JP2010101857A - Scanning probe microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a scanning probe microscope that observes the surface of a sample even when a probe does not contact the sample irrespective of a Q value of a cantilever. <P>SOLUTION: An ultrasonic wave of a sample side frequency f1 is emitted to a sample, and an ultrasonic wave of a lever side frequency f2 is emitted to the cantilever 3. An extraction unit 25 extracts a specific frequency component changing depending on the distance between a probe in a non-contact state and the sample from a vibration signal of the cantilever 3. For example, the specific frequency component is a frequency component of an absolute value of the difference between the sample side frequency f1 and the lever side frequency f2, or the component of the sample side frequency f1. An amplitude detection unit 27 detects the amplitude of the specific frequency component. A feedback control unit 29 performs feedback control of a scanner 7 by using the amplitude of the specific frequency component. A target value of the feedback control is set at the amplitude of the specific frequency component when the cantilever 3 and the sample approximate to each other in a non-contact state. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a non-contact scanning probe microscope using ultrasonic waves.

  Conventionally, for example, an atomic force microscope (AFM) is known as a scanning probe microscope (SPM). The AFM can measure the mechanical behavior (change in amplitude, phase, and resonance frequency) of the cantilever caused by the interaction between the cantilever probe and the sample, and can obtain the shape and physical property map of the sample based on the measurement.

  As one of the AFMs, a non-contact mode AFM is known. The non-contact type AFM is used in a state where the probe and the sample are not in contact with each other. Conventional non-contact AFM is mainly used in a vacuum. In a vacuum, the Q value of the cantilever can be increased, and as a result, the interaction between the probe and the sample can be detected with high sensitivity, so that imaging is possible even when the probe is not in contact with the sample. is there. On the other hand, since the Q value is low in the liquid, imaging can be performed only in a situation where a repulsive force acts between the probe and the sample (that is, a contact state).

  Here, the Q value (Quality Factor) is a quantity representing the sharpness of the resonance spectrum. The Q value increases as the viscous resistance applied to the resonance system decreases, and conversely, the Q value decreases as the viscous resistance increases. The sensitivity increases as the Q value increases.

  When measuring a soft and brittle sample such as a biological sample, the sample may be destroyed by contact with the probe. In addition, an extremely soft surface such as a living cell membrane is greatly deformed by contact with a probe, so that the fine structure on the surface cannot be observed. Therefore, it is desirable to measure such a sample in a state where the probe and the sample are not in contact. However, the conventional non-contact type AFM is used exclusively in a vacuum because the use conditions are limited by the Q value. Therefore, it has been difficult to observe the biological sample as described above.

  Recently, Shekhawat et al. Have shown that the internal structure (subsurface structure) of a sample can be observed by combining AFM and ultrasonic waves (Patent Document 1). As shown in FIG. 1, an ultrasonic wave having a frequency f1 is emitted from below the sample stage, and an ultrasonic wave having a frequency f2 is emitted from the cantilever support. These ultrasonic waves interfere non-linearly (by the cantilever probe portion), and a sound having a frequency | f1-f2 | is generated. If this frequency difference is matched with the resonance frequency of the cantilever, the cantilever resonates. The amplitude and phase of the ultrasonic wave emitted from under the sample stage are modulated by an object under the sample surface. The amplitude and phase of the cantilever resonance are also affected by the presence of the object. Therefore, when the sample and the cantilever are two-dimensionally scanned in the in-plane direction, the distribution of the object below the sample surface can be imaged. By using this principle, it is possible to observe infected bacteria and cell nuclei inside the cell.

  However, the conventional ultrasonic SPM cannot be used to measure the shape of a fine object such as a biomolecule in a non-contact mode for the following reason.

  First, the conventional ultrasonic SPM measures the modulation of ultrasonic waves by an object inside the sample. The degree of modulation represents, for example, the sum of the thicknesses a, b, and c shown in FIG. Therefore, the information obtained by the prior art is a projection image in the depth direction, and is a light and shade distribution of the object. The prior art also does not scan in the height direction. Therefore, the prior art cannot obtain height information and cannot be used for measuring the uneven shape of the object surface.

  The reason why the ultrasonic wave is modulated by the object inside the sample is that the object is considerably large. This means that the prior art can only observe large objects such as bacteria and cell nuclei as described above. Therefore, it cannot be used for observing minute objects such as biomolecules.

  Further, in the conventional ultrasonic SPM, a probe and a sample are brought into contact with each other in order to measure the modulation of the ultrasonic wave. Therefore, the shape of a soft object such as a living body cannot be measured in a non-contact state.

As described above, the conventional non-contact type AFM is mainly used in a vacuum because the use conditions are limited by the Q value. Therefore, for example, it is difficult to observe a biological sample in a liquid. Further, the conventional ultrasonic SPM cannot be used for observing the shape of a fine object such as a biomolecule in a non-contact mode.
Gajendra S. Shekhawat and Vinayak P. Dravid, Nanoscale Imaging of Buried Structures via Scanning Near-Field Ultrasound Holography, Science, USA, the American Association for the Advancement of Science, (October 7, 2005) VOL310, Pages 89-92.) .

