Atomic force microscope and distance control method

Abstract

Provided is an atomic force microscope, etc., suited for keeping a force reduced between a probe and a sample. An atomic force microscope 1 is configured to understand the surface structure of a sample 9 by vertically moving (feedbacking) the sample 9 through use of interaction between a cantilever 3 and the sample 9. A displacement detector 15 detects a change of a physical amount generated in the cantilever 3, and outputs a detection signal. A thermal fluctuation detection device 17 extracts, from the detection signal, a thermal fluctuation signal indicating thermal fluctuation generated in the cantilever 3. A distance control unit 21 vertically moves the sample 9 by controlling a Z piezo 11 through use of the thermal fluctuation signal.

Description

[Technical field]

[0001] The present invention relates to an atomic force microscope and a distance control method, and more particularly to an atomic force microscope for acquiring the surface structure of a sample. [Background technology]

[0002] Patent Document 1 describes setting control parameters of a control circuit in an atomic force microscope (AFM). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2019-117110 A Summary of the Invention [Problem to be solved by the invention]

[0004] An atomic force microscope is a device that detects the interaction between a probe and a sample to image the surface structure. Therefore, it is unavoidable that the probe exerts a certain amount of force on the sample. Samples that are soft and vulnerable to external forces may be deformed or destroyed by the force exerted by the probe.

[0005] In atomic force microscopy, in order to obtain reliable structural information for soft and fragile samples, it is important to keep the force between the tip and the sample generated during AFM measurement as small as possible.

[0006] However, the technology described in Patent Document 1 is related to the initial setting of control parameters, and does not describe or suggest anything about keeping the force between the probe and the sample small.

[0007] SUMMARY OF THE PRESENT EMBODIMENTS It is therefore an object of the present invention to provide an atomic force microscope and the like that is suitable for keeping the force between the probe and the sample small. [Means for solving the problem]

[0008] A first aspect of the present invention is an atomic force microscope for acquiring the surface structure of a sample, comprising a cantilever that traces the surface of the sample, a displacement detection unit that measures changes in physical quantities occurring in the cantilever, and a distance control unit that controls the distance between the sample and the cantilever, wherein the displacement detection unit measures thermal fluctuations occurring in the cantilever, and the distance control unit uses the measured thermal fluctuations to control the distance between the sample and the cantilever.

[0009] A second aspect of the present invention is an atomic force microscope according to the first aspect, comprising a thermal fluctuation detection unit, the displacement detection unit measures at least the thermal fluctuation occurring in the cantilever and the deflection due to the interaction between the cantilever and the sample, the thermal fluctuation detection unit removes at least the deflection due to the interaction between the cantilever and the sample from the measurement results of the displacement detection unit, and the distance control unit controls the distance between the sample and the cantilever using the measurement results of the displacement detection unit from which the deflection due to the interaction between the cantilever and the sample has been removed.

[0010] A third aspect of the present invention is the atomic force microscope of the second aspect, wherein the distance control unit controls the distance between the sample and the cantilever so that the measurement result of the displacement detection unit, in which deflection due to interaction between the cantilever and the sample is removed, approaches a target value.

[0011] A fourth aspect of the present invention is a distance control method in an atomic force microscope for acquiring a surface structure of a sample, the atomic force microscope comprising a cantilever that traces the surface of the sample, a displacement detection unit that measures a change in a physical quantity occurring in the cantilever, and a distance control unit that controls the distance between the sample and the cantilever, the method including the steps of: the displacement detection unit measuring thermal fluctuations occurring in the cantilever; and the distance control unit controlling the distance between the sample and the cantilever using the measured thermal fluctuations.

