JP7152706B2 - Method for measuring mechanical properties of yeast cells, and screening method for yeast cells based on mechanical properties - Google Patents

Description

The present invention relates to a method for measuring the mechanical properties of yeast cells using an atomic force microscope, and a screening method for yeast cells based on the mechanical properties.

Atomic Force Microscopy (AFM) is a technique capable of measuring mechanical properties such as rigidity of microbial cells such as yeast in single cell units (Non-Patent Documents 1 and 2).

Not only yeast cells but also living cells, even cells of the same type, have great variations in individual cell characteristics (eg, morphology, function and mechanical properties). Therefore, it is expected that it will become possible to identify the cell state from the mechanical properties of the cell. Currently, there is a research report using animal cells (mechanical discrimination between normal cells and cancer cells) for dynamic discrimination in cell units (Non-Patent Document 3).

In order to perform dynamic discrimination on a cell-by-cell basis with high accuracy, it is necessary to improve the accuracy of quantification of deviations derived from the individuality of individual cells. In animal cells that do not have a cell wall, recently, using AFM, we have succeeded in removing measurement errors from the measurement values of the mechanical properties of many single cells and extracting deviations derived from the original individuality of the cells. (Non-Patent Document 4). On the other hand, in yeast cells that have a cell wall and have an elastic modulus that is several orders of magnitude greater than that of animal cells, the mechanical properties of many single cells have been successfully measured using AFM (Non-Patent Document 5). , has not yet been able to quantify deviations derived from cell innate individuality.

Applied and Environmental Microbiology, 2016, 82(15), pp. 4789-4801 Nature protocols, 2008, 3(7), pp. 1132-1138 Nature Nanotechnology, 2007, 2(12), pp. 780-783 Biophysical Journal, 2013, 105, pp. 1093-1102 Nature Protocols, 2015, 10(1), pp. 199-204

In order to quantify the deviation derived from the original individuality of the cell, the deviation derived from the original individuality of the cell and the deviation due to measurement (measurement error) are separated as described in Non-Patent Document 4, or It is necessary to minimize deviations (measurement errors).

An object of the present invention is to provide a method for measuring the mechanical properties of yeast cells using an atomic force microscope, in which deviation (measurement error) due to measurement is sufficiently reduced. Another object of the present invention is to provide a screening method for yeast cells based on mechanical properties, in which the accuracy of mechanical discrimination on a cell-by-cell basis is enhanced.

The present invention is a method for measuring the mechanical properties of yeast cells using an atomic force microscope, comprising the steps of preparing yeast cells immobilized on the surface of a support; A step of measuring the height and mechanical properties of the yeast cells in each measurement target area by performing measurement using an atomic force microscope, and the measurement target area where the inclination angle of the yeast surface is 20 ° or less Among them, the measured value of the mechanical properties in any one measurement target region is set as the mechanical properties of the yeast cell, or the statistical value of the measured values of the mechanical properties in any two or more measurement target regions is set as the mechanical properties of the yeast cell and determining the mechanical properties of yeast cells.

The present invention found that, in measuring the mechanical properties of yeast cells using an atomic force microscope, the variation in the measured values of the mechanical properties depends on the cell shape (inclination of the cell surface) in the measurement target area. based on. That is, the measurement method according to the present invention measures the height (vertical distance between the cell surface and the support) and mechanical properties in a plurality of measurement target regions for a single yeast cell, and the obtained measurement Among the values, the measurement values in the measurement target area where the tilt angle of the yeast surface is a predetermined value or less are selected to determine the mechanical properties of the yeast cells, so the deviation (measurement error) due to the measurement is sufficiently reduced. be.

In the measuring method according to the present invention, the statistical value may be an average value. By adopting the average value of the measured values in a plurality of measurement target regions where the tilt angle of the yeast surface is equal to or less than a predetermined value, the mechanical properties of yeast cells can be determined with higher accuracy.

In the measuring method according to the present invention, the mechanical property may be either one or both of the elastic modulus and the adsorption force. Elastic modulus is a measure of the elasticity (eg, ability to maintain shape) of yeast cells. Adsorptivity is an indicator of yeast cell surface physical properties (eg, capacity for cell-cell interactions).