  The present invention has been made under the above-mentioned background, and its object is to provide a scanning probe microscope capable of observing a sample surface even when the probe does not contact the sample, regardless of the Q value of the cantilever. It is to provide.

  One embodiment of the present invention is a scanning probe microscope, which includes a cantilever having a probe, a scanner that performs relative scanning of the cantilever and the sample, and sample-side ultrasonic waves that emit ultrasonic waves having a sample-side frequency with respect to the sample. Non-contact state from the launcher, the lever-side ultrasound launcher that launches ultrasonic waves with a lever-side frequency different from the sample-side frequency to the cantilever, the sensor that detects the lever vibration signal that represents the vibration of the cantilever, and the lever vibration signal An extraction unit that extracts a specific frequency component that changes according to the distance between the probe and the sample, an amplitude detection unit that detects the amplitude of the specific frequency component, and the amplitude of the specific frequency component matches a predetermined target value A feedback control unit that performs feedback control of the scanner, and the target value of the feedback control is close to the probe and the sample in a non-contact state Kino is set to an amplitude of the specific frequency components.

  One embodiment of the present invention is a sample observation method using a scanning probe microscope, in which a cantilever having a probe and a sample are placed close to each other, ultrasonic waves having a sample-side frequency are emitted to the sample, and are different from the sample-side frequency. Lever-side ultrasonic waves are emitted to the cantilever, a lever vibration signal representing the vibration of the cantilever is detected, and a specific frequency component that changes according to the distance between the probe and the sample in a non-contact state is detected from the lever vibration signal. Extract and detect the amplitude of the specific frequency component, and perform feedback control of the distance between the cantilever and the sample so that the amplitude of the specific frequency component matches the predetermined target value. It is set to the amplitude of the specific frequency component when close in a non-contact state.

  In the present invention, the signal of the specific frequency component may be an interference signal that is a frequency component of an absolute value of a difference between the sample side frequency and the lever side frequency. Alternatively, the signal of the specific frequency component may be a signal of the sample side frequency component. Alternatively, the signal of the specific frequency component may be a signal of the lever side frequency component.

  The scanning probe microscope of the present invention can observe the sample surface even if the probe does not contact the sample, regardless of the Q value of the cantilever.

  The detailed description of the present invention will be described below. The following detailed description and the accompanying drawings do not limit the invention. Instead, the scope of the invention is defined by the appended claims.

  According to the present invention, as described above, the ultrasonic wave of the sample side frequency is emitted to the sample, and the ultrasonic wave of the lever side frequency different from the sample side frequency is emitted to the cantilever. Then, a specific frequency component is extracted from the lever vibration signal. The specific frequency component is a predetermined frequency component that is included in the lever vibration signal and changes according to the distance between the probe and the sample in a non-contact state. Further, the amplitude of the specific frequency component is detected, and the feedback control of the scanner is performed so that the amplitude of the specific frequency component matches a predetermined target value. The target value of the feedback control is set to the amplitude of the specific frequency component when the probe and the sample are close to each other in a non-contact state. The signal of the specific frequency component may be an interference signal that is the absolute frequency component of the difference between the sample-side frequency and the lever-side frequency, a sample-side frequency component signal, or a lever-side frequency component signal.

  As described above, the present invention emits ultrasonic waves of the sample side frequency and the lever side frequency to the sample and the cantilever, respectively, and detects the amplitude of the specific frequency component from the lever vibration signal. Then, feedback control is performed so that the amplitude of the specific frequency component matches the target value. The present invention pays attention to the fact that the amplitude of the specific frequency component changes according to the distance between the probe and the sample even if the probe and the sample are not in contact with each other, and the target value of the feedback control is set as a cantilever (probe). It is set to the amplitude value of the specific frequency component when the sample is close in a non-contact state. The change in the amplitude of the specific frequency component occurs regardless of the Q value of the cantilever, for example, even in the liquid. Therefore, according to the present invention, the sample surface can be observed even if the probe does not contact the sample, regardless of the Q value of the cantilever.

  Further, the absolute value of the difference between the sample side frequency and the lever side frequency may be set as the resonance frequency of the cantilever. This is advantageous because the cantilever resonates and the amplitude due to interference increases, and more accurate observation is possible.

  Further, the lever side frequency may be set smaller than the sample side frequency. Thereby, the change of the amplitude according to the distance between the probe and the sample appears more clearly. Therefore, more accurate observation can be performed.

  As described above, the specific frequency component may be an interference signal that is a frequency component of an absolute value of a difference between the sample side frequency and the lever side frequency. The extraction unit may extract an interference signal as a signal of a specific frequency component, the amplitude detection unit may detect an interference amplitude that is the amplitude of the interference signal, and the target value for feedback control is that the probe and the sample are not It may be set to the interference amplitude when close in contact. A preferred configuration of feedback control when using interference amplitude will be described below.