[0012] A fifth aspect of the present invention is the distance control method of the fourth aspect, wherein the atomic force microscope is equipped with a thermal fluctuation detection unit, and the displacement detection unit measures at least the thermal fluctuation occurring in the cantilever and the deflection due to the interaction between the cantilever and the sample, and the thermal fluctuation detection unit removes at least the deflection due to the interaction between the cantilever and the sample from the measurement results of the displacement detection unit, and in the control step, the distance control unit controls the distance between the sample and the cantilever using the measurement results of the displacement detection unit from which the deflection due to the interaction between the cantilever and the sample has been removed.

[0013] A sixth aspect of the present invention is a distance control method according to the fifth aspect, wherein in the control step, the distance control unit controls the distance between the sample and the cantilever so that the measurement result of the displacement detection unit, from which deflection caused by interaction between the cantilever and the sample has been removed, approaches a target value.

[0014] In each aspect of the present invention, the thermal fluctuation detection unit includes a filter unit, and the detection signal z of the deflection measured by the displacement detection unit is DC +Δz AC Deflection signal z DC It may be one that removes. Effect of the Invention

[0015] According to each aspect of the present invention, the force applied to the sample can be kept small by controlling the distance between the cantilever and the sample using the thermal fluctuations generated in the cantilever. In other words, while the conventional technology uses the deflection or amplitude of the cantilever by a mechanical approach, the present invention uses the thermal fluctuations of the cantilever to detect interactions by thermo-statistical mechanics. The present invention can be basically realized by incorporating a thermal fluctuation detection circuit into a conventional atomic force microscope device. This allows the force acting on the sample to be as small as a fraction of that of the conventional one, and even proteins with fragile structures can be observed almost non-destructively. Note that each aspect of the present invention may be understood as the distance control unit moving the sample up and down to control the distance between the cantilever and the sample. [Brief description of the drawings]

[0016] [Figure 1] FIG. 1 is a diagram for explaining an outline of an atomic force microscope. [Diagram 2] 1 is a block diagram showing an example of the configuration of an atomic force microscope 1 according to an embodiment of the present invention. [Diagram 3] 1A and 1B are diagrams for explaining the difference between the feedback utilizing thermal fluctuation of the present invention and the conventional tapping mode and contact mode. [Figure 4] FIG. 13 is a diagram showing an example of a spectrum of a cantilever thermal fluctuation signal. [Diagram 5] FIG. 1 is a diagram showing force curve data for estimating the average value of the magnitude of a load force acting on a sample in the present invention. [Figure 6] FIG. 1 shows a comparison of measurement examples of protein samples. [Figure 7] FIG. 13 shows another example of observation of a protein sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, examples of the present invention will be described with reference to the drawings. However, the present invention is not limited to the following examples. EXAMPLES

[0018] FIG. 1 is a diagram for explaining an outline of an atomic force microscope.

[0019] FIG. 1(a) is a photograph showing the appearance of an actual atomic force microscope.

[0020] FIG. 1(b) shows an outline of an example of the configuration of an atomic force microscope. The sample 51 is, for example, a mica substrate. The surface of the sample 51 is traced in the xy direction with a cantilever 53. The cantilever 53 is a micro probe attached to the tip of a leaf spring. The atomic force microscope utilizes the interaction between the sample 51 and the cantilever 53, so a change in physical quantity occurs in the cantilever 53. The displacement detector measures the change in physical quantity occurring in the cantilever 53. In FIG. 1(b), the displacement detector includes a laser irradiator 55, a lens 57, and a detector 59. The laser irradiator 55 irradiates the cantilever 53 with laser light by transmitting it through the lens 57. The laser light is reflected by the cantilever 53. The detector 59 detects the displacement of the reflected light from a reference position. By moving the sample 51 up and down based on the displacement detected by the detector 59, the interaction between the sample 51 and the cantilever 53 is made constant (the fluctuation in the interaction is made small). The up and down movement of the sample 51 indicates a change in the height of the surface of the sample 51. This allows the atomic force microscope to obtain the surface structure of the sample 51. The surface structure obtained by scanning is usually imaged and displayed.