In the measuring method according to the present invention, it is preferable that the plurality of measurement target regions be regions obtained by dividing a square region with a side of 6 to 10 μm into a plurality of square regions with a side of 1 to 3 μm. This makes it possible to more efficiently obtain the measured values of the yeast cell height and mechanical properties in each measurement target region.

The present invention also provides a method for screening yeast cells based on mechanical properties, comprising the steps of preparing a sample in which a plurality of test yeast cells are immobilized on a support surface; A method of screening yeast cells based on mechanical properties, comprising the steps of: determining the mechanical properties of each test yeast cell by performing such a measurement method; and selecting the desired yeast cells based on the determined mechanical properties. I will provide a.

Since the screening method according to the present invention employs the measurement method according to the present invention when determining the mechanical properties of each test yeast cell, the value of the mechanical properties of each test yeast cell is a deviation due to measurement (measurement error) is sufficiently reduced, and the deviation derived from the original individuality of each test yeast cell is fully reflected. Therefore, the screening method according to the present invention further enhances the accuracy of mechanical identification on a cell-by-cell basis.

The screening method according to the present invention preferably further comprises a step of isolating the selected yeast cells by means of cell collecting means attached to the atomic force microscope. The isolated yeast cells can be used for any desired purpose.

ADVANTAGE OF THE INVENTION According to this invention, the measurement method by which the deviation (measurement error) by a measurement is fully reduced can be provided in the measurement of the mechanical property of a yeast cell using an atomic force microscope. Moreover, according to the present invention, it is possible to provide a screening method for yeast cells based on mechanical properties, in which the accuracy of mechanical identification on a cell-by-cell basis is further enhanced.

FIG. 4 is a schematic diagram for explaining a method for measuring mechanical properties of yeast cells using AFM. FIG. 4 is a schematic diagram for explaining a method of measuring the height and mechanical properties of a yeast cell in a plurality of measurement target regions of a single yeast cell. FIG. 4 is a schematic diagram showing an example of a method for setting a plurality of measurement target areas; Elastic modulus (logarithmic representation of Young's modulus E (Pa) Log E), standard deviation of Log E (σ _E (Pa)) and yeast surface slope angle (Slope (°)) 2 is a graph plotted as a function of distance (height in the vertical direction and distance in the plane perpendicular to the vertical direction) from the point). FIG. 3 is a schematic diagram showing a cell collecting means according to one embodiment; FIG. 10 is a diagram showing the results of Test Example 2; FIG. 10 is a diagram showing the results of Test Example 3; FIG. 10 is a diagram showing the results of Test Example 4;

DETAILED DESCRIPTION OF THE INVENTION Embodiments for carrying out the present invention will be described in detail below. However, the present invention is not limited to the following embodiments.

[Method for measuring mechanical properties of yeast cells]
The method for measuring the mechanical properties of yeast cells according to the present embodiment uses an atomic force microscope (AFM), and includes the steps of preparing yeast cells immobilized on the surface of a support, and A step of performing measurement using an atomic force microscope on a plurality of measurement target regions to obtain measured values of the height and mechanical properties of the yeast cells in each measurement target region; Among the measurement target regions, the measured value of the mechanical properties in any one measurement target region is set as the mechanical properties of the yeast cell, or the statistical value of the measurement values of the mechanical properties in any two or more measurement target regions and determining as a mechanical property of the yeast cell.

In the measurement method according to this embodiment, the yeast cells to be measured are immobilized on the surface of a support. The strength of immobilization on the surface of the support may be such that the yeast cells do not migrate during AFM measurement. Moreover, when further culturing or isolating the yeast cells after the measurement, it is preferable that the strength is such that the survival of the yeast cells is not affected.

A support for immobilizing yeast cells is not particularly limited, and may have any shape and may be made of any material. Examples of the shape of the support include a flat plate (culture dish, etc.), and a plate in which a large number (eg, 100 to 1,000) of wells having a predetermined pitch (eg, 15 μm) and a predetermined depth (eg, 5 μm) are formed. (microarray plate). Materials for forming the support include, for example, glass, plastics commonly used for cell culture instruments (eg, polypropylene, polystyrene), and rubbers (eg, silicone, polydimethylsiloxane).