  The feedback gain when the interference amplitude is larger than the target value may be larger than when the interference amplitude is smaller than the target value. When the probe and the sample are brought close to each other, the interference amplitude increases as the distance between the probe and the sample decreases. However, the interference amplitude decreases after the probe contacts the sample. When the interference amplitude falls below the target value on the contact side, normal measurement becomes difficult. The present invention increases the feedback gain when the interference amplitude is larger than the target value, compared to when the interference amplitude is smaller than the target value. Thereby, it can aim at maintaining the non-contact state of a probe and a sample. In addition, even when the probe and the sample are in contact, the probe and the sample can be separated earlier, that is, the non-contact state can be recovered earlier.

  Further, when the interference amplitude is larger than the target value, the feedback gain may be increased according to the difference between the interference amplitude and the target value. The feedback gain may increase in proportion to the deviation between the interference amplitude and the target value. Thereby, when the deviation between the interference amplitude and the target value is small, the adjustment amount of the feedback gain can be reduced. In addition, the adjustment effect can be increased by gradually increasing the adjustment amount as the deviation increases. By the above control, it is also possible to avoid a sudden change in feedback gain when the sign of the deviation is switched. In addition, the feedback gain can be increased as the probe and the sample are closer, and the non-contact state can be maintained as much as possible. In this way, smooth and stable control for gradually increasing the gain adjustment amount can be performed.

  Further, the bending of the cantilever may be detected from the lever vibration signal. The feedback control may perform a process of separating the cantilever and the sample based on the deflection. As already described, when the probe and the sample are brought close to each other, the interference amplitude increases in the non-contact area and subsequently decreases in the contact area. When the interference amplitude falls below the target value on the contact side, normal measurement becomes difficult. The present invention focuses on the bending of the cantilever in order to avoid excessive contact between the probe and the sample. Here, the bend is an average bend and corresponds to a DC displacement of cantilever vibration, and the bend occurs when the probe contacts the sample. Therefore, by separating the cantilever from the sample when bending occurs, the probe can be prevented from excessively contacting the sample, and the probe and the sample can be returned to a non-contact state.

  In the present invention, the detection signal of the detected interference amplitude may be adjusted to increase based on the bending of the cantilever. Feedback control may be performed using the adjusted interference amplitude. With this configuration, by adjusting the interference amplitude detection signal in the increasing direction before the feedback signal is generated, the feedback control can be strengthened, and the probe and the sample can be suitably separated.

  Further, in the present invention, the detection signal of the interference amplitude is at least one of when the deflection is larger than the predetermined amplitude adjustment threshold deflection and when the interference amplitude is larger than the predetermined amplitude adjustment threshold amplitude. May be adjusted. In this configuration, an interference amplitude threshold is used in addition to the deflection threshold. Thereby, it is possible to maintain a non-contact state between the probe and the sample, and even when the probe and the sample are in contact with each other, they can be returned to the non-contact state earlier.

  In the present invention, the feedback signal may be adjusted based on the bending of the cantilever so that the cantilever and the sample are separated from each other. If the deflection is greater than a predetermined feedback signal adjustment threshold deflection, the feedback signal may be adjusted to a value that forces the cantilever and sample apart. In this configuration, by adjusting and increasing the feedback signal, when the probe and the sample are in contact with each other, they can be returned to the non-contact state more quickly.

  In the above description, the specific frequency component is the interference signal as the frequency component of the absolute value of the difference between the sample side frequency and the lever side frequency. On the other hand, the specific frequency component may be a signal of the sample side frequency component or the lever side frequency component. The extraction unit may extract the signal of the sample side frequency component or the lever side frequency component, the amplitude detection unit may detect the amplitude of the sample side frequency component or the lever side frequency component, and the target value of the feedback control is The amplitude of the sample-side frequency component or the lever-side frequency component when the probe and the sample are close to each other in a non-contact state may be set. A preferred configuration of feedback control in this case will be described below.

  The feedback gain when the amplitude of the sample side frequency component or the lever side frequency component is smaller than the target value may be larger than when the amplitude of the sample side frequency component or the lever side frequency component is larger than the target value. When the probe and the sample are brought closer, the amplitude of the sample-side frequency component or the lever-side frequency component decreases as the distance between the probe and sample decreases. The present invention increases the feedback gain when the amplitude of the sample-side frequency component or the lever-side frequency component is smaller than the target value. Thereby, it can aim at maintaining the non-contact state of a probe and a sample. Further, even when the probe and the sample are in contact, the probe and the sample can be separated earlier, that is, the non-contact state can be recovered earlier.

  Further, when the amplitude of the sample side frequency component or the lever side frequency component is smaller than the target value, the feedback gain may be increased according to the difference between the amplitude of the sample side frequency component or the lever side frequency component and the target value. The feedback gain may increase in proportion to the deviation between the amplitude of the sample-side frequency component or the lever-side frequency component and the target value. Accordingly, when the deviation between the amplitude of the sample-side frequency component or the lever-side frequency component and the target value is small, the feedback gain adjustment amount can be reduced. In addition, the adjustment effect can be increased by gradually increasing the adjustment amount as the deviation increases. By the above control, it is also possible to avoid a sudden change in feedback gain when the sign of the deviation is switched. In addition, the feedback gain can be increased as the probe and the sample are closer, and the non-contact state can be maintained as much as possible. In this way, smooth and stable control for gradually increasing the gain adjustment amount can be performed.