[0021] Figure 1(c) shows an example of processing when the surface of sample 51 in Figure 1(b) is scanned in the direction of the arrow. From the top, it shows the cantilever signal (detection signal by detector 59), the cross-sectional structure of sample 51 indicated by the arrow, and the up-and-down movement of sample 51 (AFM structure data). When sample 51 is scanned, the cantilever signal changes at points where the surface structure of sample 51 changes. By moving sample 51 up and down in accordance with this, AFM structure data can be obtained.

[0022] An atomic force microscope is a microscope that uses a cantilever to trace the surface of a sample in the xy direction (horizontal plane), moves the sample up and down in the z direction (vertical direction) so that the interaction between the sample and the probe is constant, and obtains the magnitude of the up and down movement as the surface structure to image the surface structure. The up and down movement of the sample is also called "feedback." Atomic force microscopes can measure samples in aqueous solutions with nanometer-order spatial resolution, so they are widely used for observing flexible biological samples such as proteins.

[0023] The interaction between the tip and the sample is detected as a change in the physical quantity of the cantilever. There are many measurement methods for atomic force microscopes. For example, tapping mode and contact mode are available for measuring samples in liquid. In tapping mode, the vibration amplitude of the cantilever is measured as a change in physical quantity. In contact mode, the deflection of the cantilever is measured as a change in physical quantity.

[0024] Since an atomic force microscope utilizes the interaction between a probe and a sample, it is unavoidable that the probe exerts a certain amount of force on the sample.

[0025] However, samples that are particularly soft and fragile may be deformed or destroyed by the force applied by the probe. In order for atomic force microscopes to obtain reliable structural information on soft and fragile samples, it is important to keep the interaction between the probe and the sample during measurements as small as possible.

[0026] The present invention utilizes thermal fluctuations as a change in a physical quantity occurring in the cantilever, and moves the sample up and down (feedback) in the z direction so that the magnitude of the thermal fluctuations remains constant. As shown in Figure 5, the force between the probe and the sample can be made extremely small. Furthermore, if the amplitude of the thermal fluctuations is taken into consideration, changes in the thermal fluctuation signal can occur even when the average position of the probe is in front of the sample surface. Therefore, it is expected that damage to the sample can be reduced by moving the sample up and down when the average position of the probe is in front of the sample surface.

[0027] FIG. 2 is a block diagram showing an example of the configuration of an atomic force microscope 1 according to an embodiment of the present invention.

[0028] 2(a), atomic force microscope 1 includes cantilever 3 (one example of the "cantilever" of the present invention), sample stage 7, Z piezo 11, XY piezo 13, displacement detector 15 (one example of the "displacement detector" of the present invention), thermal fluctuation detector 17, calculator 19, distance controller 21 (one example of the "distance controller" of the present invention), information processor 23, and XY scan unit 25. Cantilever 3 includes probe 5 at its tip. Sample 9 is placed on sample stage 7.

[0029] The Z piezo 11 moves the sample stage 7 up and down according to an instruction from the distance control unit 21 to change the height of the surface of the sample 9 .

[0030] The information processing device 23 instructs the detection position. The information processing device 23 grasps the detection position in the x and y directions on the sample 9. The XY scan unit 25 moves the sample stage 7 horizontally in the x and y directions relative to the XY piezo 13 according to the detection position instruction by the information processing device 23. The XY piezo 13 moves the sample stage 7 horizontally in the x and y directions according to the instruction of the XY scan unit 25.

[0031] The displacement detector 15 detects the deflection of the cantilever 3 caused by the probe-sample interaction and thermal fluctuations (fluctuations mainly caused by heat). DC is the deflection signal that indicates the deflection due to the tip-sample interaction. AC is a fluctuation signal, and includes a thermal fluctuation signal indicating thermal fluctuation. The displacement detector 15 detects the detection signal z DC +Δz AC A specific configuration of the displacement detector 15 is, for example, a laser irradiator 55, a lens 57, and a detector 59 shown in FIG.