The surface of the support may be one subjected to a surface treatment that is widely used in this technical field for adhesion and fixation of cells and tissues. Specific examples of surface treatments include gelatin coating, poly-L-lysine coating, and adhesive protein coating.

In addition to the strength to the extent that the yeast cells do not move during measurement with AFM, the strength that does not affect the survival of the yeast cells can be obtained. A treated microarray plate is preferred, and a poly-L-lysine-coated glass microarray plate is more preferred. Since the microarray plate is more suitable for immobilizing yeast cells, it is preferably a plate having a large number of wells with a pitch of 10 to 20 μm and a depth of 3 to 8 μm.

In the measurement method according to the present embodiment, yeast cells immobilized on the surface of a support are subjected to measurement using an atomic force microscope for a plurality of measurement target regions of a single yeast cell, and each measurement target Obtain measurements of yeast cell height and mechanical properties in the area.

In the measurement using AFM, the attractive force or repulsive force acting between the probe at the tip of the AFM cantilever and the yeast cell is measured from the deflection amount of the cantilever. The amount of deflection of the cantilever can be measured by a known displacement detection method (for example, irradiating the cantilever with laser light and measuring the displacement of the reflected light).

FIG. 1 is a schematic diagram for explaining a method for measuring mechanical properties of yeast cells using AFM. In the AFM 100 shown in FIG. 1, a dish 6 is mounted on a sample mounting section composed of a Z scanner 1, a Y scanner 2 and an X scanner 3 in the measurement section. A number of wells 7 are formed in the bottom of the dish 6 , and yeast cells 8 are immobilized on the surface within each well 7 . By scanning the immobilized yeast cells 8 in the Z direction with a probe 5 at the tip of the cantilever 4, the height and mechanical properties of the yeast cells are measured. Although FIG. 1 shows a mode in which the sample mounting portion moves in the XYZ directions, the cantilever 4 and the probe 5 may move in the XYZ directions.

The yeast cell height measurement value can be obtained, for example, by measuring an AFM image of unevenness on the yeast cell surface in contact mode, non-contact mode, intermittent contact mode, or force mode. The measured value of the elastic modulus of yeast cells can be obtained as Young's modulus by measuring an AFM image of the yeast cell surface in force mode according to the method described in Non-Patent Document 1, for example. In this specification, the term "elastic modulus" means "Young's modulus" unless otherwise specified. The measurement value of the adsorption force of yeast cells is obtained by, for example, using a probe 5 subjected to a surface treatment (for example, poly-L lysine coating treatment) that is widely used for surface adhesion and fixation of cells and tissues, and AFM images in force mode. It can be obtained by measuring.

In the measurement method according to the present embodiment, the height and mechanical properties of the yeast cell are measured in a plurality of measurement target regions of a single yeast cell, and a set of measurement values (height and mechanical properties) in each measurement target region is calculated. Get multiple.

FIG. 2 is a schematic diagram for explaining a method for measuring the height and mechanical properties of a single yeast cell in multiple measurement target regions. As shown in FIG. 2, first, the Z scanner 1 moves the first measurement target region of the yeast cell 8 immobilized in the well 7 to a position where the probe 5 at the tip of the cantilever 4 can be measured (FIG. 2). (A)). Next, while moving the Z scanner 1, Y scanner 2 and X scanner 3, the AFM image in the first measurement target area is measured (FIG. 2(B)). When the measurement of the AFM image of the first measurement target region is completed, the Z scanner 1, Y scanner 2 and X scanner 3 move the second measurement target region of the yeast cell 8 vertically below the probe 5 at the tip of the cantilever 4. Move the sample so that it comes (Fig. 2(C)). Then, FIGS. 2A to 2C are repeated for the second measurement target area. This is repeated until the height and mechanical properties of yeast cells are obtained in all preset measurement target regions.