  Further, the bending of the cantilever may be detected from the lever vibration signal. The feedback control may perform a process of separating the cantilever and the sample based on the deflection. When the probe and the sample are brought close to each other, the amplitude of the sample side frequency component or the lever side frequency component decreases. The amplitude continues to decrease after the probe contacts the sample. Therefore, the contact between the probe and the sample cannot be determined from the amplitude. The present invention focuses on the bending of the cantilever in order to avoid excessive contact between the probe and the sample. Here, the bend is an average bend and corresponds to a DC displacement of cantilever vibration, and the bend occurs when the probe contacts the sample. Therefore, by separating the cantilever from the sample when bending occurs, the probe can be prevented from excessively contacting the sample, and the probe and the sample can be returned to a non-contact state.

  In the present invention, the detected signal having the detected amplitude may be adjusted to decrease based on the bending of the cantilever. Feedback control may be performed using the adjusted amplitude. With this configuration, by adjusting the amplitude detection signal in a decreasing direction before the feedback signal is generated, feedback control can be strengthened, and the probe and the sample can be suitably separated.

  According to the present invention, the amplitude detection signal is adjusted at least one of when the deflection is larger than the predetermined amplitude adjustment threshold deflection and when the amplitude is smaller than the predetermined amplitude adjustment threshold amplitude. It's okay. In this configuration, an amplitude threshold is used in addition to the deflection threshold. Thereby, it is possible to maintain a non-contact state between the probe and the sample, and even when the probe and the sample are in contact with each other, they can be returned to the non-contact state earlier.

  In the present invention, the feedback signal may be adjusted based on the bending of the cantilever so that the cantilever and the sample are separated from each other. If the deflection is greater than a predetermined feedback signal adjustment threshold deflection, the feedback signal may be adjusted to a value that forces the cantilever and sample apart. In this configuration, by adjusting and increasing the feedback signal, when the probe and the sample are in contact with each other, they can be returned to the non-contact state more quickly.

  In addition, a sample-side ultrasonic wave generation source may be disposed between the sample stage and the substrate holding the sample. This configuration is suitable for measuring the surface shape of a fine sample. The material density distribution from the ultrasonic source to the sample surface causes the modulation of the ultrasonic wave, which reduces the shape measurement accuracy. In the present invention, the ultrasonic wave generation source is disposed between the sample stage and the substrate holding the sample. Therefore, a decrease in accuracy due to ultrasonic modulation can be avoided, and shape measurement can be performed with high accuracy.

  Moreover, the signal of a specific frequency component may be extracted using a Fourier filter. In the present invention, the Fourier filter reconstructs the first harmonic component of the Fourier series in the signal of the specific frequency component from the lever vibration signal. A Fourier filter multiplies a lever vibration signal by a cosine wave signal having a frequency of a specific frequency component, performs integration for one period, obtains a first DC value, and applies a sine wave signal having the same frequency as the cosine wave signal to the lever vibration. Multiply the signal and integrate for one period to obtain the second DC value, and further obtain the sum of the signal obtained by multiplying the first DC value by the cosine wave signal and the signal obtained by multiplying the second DC value by the sine wave signal. It's okay. With this configuration, a signal having a specific frequency component can be preferably extracted.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the present embodiment, the present invention is applied to an atomic force microscope (AFM). However, the present invention is not limited to AFM, and may be applied to other SPMs.

  First, the principle of the present invention will be described. Here, the specific frequency component of the present invention (the predetermined frequency component which is included in the lever vibration signal and changes according to the distance between the probe in a non-contact state and the sample) is the sample side frequency and the lever side. It is an interference signal that is the frequency component of the absolute value of the frequency difference. However, the specific frequency component may be another component. The specific frequency component may be, for example, a sample-side frequency component or a lever-side frequency component as will be described later.

  Please refer to FIG. 2 and FIG. The present inventor considered that when ultrasonic interference is used in SPM, the sample surface can be observed even if the probe is not in contact with the sample surface. In FIG. 2, a sample such as a biomolecule is placed on the substrate surface, and an ultrasonic wave with a frequency f1 is emitted from below the sample substrate. In addition, an ultrasonic wave having a frequency f2 is emitted to the cantilever. The frequency f1 is called a sample side frequency, and the frequency f2 is called a lever side frequency. The absolute value of the difference between the frequencies f1 and f2 is set almost equal to the resonance frequency of the cantilever.

  As shown in the figure, when an ultrasonic wave is emitted to the sample, the ultrasonic wavefront covers (or traces) the sample surface. In other words, the sample surface is ultrasonically vibrated up and down (in the figure, the vibration is shown by a wavy line, but a longitudinal wave is actually generated). This ultrasonic vibration interferes with the ultrasonic wave emitted from the cantilever support part at the tip of the probe. Even if the probe is not in contact with the sample, interference occurs if both are close to each other to some extent. Therefore, the sample surface can be observed without contact.