[0032] The thermal fluctuation detector 17 detects the detection signal z DC +Δz AC Deflection signal z DC and eliminate the fluctuation signal Δz ACThe thermal fluctuation signal is extracted as much as possible from the signal and output. In the example in Figure 2, the square of the extracted signal is output.

[0033] Calculator 19 and distance control unit 21 generate a control signal for controlling the up and down movement of Z piezo 11 so that the square of the extracted signal becomes a constant value. Specifically, calculator 19 sets a target value of the square of the extracted signal as a constant value and subtracts a constant value from the square of the extracted signal. Distance control unit 21 determines the direction to move up and down depending on whether the calculation result of calculator 19 is positive or negative, and determines the distance to move up and down depending on the magnitude of the calculation result.

[0034] The distance control unit 21 outputs a control signal to the Z piezo 11 to control the height of the sample stage 7, and also transmits the change in the height of the sample stage 7 to the information processing device 23. The information processing device 23 is aware of the detection position in the x and y directions, and can grasp the up and down movement in the z direction by the distance control unit 21. This enables the information processing device 23 to obtain the surface structure of the sample 9. The information processing device 23 images the surface structure of the sample 9 and displays it on a display (not shown) or the like.

[0035] 2(b) shows an example of a specific configuration of the displacement detector 15 and the thermal fluctuation detection device 17 in FIG. 2(a). The thermal fluctuation detection device 17 includes a high-pass filter 31, a first gain circuit 33, a filter circuit 35, a multiplier 37, and a second gain circuit 39.

[0036] The displacement detector 15 detects a deflection signal z DC and the fluctuation signal Δz AC The high-pass filter 31 outputs the sum of the detection signal z DC +Δz AC Deflection signal z DCThe first gain circuit 33 amplifies the signal after the removal. The filter circuit 35 is a bandpass filter, and extracts at least the portion including the thermal fluctuation signal. In FIG. 2(b), the vicinity of the main frequency component of the thermal fluctuation signal is extracted. The multiplier 37 calculates the square of the extracted signal. Since the thermal fluctuation signal obtained by the displacement detector 15 is weak, the signal is amplified by the second gain circuit 39.

[0037] FIG. 3 is a diagram for explaining the difference between the present invention utilizing thermal fluctuation and the conventional tapping mode and contact mode.

[0038] As shown in Figure 3(a), the sample GroEL (protein) is placed on a substrate, and the surface structure of the sample is scanned using a cantilever.

[0039] Figure 3(b) shows an overview of the present invention when thermal fluctuations are utilized. The cantilever is constantly colliding with surrounding molecules (such as water), and these collisions constantly generate minute vibrations. Thermal fluctuations are mainly caused by heat (for example, the average value of the position is constant macroscopically, but deviations occur microscopically).

[0040] The thermal fluctuation signal of the cantilever is denoted as Δz. Cantilevers that are typically used to measure soft samples such as biological samples have a spring constant of about 0.1 N / m.

[0041] Under conditions where there is no interaction with an external field such as a sample, the magnitude of the thermal fluctuation signal of the cantilever (<Δz 2 >) 0.5 The law of equipartition of energy is 0.5k<Δz 2 >=0.5k B T is calculated to be approximately 2 Å at room temperature.

[0042] The magnitude of the thermal fluctuations begins to decrease when the probe begins to come into contact with the sample. Here, the tip of the probe is constantly moving up and down by about 2 Å from the equilibrium position. As a result, a change occurs in the thermal fluctuation signal when the sample surface is about 2 Å before the average position of the probe (the equilibrium position). If the sample surface is further moved along the z-axis direction toward the cantilever, the thermal fluctuations decrease further and the cantilever begins to deflect significantly when the sample surface reaches the equilibrium position of the cantilever. The amount of cantilever deflection multiplied by the spring constant is the load force applied by the probe to the sample.