FIG. 3 is a schematic diagram showing an example of a method for setting a plurality of measurement target areas. FIG. 3 illustrates a case where yeast cells 8 immobilized in wells 7 are observed vertically from above or below. Only one well 7 is shown in FIG. A plurality of measurement target regions can be set randomly, but it is also possible to set a measurement target region, divide the region into a plurality of non-overlapping sub-regions, and use each of the sub-regions as the measurement target regions. For example, the area to be measured is a square area with a side of 6 to 10 μm, and the square area is further divided into a plurality of non-overlapping square areas (sub-areas) with a side of 1 to 3 μm. may be In FIG. 3, an 8 μm square square including the vicinity of the center of the yeast cell 8 (or well 7) is set as a measurement target area, and the area is divided into 64 sub-areas consisting of 1 μm squares. It is used as a measurement target area (there are 64 measurement target areas). The area to be measured is not limited to a square, but is preferably set to an area including the vicinity of the center of the yeast cell 8 (or well 7). The term “near the center” as used herein means that when the yeast cell 8 (or well 7) is observed vertically from above or below, the distances from each point on the contour of the yeast cell 8 (or well 7) are approximately equal. It is a point. The region including the vicinity of the center is likely to include the apex (the point where the height is maximum) of the yeast cell 8, so that the measurement deviation (measurement error) is further reduced.

The number of measurement target regions may be appropriately set according to the type of yeast cell, the type of mechanical properties to be measured, and the like. From the viewpoint of further reducing deviation (measurement error) due to measurement, the number of measurement target regions is preferably 5 or more, more preferably 10 or more, and even more preferably 30 or more. From the viewpoint of reducing the measurement load, the number of measurement target regions is preferably 100 or less, more preferably 70 or less.

In the measurement method according to this embodiment, the mechanical properties of yeast cells are determined according to the following procedure based on the plurality of measured values obtained as described above. First, of the plurality of measurement target regions, only the measurement target regions where the tilt angle of the yeast surface is 20° or less (hereinafter also referred to as “20° or less region”) are used to determine the mechanical properties of yeast cells. The tilt angle of the yeast surface can be obtained from the slope of adjacent measurement points in the measurement target area. The tilt angle of the surface of yeast may be obtained by the method each time measurement is performed, or a measurement target region in which the tilt angle of the surface of yeast is 20° or less may be obtained and set in advance. For example, when the measurement target areas are determined as shown in FIG. 3, the measurement target areas whose height is in the top 25% of the 64 measurement target areas are the 20° or less areas.

Next, from the measured values of the mechanical properties in the 20 ° or less region, (i) any one of the measured values of the mechanical properties in the 20 ° or less region is determined as the mechanical properties of the yeast cell, or (ii) any two A statistic of mechanical property measurements in one or more sub-20° regions can be determined as the mechanical property of the yeast cell. Determining the mechanical properties of yeast cells in this way can sufficiently reduce the deviation (measurement error) due to measurement. The statistical value in (ii) includes, for example, a median value and an average value, and the average value is preferable because the measurement error can be further reduced.

The principle of the measuring method according to this embodiment will be described with reference to FIG. FIG. 4 shows the elastic modulus of yeast cells (logarithmic representation of Young's modulus E (Pa) Log E), the standard deviation of Log E (σ _E (Pa)), and the slope angle of the yeast surface (Slope (°)). Fig. 3 is a graph plotted as a function of distance (height in the vertical direction and distance in a plane perpendicular to the vertical direction) from (the point of maximum height); At measurement points where the vertical distance is in the range of -1 μm from the apex (0 μm) of the yeast cell, both the elastic modulus and the measurement error are stable, while the vertical distance from the apex exceeds -1 μm. It can be seen that the elastic modulus is underestimated as the distance increases, and the measurement error increases (Figs. 4(A) and 4(B)). In addition, at the measurement points where the distance on the plane perpendicular to the vertical direction is in the range of 1 μm to 3 μm, the values of both the elastic modulus and the measurement error are stable, while the distance on the plane perpendicular to the vertical direction is more than 3 μm. It can be seen that the elastic modulus is underestimated and the measurement error increases as the temperature increases (FIGS. 4(D) and 4(E)). From FIGS. 4(C) and 4(F), it can be seen from the measurement points that the distance in the vertical direction is in the range of 0 μm to −1 μm, and that the distance in the plane perpendicular to the vertical direction is in the range of 1 μm to 3 μm. All surface inclination angles are 20° or less (other measurement points exceed 20°). Therefore, it can be seen that the measurement error can be minimized by using the measurement values at the measurement point (measurement target area) where the inclination angle of the yeast surface is 20° or less for determining the mechanical properties of yeast cells.