  The interference described above is nonlinear and appears as a signal having a frequency Δf (= | f1-f2 |, the absolute value of the difference between the sample-side frequency f1 and the lever-side frequency f2). In the present invention, this frequency Δf signal is called an interference signal, and the amplitude of the interference signal is called an interference amplitude. The frequency Δf is set to the resonance frequency of the cantilever, and the cantilever resonates at the frequency Δf.

  FIG. 3 shows the measurement result of the interference amplitude when the cantilever is brought close to the sample substrate in the arrangement of FIG. FIG. 3 also shows the deflection of the cantilever. In the present invention, the bending of the cantilever is an average amount of bending and corresponds to a DC displacement of cantilever vibration. In FIG. 3, the horizontal axis represents the probe sample distance (distance between the probe and the sample), and the vertical axis represents the deflection (left side) and the interference amplitude (right side).

  First, the probe sample distance on the horizontal axis will be described. The vibration of the cantilever is a complex vibration including a component of the interference signal Δf, a component of the sample side frequency f1, and a component of the lever side frequency f2. When the probe contacts the sample at the lowest point of the composite signal, the probe sample distance is zero. Contact between the probe and the sample is determined from the deflection. Deflection begins to occur when the probe approaches and contacts the sample. The bending start point corresponds to the probe sample distance = 0, and becomes a reference point for the probe sample distance. The amount of movement from the reference point is the probe sample distance.

  Next, referring to FIG. 3, the characteristics of the interference amplitude will be examined. As shown in FIG. 3, when the probe was brought close to the sample, the cantilever began to vibrate at a frequency Δf from the point where the probe was about 2 nm away from the sample, and an interference signal appeared. As the probe was moved closer, the interference amplitude increased. FIG. 3 clearly shows that even when the probe and the sample are completely non-contact, the ultrasonic interference amplitude changes according to the probe sample distance, and the sample surface shape can be detected. became. The interference amplitude decreases when the probe contacts the sample. This will be discussed later.

  Based on the above principle, the present invention performs feedback control in the height direction (Z direction) of the sample and the cantilever. As shown in FIG. 3, the set point (target value) of the feedback control is set to the interference amplitude when the cantilever and the sample are close to each other in a non-contact state. This enables feedback control that keeps the distance between the cantilever and the sample constant.

  The nonlinear interference due to the ultrasonic waves can be measured regardless of the Q value of the cantilever. Therefore, according to the present invention, the sample surface can be observed even if the probe does not contact the sample, regardless of the Q value of the cantilever. The present invention can enable measurement that has been difficult in the past. For example, it is possible to observe the shape of a biomolecule in a liquid without contact.

  Furthermore, according to the present invention, a soft object such as a living body can be measured in a non-contact state. This means that the cantilever can be made harder, whereby the resonance frequency can be increased and the SPM can be speeded up.

  Further, as described above, in the present invention, Δf (the absolute value of the difference between the sample-side frequency f1 and the lever-side frequency f2) is suitably set so as to match the resonance frequency of the cantilever. This frequency setting is advantageous because it resonates the cantilever by ultrasonic interference and increases the amplitude due to the interference, increasing sensitivity and allowing more accurate observation. However, within the scope of the present invention, the frequency Δf may not coincide with the resonance frequency of the cantilever. For example, the frequency Δf may be set to be somewhat smaller than the resonance frequency. Even in this case, although the sensitivity is lowered, the shape measurement using ultrasonic interference is possible.

  The present invention is completely different from the conventional ultrasonic SPM. Conventionally, a probe and a sample are brought into contact with each other to measure the modulation of ultrasonic waves by the sample. What is measured is the density distribution inside the sample, and height information of the surface shape cannot be obtained. There is no feedback control to obtain height information. In addition, since the modulation of ultrasonic waves occurs only in large objects such as bacteria and cell nuclei inside the object, conventionally only large objects can be measured.

  The present invention measures non-linear interference due to ultrasonic vibration of the object surface in a non-contact state, not modulation of the ultrasonic wave inside the object. For this measurement, the present invention sets the interference amplitude in the non-contact state as a feedback target value, performs feedback control in the height direction, and obtains height information. Thereby, the present invention measures the sample surface shape, and in particular, measures fine shapes such as biomolecules.

  The prior art cannot measure such a fine surface shape. This is because there is no measurable ultrasonic modulation depending on the fine surface shape.

  In addition, the modulation of ultrasonic waves by an object below the sample surface used in the prior art is rather an obstacle to shape measurement in the present invention. Therefore, in the example of FIG. 2, the substrate is placed on the ultrasonic wave generation source, and the sample is placed on the substrate. By using a glass substrate or the like having no density distribution, modulation can be avoided. By measuring in a situation where no modulation occurs, the surface shape of a fine object can be measured.