[0043] Figure 3(c) shows an overview of the conventional tapping mode. In the tapping mode, the cantilever is forcibly vibrated while feedback is performed to keep the amplitude value constant. In the tapping mode, the cantilever is forcibly vibrated and the tip periodically contacts the sample. The amplitude decreases where a sample is present. The change in amplitude is used to obtain the surface structure of the sample. In the tapping mode, the average force acting between the tip and the sample is usually estimated to be about 20pN-30pN. The cantilever continuously hits the sample, and there is a high possibility that the sample will dissociate (destroy).

[0044] Figure 3(d) shows an overview of the conventional contact mode. In contact mode, the tip of the cantilever is rubbed against the surface of the sample. The cantilever bends where the sample is present. The deflection of the cantilever is used to obtain the surface structure of the sample. In contact mode, feedback is performed to keep the load force constant. In contact mode, measurements are taken while keeping the deflection of the cantilever constant in the region beyond thermal fluctuation. A deflection large enough to be measurable is required, so a force of 50pN-100pN or more is usually required. The cantilever continues to rub against the sample, and there is a high possibility that the sample will dissociate (destroy).

[0045] Figure 4 shows an example of the spectrum of the cantilever thermal fluctuation signal. The range T is the main component of the thermal fluctuation (Δz AC ) The filter circuit (high-pass filter 31, band-pass filter 35, etc.)DC ) and remove the fluctuation component (Δz AC ) is obtained.

[0046] FIG. 5 shows force curve data for estimating the average magnitude of the load force acting on the sample in the present invention. In the present invention, feedback is performed so that the decay rate of thermal fluctuation becomes constant (approximately 0.7-0.8). The average magnitude of the load force acting on the sample is estimated by measuring the force curve. A force curve is a curve plotting the relationship between the distance between the probe and the sample when the sample is moved up and down and the force acting on the cantilever. In this measurement, the force acting on the cantilever and the decay curve of thermal fluctuation were measured simultaneously.

[0047] In Fig. 5(a), the horizontal axis is the sample position (nm) and the vertical axis is the force (nN). As the sample position moves from right to left on the horizontal axis, the distance between the probe and the sample becomes closer, and the probe comes into contact with the sample at around 0 nm. When the sample is moved further, the cantilever bends. The magnitude of the force can be estimated by multiplying the amount of cantilever deflection by the spring constant of the cantilever. Fig. 5(a) shows the force curve data. Fig. 5(b) shows the thermal fluctuation signal (signal passed through a 10 kHz low-pass filter) acquired at the same time as Fig. 5(a). The horizontal axis is the sample position (nm) and the vertical axis is the voltage (V). Fig. 5(c) and (d) show the force curve data and the average of 100 thermal fluctuation signal curves, respectively.

[0048] From Figure 5, we can see that the thermal fluctuations begin to decay before the cantilever is lifted by the force from the sample. In addition, the average load force in the thermal fluctuation feedback measurement is well below 50 pN. Even taking noise into account, it is expected to be less than 10 pN. The interaction between the tip and the sample is greatly reduced.

[0049] Figure 6 shows a comparison of measurement examples of protein samples. The sample is GroEL, which has a double ring structure. Figure 6(a) shows the method of the present invention using thermal fluctuation for feedback. Figure 6(b) shows the tapping mode. Figure 6(c) shows the contact mode. The scan range is 150 nm x 150 nm in both cases. The numbers above each image are the frame numbers. Figures 6(a) and (b) were measured with the same cantilever and probe. The black parts in Figure 5 indicate that the bonds between the GroEL rings have been broken, and the ring facing the cantilever has come off.

[0050] As shown in Fig. 6(c), in the contact mode, the GroEL is rapidly destroyed after the tip starts to contact the sample, and as shown in Fig. 5(b), in the tapping mode, the bonds between the GroEL rings are also destroyed.