[Screening method for yeast cells based on mechanical properties]
The yeast cell screening method based on the mechanical properties according to the present embodiment includes the steps of preparing a sample in which a plurality of test yeast cells are immobilized on the surface of a support, and Performing the method includes determining the mechanical properties of each test yeast cell, and selecting the desired yeast cells based on the determined mechanical properties.

In the screening method according to this embodiment, the target yeast cell group is not particularly limited, and any yeast cell group can be used. The target yeast cell group may be any yeast capable of producing alcohol, and specific examples thereof include beer yeast, wine fermenting yeast, sake yeast, shochu yeast, and any combination thereof. .

Supports for immobilizing target yeast cell populations include those described above. Since the mechanical properties of each yeast cell can be continuously measured and the time required for selection can be shortened, the support is a plate (microarray plate) in which a large number of wells with a predetermined pitch and a predetermined depth are formed. Preferably. More preferably, the microarray plate is coated with poly-L lysine on the yeast cell-immobilizing surface. The pitch at which the wells are formed is preferably in the range of 10-20 μm, more preferably 15 μm. A pitch within this range results in immobilization of one yeast cell per well, which is more suitable for continuous measurement. The well depth is preferably in the range of 3 to 8 μm, more preferably about 5 μm. When the well depth is within this range, the AFM probe can easily access the yeast cell surface.

The step of determining the mechanical properties of each test yeast cell can be performed by the above-described measurement method according to the present invention. The measurement method according to the present invention has sufficiently reduced deviation (measurement error) due to measurement, and can more accurately quantify the deviation (individual difference) derived from the original individuality of each yeast cell. Therefore, the screening method according to the present embodiment further enhances the accuracy of mechanical identification on a cell-by-cell basis.

In the screening method according to this embodiment, desired yeast cells are selected based on the determined mechanical properties of each yeast cell. The yeast cells to be selected may be arbitrarily set according to the purpose. For example, when the purpose is to select yeast cells with excellent rigidity, yeast cells with elastic modulus in the top 10% may be selected from the target yeast cell group. Yeast cells in the top 5% of sizes may be selected, and yeast cells with the largest modulus of elasticity may be selected.

The screening method according to this embodiment preferably further comprises a step of isolating the selected yeast cells by means of cell collecting means attached to the atomic force microscope. The isolated yeast cells can be used for any desired purpose.

The cell collecting means may be any means capable of detaching and collecting the yeast cells from the support, and specific examples include a micropipette connected to a pump (collecting the yeast cells by the attractive force of the pump).

FIG. 5 is a schematic diagram showing a cell collecting means according to one embodiment. The AFM 200 shown in FIG. 5 includes a sample mounting section 50 composed of a Z scanner 1, a Y scanner 2 and an X scanner 3, a sample measuring section 60 composed of a cantilever 4 having a probe 5 at its tip, and cell sampling means. 70. The cell collecting means 70 includes a micropipette 9 for aspirating the yeast cells to be isolated together with the culture medium, a pump 11 for applying a suction force to the micropipette 9, and a suction force generated by the pump 11 connected to the micropipette 9 to a micropipette. a tube 10 leading to a pipette 9;

Any micropipette 9 can be used as long as the diameter of the tip allows selective collection of desired yeast cells. For example, a disposable pipette or the like connected to a push-button liquid microvolume meter can be used. The micropipette 9 may be made of plastic or glass.

The AFM 200 first measures the height and mechanical properties of the yeast cells 8 for all the wells 7 by operating the sample placement section 50 and the sample measurement section 60 . Next, the measured values are analyzed on a computer (not shown), and yeast cells 8 having desired mechanical properties are identified and selected as yeast cells to be isolated. After that, the cell collecting means 70 attached to the position of the probe 5 at the tip of the cantilever 4 operates the Y scanner 2 and the X scanner 3 of the sample mounting section 50 under the control of the AFM 200 to extract the yeast cells to be isolated. The contained well 7 moves vertically below the micropipette 9 . Then, the well 7 containing the yeast cells to be isolated is moved vertically upward by the operation of the Z scanner 1 of the sample placement unit 50 , and the tip of the micropipette 9 enters the culture medium in the well 7 . Yeast cells to be isolated are collected into the micropipette 9 together with the culture medium by the suction force generated by the operation of the pump 11 . The harvested yeast cells are transferred, for example, to another container containing culture medium.