  In short, in the prior art and the present invention, the phenomenon to be measured is different and therefore the configuration is also different. The prior art measures the density distribution inside the object and, for that, measures the modulation of the ultrasonic waves. The present invention measures the ultrasonic vibration of the object surface by feedback control in the sample height direction and obtains information on the surface shape of the sample, which is completely different from the prior art.

  The inventor has actually investigated whether or not a sample placed on a substrate can be imaged in a non-contact manner. The sample is myosin V molecule (protein) placed on a mica substrate. The resonance frequency of the cantilever was 590 kHz (underwater), the sample side frequency f1 was set to 1600 kHz, and the lever side frequency f2 was set to 1010 kHz. As shown in FIG. 3, a set point is set for the amplitude value of the interference signal having the frequency Δf. Then, during the XY scanning of the sample stage, feedback scanning in the Z direction of the sample stage was performed so that the interference amplitude coincided with the set point. The scanning amount in the Z direction was used as sample height information. As a result, an image of myosin V was observed, indicating that non-contact imaging is possible.

"Improvement of feedback control"
In the present invention, feedback control using ultrasound is preferably improved as further described below.

  FIG. 4 is a simplified diagram of the interference amplitude curve and the deflection curve of FIG. As shown in FIGS. 3 and 4, the interference amplitude as a function of the probe sample distance is biphasic. That is, in the process of decreasing the probe sample distance, the interference amplitude increases and then decreases. More specifically, the interference amplitude increases in the non-contact area and decreases in the contact area. Therefore, the interference amplitude coincides with the feedback control set point at two points, a point a in the non-contact area and a point b in the contact area.

  The feedback control can apply feedback in the correct direction as long as the probe sample distance is in the contact area. Even if the probe sample distance enters the contact area, if the distance is larger than the point b, the feedback works in the correct direction.

  However, for example, when a tall sample is imaged, the probe may come into strong contact with the sample beyond the position of point b. When such a phenomenon occurs, feedback scanning is not performed in the correct direction, the contact between the probe and the sample becomes stronger, and imaging becomes impossible.

  In order to avoid such a situation, it is desirable to keep the non-contact state as much as possible. Further, even when a contact state occurs, it is desired to quickly return to the non-contact state. The present invention further improves feedback control to meet such demands.

(1) Variable control of feedback gain The present invention increases the feedback gain when the interference amplitude is larger than the target value, compared to when the interference amplitude is smaller than the set point. Thereby, when the interference amplitude becomes larger than the set point, the probe and the sample can be separated earlier.

  Preferably, when the interference amplitude is smaller than the target value, the feedback gain may be fixed. When the interference amplitude is larger than the target value, the feedback gain is increased according to the difference between the interference amplitude and the target value. The feedback gain may increase in proportion to the difference between the interference amplitude and the target value.

  Thereby, when the deviation between the interference amplitude and the target value is small, the adjustment amount of the feedback gain can be reduced. In addition, the adjustment effect can be increased by gradually increasing the adjustment amount as the deviation increases. By the above control, it is also possible to avoid a sudden change in feedback gain when the sign of the deviation is switched. Further, the closer the probe and the sample are, the larger the feedback gain can be maintained as much as possible. In this way, smooth and stable control for gradually increasing the gain adjustment amount can be performed.

(2) Control using cantilever deflection When the interference amplitude is in the vicinity of the set point, the non-contact state is maintained as much as possible by the above control. However, the interference amplitude may be away from the set point due to the sample shape or the like. In such a case, the following control functions effectively.

  The present invention focuses on the average deflection of the cantilever. Deflection occurs when the cantilever contacts the sample. Therefore, the probe can be prevented from excessively contacting the sample by separating the cantilever from the sample when bending occurs.

  As a specific configuration, the detected value of the interference amplitude may be adjusted, and a feedback signal may be generated using the adjusted interference amplitude. Further, the feedback signal may be adjusted during or after the generation of the feedback signal. Here, the feedback signal is a control signal for driving the actuator (scanner) so as to keep the interference amplitude at a target value. The following control can be considered as a specific example. The configuration for realizing these controls will be described in detail in an embodiment described later.

(2.1) Upper limit amplitude A1, first upper limit deflection D1
As shown in FIG. 4, the upper limit amplitude A1 is set as the upper limit set value of the interference amplitude. Further, the first upper limit deflection D1 is set as the upper limit set value for the first stage of deflection. The upper limit amplitude A1 corresponds to the threshold amplitude for amplitude adjustment of the present invention, and the first upper limit deflection D1 corresponds to the threshold deflection for amplitude adjustment of the present invention. As shown in the drawing, the first upper limit deflection D1 is set to a small value corresponding to immediately after the contact of the probe.

  According to the present invention, when the interference amplitude becomes larger than the upper limit amplitude A1, or when the deflection becomes larger than the first upper limit deflection D1, the interference amplitude detection signal is adjusted to increase. Thereby, increase adjustment of the amplitude detection signal is performed immediately before or immediately after the contact of the probe. Therefore, it is possible to maintain the non-contact state between the probe and the sample, and even when the probe and the sample are in contact with each other, the non-contact state can be quickly restored.