[0051] Comparing FIG. 6(a) and FIG. 6(b), it can be seen that when the thermal fluctuation signal of the present invention is used for feedback, the destruction of GroEL is suppressed as compared to the tapping mode.

[0052] Figure 7 shows another example of observing a protein sample. Figure 7(a) shows the method of the present invention using thermal fluctuations for feedback. Figure 7(b) shows the tapping mode. In both Figures 7(a) and (b), the structure of GroEL consisting of two rings can be clearly observed. Figure 7 shows that the resolution of the present invention is equivalent to that of the tapping mode.

[0053] The present invention uses a thermal fluctuation signal for feedback, making it possible to measure samples that are flexible and vulnerable to external forces while keeping the force applied to the sample small during measurement. [Explanation of symbols]

[0054] 1 atomic force microscope, 3 cantilever, 5 probe, 7 sample stage, 9 sample, 11 Z piezo, 13 XY piezo, 15 displacement detector, 17 thermal fluctuation detector, 19 calculator, 21 distance control unit, 23 information processing unit, 25 XY scan unit, 31 high-pass filter, 33 first gain circuit, 35 filter circuit, 37 multiplier, 39 second gain circuit, 51 sample, 53 cantilever, 55 laser irradiator, 57 lens, 59 detector

Claims

1. An atomic force microscope for acquiring a surface structure of a sample, comprising: A cantilever that traces the surface of the sample; a displacement detection unit for measuring a change in a physical quantity occurring in the cantilever; a distance control unit for controlling the distance between the sample and the cantilever; An information processing device is provided, The displacement detection unit measures thermal fluctuations occurring in the cantilever, the distance control unit controls the distance between the sample and the cantilever by using the measured thermal fluctuation; The information processing device acquires a surface structure of the sample using the distance controlled by the distance control unit.

2. A thermal fluctuation detection unit is provided, the displacement detection unit measures at least a thermal fluctuation occurring in the cantilever and a deflection caused by an interaction between the cantilever and the sample; the thermal fluctuation detection unit removes, from the measurement result of the displacement detection unit, at least a deflection caused by an interaction between the cantilever and the sample; 2. The atomic force microscope according to claim 1, wherein the distance control unit controls the distance between the sample and the cantilever using a measurement result of the displacement detection unit in which deflection caused by an interaction between the cantilever and the sample has been removed.

3. 3. The atomic force microscope according to claim 2, wherein the distance control unit controls the position of the sample so that the measurement result of the displacement detection unit, from which deflection caused by interaction between the cantilever and the sample has been removed, approaches a target value.

4. A distance control method for an atomic force microscope for acquiring a surface structure of a sample, comprising the steps of: The atomic force microscope comprises: A cantilever that traces the surface of the sample; a displacement detection unit for measuring a change in a physical quantity occurring in the cantilever; a distance control unit for controlling the distance between the sample and the cantilever; An information processing device is provided, The displacement detection unit measures thermal fluctuations occurring in the cantilever, a control step in which the distance control unit controls the distance between the sample and the cantilever by using the measured thermal fluctuation; The distance control method includes an acquisition step in which the information processing device acquires a surface structure of the sample using the distance controlled by the distance control unit.

5. the atomic force microscope includes a thermal fluctuation detection unit, the displacement detection unit measures at least a thermal fluctuation occurring in the cantilever and a deflection caused by an interaction between the cantilever and the sample; the thermal fluctuation detection unit removes, from the measurement result of the displacement detection unit, at least a deflection caused by an interaction between the cantilever and the sample; 5. The distance control method according to claim 4, wherein in the control step, the distance control unit controls the distance between the sample and the cantilever using the measurement result of the displacement detection unit in which deflection caused by interaction between the cantilever and the sample has been removed.

6. The distance control method according to claim 5, wherein in the control step, the distance control unit controls the position of the sample so that the measurement result of the displacement detection unit, in which deflection caused by interaction between the cantilever and the sample is removed, approaches a target value.

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