EXAMPLES The present invention will be specifically described below based on Examples, etc., but the present invention is not limited to these.

[Test Example 1: Measurement of elastic modulus of yeast cells, measurement error, and tilt angle of yeast surface]
The results shown in the graph of FIG. 4 were measured by the following method.

2 mL of yeast cells (Saccharomyces cerevisiae) adjusted to 1×10 ⁷ cells/mL were coated on a poly-L-lysine (PLL)-coated (40 μg/cm ² ) microarray glass plate (Nunc Live Cell Array Slide 15 μm Well, Nunc). product) and allowed to stand for 10 minutes to immobilize the yeast cells on the surface. Wells having a pitch of 15 μm and a depth of about 5 μm are formed in the microarray glass plate.

Place a microarray glass plate on which yeast cells are immobilized on the AFM sample mounting unit, use a cantilever with a 10 μm diameter bead (probe) at the tip, and measure at a measurement speed of 8 μm / sec at the center of each well. A region of 8 μm×8 μm was set as a measurement target region, and the region was divided into 64 sub-regions each having a size of 1 μm×1 μm, and measurements were performed in all of the sub-regions. The measurement was performed in the force mode to obtain a force curve (relationship between the probe position and the force applied to the probe while the probe was brought close to the sample and pressed into contact with the sample) at each measurement point.

From the obtained force curve, fitting is performed based on Hertz's elastic contact theory to derive the elastic modulus (Young's modulus: LogE (Pa)), and from the piezo position when force starts to be applied to the probe, the measurement position derived the height of Based on this value, the standard deviation (σ _E (Pa)) of the elastic modulus (Log E) and the slope angle (Slope (°)) of the yeast surface are calculated from the top of the yeast cell (the point where the height is maximum). Plotted as a function of (vertical distance (Height) and distance in the plane perpendicular to the vertical direction (Distance)).

[Test Example 2: Simultaneous measurement of elastic modulus and adsorption force of yeast cells]
10 μl of PLL (100 μg/ml) was dropped on a bead with a diameter of about 10 μm that was adhered to the cantilever probe, and the probe was dried to apply a PLL coating process. The measurement was performed in the same manner as in Test Example 1, except that it was obtained not only during pressing but also during retraction.

The adsorption force (nN) was derived by obtaining the maximum value of the force applied to the cantilever when the probe was retracted from the obtained force curve during retraction. The yeast cell height and elastic modulus (Young's modulus: LogE (Pa)) were derived in the same manner as in Test Example 1. FIG. 6 shows a height image (FIG. 6(A)), an elastic modulus image (FIG. 6(B)), and an adsorption force image (FIG. 6(C)) of each well. As shown in FIG. 6, an adsorption force characteristic of individual cells was confirmed. This also means that elastic modulus and adsorption force can be measured simultaneously.

[Test Example 3: Measurement of elastic modulus of yeast cells before and after ethanol treatment]
Measurement was performed in the same manner as in Test Example 1, except that top-fermenting yeast (Saccharomyces cerevisiae) was used. From the measured values obtained, the measured value of the elastic modulus (Young's modulus: LogE (Pa)) at the highest measurement point for each yeast cell was defined as the elastic modulus of the yeast cell (elastic modulus before ethanol treatment ).

After the above measurement, 99.9% ethanol was added to the dish to make the final concentration 12%. After adding ethanol, the mixture was allowed to stand at 37° C. for 2 hours. After that, the elastic modulus of the yeast cells was measured and analyzed in the same manner as the elastic modulus before ethanol treatment, and was defined as the elastic modulus after ethanol treatment.