  Here, the detection signal of the interference amplitude is adjusted, but as described above, the feedback signal may be adjusted instead of the interference amplitude.

(2.2) Second upper limit deflection D2
Further, as shown in FIG. 4, the second upper limit deflection D2 is set as the upper limit set value for the second stage of deflection. The second upper limit deflection D2 is set to be larger than the first upper limit deflection D1, and is set to be a deflection when the contact state is surely generated even when noise or the like is taken into consideration. The second upper limit deflection D2 corresponds to the threshold deflection for adjusting the feedback signal of the present invention.

  According to the present invention, when the deflection of the cantilever is larger than the second upper limit deflection D2, the feedback signal is adjusted to a value for forcibly separating the cantilever and the sample. Thereby, when a probe and a sample have contacted, a non-contact state can be recovered more quickly. A situation in which the probe and the sample come into contact beyond the point b in FIG. 4 can be suitably avoided.

  Although the feedback signal is adjusted here, as described above, the detection signal of the interference amplitude may be adjusted instead of the feedback signal.

  The principle of the present invention has been described above. Next, a specific configuration example of the SPM of the present invention will be described. As already described, in the present embodiment, the present invention is applied to AFM. Moreover, in this Embodiment, the specific frequency component of this invention is the above-mentioned interference signal.

  FIG. 5 shows the configuration of the AFM according to the present embodiment. The AFM 1 includes a cantilever 3 and a sample stage 5 on which a sample is placed. The sample stage 5 is mounted on a scanner 7 in XYZ directions.

  The cantilever 3 is made of silicon nitride, has a probe at a free end, and is attached to the lever side piezoelectric body 9. A sample-side piezoelectric body 11 is placed on the sample stage 5, a sample substrate 13 is placed on the sample-side piezoelectric body 11, and a sample is placed on the sample substrate 13.

  The sample-side piezoelectric member 11 and the lever-side piezoelectric member 9 correspond to the lever-side ultrasonic emission unit and the sample-side ultrasonic emission unit of the present invention, and the ultrasonic waves having the sample-side frequency f1 and the lever-side frequency f2 are sampled (substrate). ) And cantilever 3 respectively. The sample-side frequency f1 and the lever-side frequency f2 are different, and the absolute value Δf (see FIG. 2) of the difference between the frequencies f1 and f2 is set as the resonance frequency of the cantilever 3. In the present embodiment, f1> f2.

  The sample-side piezoelectric body 11 is a thin plate-like piezoelectric body that can generate ultrasonic waves, and is different from the piezoelectric body used in the scanner 7. Similarly, the lever-side piezoelectric body 9 is also a thin plate-like piezoelectric body that can generate ultrasonic waves, and is different from a general lever-exciting piezoelectric body.

  The sample-side piezoelectric body 11 and the lever-side piezoelectric body 9 are connected to an oscillator 15 and generate an ultrasonic wave by vibrating according to a signal supplied from the oscillator 15. More specifically, the oscillator 15 supplies excitation signals having ultrasonic frequencies f1 and f2 to be emitted to the driver on the sample side and the driver on the lever side. Then, the sample side driver and the lever side driver amplify the respective excitation signals and supply them to the sample side piezoelectric body 11 and the lever side piezoelectric body 9.

  The AFM 1 further includes a laser unit 21, a sensor 23, an extraction unit 25, an amplitude detection unit 27, a feedback control unit 29, a computer 31, and a monitor 33.

  The sensor 23 and the laser unit 21 constitute an optical lever type displacement sensor. The laser unit 21 irradiates the cantilever 3 with laser light. The laser light is reflected by the cantilever 3 and reaches the sensor 23. The sensor 23 is composed of a photodiode and outputs a displacement signal of the cantilever 3. The displacement signal represents the vibration of the cantilever 3 and is processed as the lever vibration signal of the present invention. In the figure, the configuration of an optical system such as a lens related to the sensor is omitted.

  The extraction unit 25 extracts an interference signal from the lever vibration signal of the cantilever 3. In the present invention, as already described, the interference signal is a signal of the frequency component of the absolute value of the difference between the sample-side frequency f1 and the lever-side frequency f2, that is, a signal having a frequency Δf (= | f1-f2 |). It is. As described as the principle of the present invention, when an ultrasonic wave is emitted to the sample, the wavefront of the ultrasonic wave covers the sample surface, and the sample surface vibrates ultrasonically. This ultrasonic vibration interferes non-linearly with the ultrasonic wave emitted to the cantilever 3. This interference causes an interference signal. The extraction unit 25 may be a low pass filter. More preferably, the extraction unit 25 includes a Fourier filter described later.

  The amplitude detector 27 detects the amplitude of the interference signal, that is, the interference amplitude. The interference amplitude is supplied to the feedback control unit 29. The feedback control unit 29 controls the scanner 7 in the Z direction so that the interference amplitude continues to coincide with a preset set point (target value). The feedback control unit 29 includes a subtracter that generates a deviation signal by subtracting the set point from the interference amplitude, and a PID circuit that amplifies the deviation signal, and generates a feedback signal for controlling the scanner 7. The configuration of the feedback control unit 29 will be described in more detail later.

