DNA-nanoparticles 4: 1 mixed with the nanoparticles and 8-kb DNA is a cell To be introduced inside.

- Experiment result

Figure 4 is a microscopic image of the non-intrusive effect of the measured cells after incubating living cells on the glass cover glass of various kinds of prisms and general glass materials. 4A is an image of living cells obtained using the cover glass / cover glass mainly used for detecting existing fixed cells. Fig. 4B is an image on a triangular prism which is the device of the first embodiment. 4C is a cell image on the dove prism, the device of Example 2. FIG. FIG. 4B shows a comparison of FIG. 4A with the introduction of a DIC light source at an oblique oblique angled surface of the prism, so that light passes through the sample to some extent, and the image is relatively compared to the image measurement of the cell using the cover glass. It can be seen that it is not clear. However, in the case of Figure 4c, it showed a similar shape to the image detected on the cover glass that is commonly used in the past, it was possible to measure the image of the cell significantly superior to Figure 4b of the case of Example 2.

FIG. 5 shows images measured using a 100 objective lens with cells cultured on a cover glass on a dove prism having a flat top and bottom surfaces. Figure 5a is a conventional fluorescence (epifluorescence) method, Figure 5b is a technique using a total reflection fluorescence microscopy technique, Figure 5c is a measurement of each of the living cells by using a differential interference microscopy technique, to culture the cells Four hours before the procedure, the cells were cultured by introducing DNA-nanobeads mixed with nanobeads and DNA in a ratio of 4 to 1, in which fluorescent derivatives were bound. In the case of Figure 5c it was measured as a circle because the time that the cells start to differentiate. 5b, it can be seen that about 4 hours is sufficient time for DNA-nanobed to fully enter the cell. In addition, Fig. 5a, which is an image measured by a general fluorescence method, epifluorescence, shows that fluorescence simultaneously detects DNA-beads present in all regions inside and outside of a cell, thereby accurately checking whether DNA-nanobeads penetrate the cell membrane and exist in the cell. Or it is difficult to determine the exact distribution that exists on the outside of the cell surface. However, from FIG. 5B, total reflection fluorescence microscopy is possible by using total reflection fluorescence microscopy because accurate detection of DNA-nanobeads in living cells is possible by detecting single molecules in ELF regions generated in a region of about 150 nm. The image shows that the DNA has been inserted into the cell.

6A is a superimposed image of FIGS. 5A and 5C, and FIG. 6B is a superimposed image of FIGS. 5B and 5C. Referring to Figure 6b it can be seen that the precise location of the DNA-nanobeads in the cell can be measured.

<Example 3>

Example 3 uses the single molecule detection device shown in FIG. 3, which combines an undifferentiated effect microscope technique and a total reflection fluorescence microscopy technique using a walking prism in a transparent rhombus shape, to detect DNA in cells according to cell culture differences. It is detected.

Sample preparation

It is the same as Example 1 and Example 2 mentioned above.

-Synthesis of DNA-Nanovides

Same as Example 2 above.

-Cell culture

Same as Example 2 above. However, in Example 2, unlike the experiment by treating Trysin-EDTA so that the cells fall from the cell culture dish, Example 3 omits the Trysin-EDTA treatment and allows the cells to be naturally cultured.

- Experiment result

FIG. 7 shows images measured using the apparatus shown in FIG. 3 of the cultured cells after DNA-nanoparticles were mixed with cells and cultured for 16 hours. Figure 7a is a conventional fluorescence (epifluorescence) method, Figure 7b is a technique using a total reflection fluorescence microscopy technique, Figure 7c is a differential interference effect microscope technology. It can be easily seen that the cells are gradually differentiated as compared to FIG. 5, which is a circular cell image when treated with Trysin-EDTA. In this process, DNA molecules in living cells can be detected and the exact location distribution can be known.

FIG. 8A overlaps the images of FIGS. 7A and 7C, and FIG. 8B overlaps the images of FIGS. 7B and 7C. Referring to FIG. 8B, it can be seen that DNA-nanobeads can be accurately measured in which positions of cells.

The method and apparatus for detecting a single molecule in a cell using a non-interference effect microscope technique and a total reflection fluorescence microscope technique according to the present invention can be variously modified according to the total reflection fluorescence microscope technique. In particular, various modifications are possible when using light of multiple wavelengths derived from two or more light sources.

9 to 11 is a device combining a differential interference effect microscope technique and a dove prism type multi-wavelength total reflection fluorescence microscope technique, and FIG. 9 is a case where two wavelengths derived from two light sources are used in opposite directions. FIG. 10 shows light of two or more different wavelengths in one direction, and FIG. 11 shows light of two or more different wavelengths in both directions.

Figure 12 illustrates various prisms that can be used in the present invention.

As described above, according to the present invention, it is possible to measure single biomolecules such as one or more genes or proteins in living cells in a solution in real time, thereby measuring the reaction mechanisms, reaction rates, and dynamics of single biomolecules. In addition, application of detection and characterization of single biomolecules in vivo or in living cells becomes possible.

Claims (8)

Acquiring an image of a single molecule in a sample using differential interference microscopy; Intracellular single molecule using a differential interference microscopy technique and a total reflection fluorescence microscopy technique comprising the step of detecting light generated from a single molecule using a total reflection fluorescence microscopy technique without moving the sample Detection method. The method according to claim 1, The non-interference effect microscope technique is a method of detecting a single molecule in a cell using a non-interference effect microscope technique and a total reflection fluorescence microscope technique characterized in that using the transmitted light. The method according to claim 1, The step of using the total reflection fluorescence microscopy technique, a single molecule in the cell using a non-differentiation effect microscopy technique and a total reflection fluorescence microscopy technique characterized in that using the light of one or more wavelengths derived from one or more light sources Detection method. The method according to claim 1, The method using the total reflection fluorescence microscopy method is a method for detecting a single molecule in a cell using a non-interference effect microscopy technique and a total reflection fluorescence microscopy technique characterized in that the light is irradiated only when measuring a single molecule in order to reduce the light whitening effect . Means for obtaining an image of a single molecule in a sample using differential interference microscopy; Apparatus for detecting a single molecule in cells using a non-interference effect microscopy technique and a total reflection fluorescence microscopy technique comprising a means for detecting light generated from a single molecule using a total reflection fluorescence microscopy technique without moving the sample . The method according to claim 5, Means using the total reflection fluorescence microscopy technique without moving the sample, the cell using a non-interference effect microscopy technique and a total reflection fluorescence microscope technique characterized in that it comprises a prism through which light is transmitted from all sides up and down Single molecule detection device. The method according to claim 6, The prism is any one of a triangular right-angle prism or a top- and bottom-side transparent rhombus dove prism, characterized in that the detection of a single molecule in a cell using a combination of differential interference microscopy and total reflection fluorescence microscopy Device. The method according to claim 5, The means using the total reflection fluorescence microscopy technique further comprises a shutter device so that light is irradiated only when measuring a single molecule in order to reduce the light bleaching effect. Single molecule detection device.

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