After measuring the elastic modulus after the ethanol treatment, the ethanol solution in the dish was completely removed with a pipette, 2.5 mL of methylene blue staining solution and 2.5 mL of physiological saline were added to the dish, and the dish was allowed to stand for 10 minutes. All of the methylene blue solution in the dish was removed with a pipette, and 5 mL of physiological saline was added. This was photographed, and yeast cells stained with methylene blue were distinguished from non-stained yeast cells.

The results are shown in FIG. FIG. 7(A) is a graph of the elastic modulus distribution (n=100) before ethanol treatment, and FIG. 7(B) is a graph of the elastic modulus distribution (n=100) after ethanol treatment. It is the one that was made. A comparison of FIGS. 7A and 7B reveals that the ethanol treatment reduced the elastic modulus. FIG. 7(C) is a graph comparing the amount of change in elastic modulus by dividing the yeast cells into a methylene blue-stained section (n=20) and a non-stained section (n=80). As shown in FIG. 7(C), the elastic modulus of the stained section was significantly lower than that of the unstained section (** means p<0.01). These results indicate that stress treatment reduces elastic modulus and that the degree of reduction increases with the magnitude of stress.

[Test Example 4: Measurement of elastic modulus of yeast cells and isolation by means of cell collection]
Elastic modulus measurement and analysis were performed in the same manner as in Test Example 1. A micropipette in which two yeast cells with a high elastic modulus are bound to an AFM cantilever (connected to a pump via a tube, and configured to isolate cells using the suction force of the pump. ) was isolated using After isolation, the yeast cells were cultured in a conventional manner, and the cell population derived from the isolated yeast cells was measured in the same manner as described above.

The results are shown in FIG. FIG. 8(A) is a photograph showing an example in which yeast cells were aspirated and collected with a micropipette. It can be seen that the yeast cells present in the wells before aspiration (left figure) do not exist (collected by aspiration) in the wells after aspiration (right figure). Isolation was possible on a single cell basis.

Figure 8 (B) shows the elastic modulus image of the yeast cell population before isolation (left) and the elastic modulus image of the yeast cell population cultured after isolation (cell population derived from the isolated yeast cells) (right). Figure). The boxed wells in the left figure correspond to yeast cells before isolation. Cultivation after isolation was possible, and the elastic modulus after isolation was generally the same as that before isolation.

DESCRIPTION OF SYMBOLS 1... Z scanner, 2... Y scanner, 3... X scanner, 4... Cantilever, 5... Probe, 6... Dish, 7... Well, 8... Yeast cell, 9... Micropipette, 10... Tube, 11 ... Pump, 50 ... Sample mounting section, 60 ... Sample measuring section, 70 ... Cell sampling means, 100, 200 ... AFM.

Claims (6)

A method for measuring mechanical properties of yeast cells using an atomic force microscope,
providing yeast cells immobilized on a support surface;
performing atomic force microscopy measurements on multiple target regions of a single yeast cell to obtain measurements of height and mechanical properties of the yeast cell in each target region;
Among the measurement target regions where the inclination angle of the yeast surface is 20 ° or less, the measured value of the mechanical properties in any one measurement target region is used as the mechanical properties of the yeast cell, or in any two or more measurement target regions determining a statistic of the mechanical property measurements as a mechanical property of the yeast cell;
A method for measuring mechanical properties of yeast cells, comprising: 2. The measuring method according to claim 1, wherein said statistical value is an average value. The measuring method according to claim 1 or 2, wherein the mechanical properties are elastic modulus and/or adsorption force. The measurement method according to any one of claims 1 to 3, wherein the plurality of measurement target regions are regions obtained by dividing a square region with a side of 6 to 10 µm into a plurality of square regions with a side of 1 to 3 µm. . A method for screening yeast cells based on mechanical properties, comprising:
providing a sample in which a plurality of yeast cells to be tested are immobilized on a support surface;
performing the measurement method according to any one of claims 1 to 3 on each test yeast cell to determine the mechanical properties of each test yeast cell;
selecting the desired yeast cells based on the determined mechanical properties;
A method for screening yeast cells based on mechanical properties, comprising: 6. The screening method according to claim 5, further comprising the step of isolating the selected yeast cells with a cell collecting means attached to an atomic force microscope.

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