  The scanner 7 includes a piezoelectric element (piezo element), and moves the sample stage 5 in the X, Y, and Z directions to scan the sample relative to the cantilever 3. The XY direction is a direction orthogonal to the horizontal plane, the Z direction is a vertical direction, and is a concave / convex direction (height direction) of the sample. Scanning in the XY directions is controlled by the computer 31, and scanning in the Z direction is controlled by the feedback control unit 29 as described above. More specifically, the control signal is supplied to the scanner driver, amplified, and supplied to the scanner 7.

  The computer 31 is composed of a personal computer or the like, and controls the entire AFM 1. The computer 31 also provides a user interface function. Various user instructions are input to the computer 31, and the computer 31 controls the AFM 1 in accordance with the user input. The computer 31 controls the oscillator 15 to control the sample side frequency f1 and the lever side frequency f2. The set point of feedback control is also supplied from the computer 31 to the feedback control unit 29. Further, the computer 31 generates an image of the sample surface and outputs it to the monitor 33.

  Next, the operation of the AFM 1 will be described. Under the control of the computer 31, the scanner 7 scans the sample stage 5 in the XY directions. During scanning in the XY directions, the oscillator 15 supplies the excitation signal having the sample side frequency f1 and the excitation signal having the lever side frequency f2 to the sample side piezoelectric body 11 and the lever side piezoelectric body 9, respectively. The piezoelectric body 9 emits an ultrasonic wave having a sample-side frequency f1 and an ultrasonic wave having a lever-side frequency f2 to the sample and the cantilever 3, respectively. In this way, the cantilever 3 and the sample are scanned relatively in the XY directions while emitting ultrasonic waves to the sample and the cantilever 3.

  During scanning in the XY directions, the sensor 23 detects a lever vibration signal of the cantilever 3 and supplies it to the extraction unit 25. The extraction unit 25 extracts the interference signal from the lever vibration signal and supplies it to the amplitude detection unit 27, and the amplitude detection unit 27 detects the interference amplitude and supplies it to the feedback control unit 29. The feedback control unit 29 performs feedback control so that the detected interference amplitude matches the set point.

  In the feedback control, a feedback signal corresponding to the difference between the interference amplitude and the set point is generated and supplied to the scanner 7. The scanner 7 operates in the Z direction according to the feedback signal (more specifically, the scanner driver drives the scanner 7 according to the feedback signal).

  As described with reference to FIG. 3, the interference amplitude changes according to the distance between the cantilever 3 and the sample. Therefore, the distance between the cantilever 3 and the sample is kept constant by feedback control.

  In this way, XY scanning is performed while keeping the distance between the cantilever 3 and the sample constant. The feedback signal is also supplied to the computer 31. The feedback signal is a signal for driving the scanner 7 in the Z direction, and corresponds to the height of the sample in the Z direction. The position in the XY direction on the sample is controlled by the computer 31. The computer 31 generates an image of the sample surface based on the XY scanning control data and the input feedback signal and displays the image on the monitor 33. A three-dimensional image is suitably generated and displayed.

"Extractor"
Next, the configuration of the extraction unit 25 in the AFM 1 in FIG. 5 will be further described. The lever vibration signal of the cantilever 3 includes a component of the lever side frequency f2, a component of the sample side frequency f1, and a component of the frequency Δf (= | f1-f2 |) generated by the nonlinear interference of the ultrasonic waves. The component of frequency Δf is the interference signal of the present invention and is extracted by the extraction unit 25. The extraction unit 25 may be a low-pass filter, but preferably the extraction unit 25 includes the following Fourier filter.

  FIG. 6 shows a configuration of the extraction unit 25 preferably used in the present invention. As described with reference to FIG. 5, the oscillator 15 is configured to cause the sample-side piezoelectric body 11 and the lever-side piezoelectric body 9 to emit ultrasonic waves to the sample and the cantilever 3, and the excitation signal sin with the sample-side frequency f <b> 1. (2πf1t) is supplied to the sample-side piezoelectric body 11, and the excitation signal sin (2πf2t) having the lever-side frequency f2 is supplied to the lever-side piezoelectric body 9.

  The extraction unit 25 has a filter oscillator 41. The filter oscillator 41 is synchronized with the oscillator 15 and generates a cosine wave signal and a sine wave signal having a frequency Δf (= | f1-f2 |) as a reference signal for extraction. The filter oscillator 41 may be configured integrally with the oscillator 15.

  The cosine wave signal cos (2πΔft) is multiplied by the lever vibration signal of the cantilever 3 by the multiplier 43 and further integrated by one period by the integrator 45 to obtain the first DC value a. Further, the sine wave signal sin (2πΔft) is multiplied by the lever vibration signal by the multiplier 47 and further integrated by one period by the integrator 49 to obtain the second DC value b.

  Next, the multiplier 51 multiplies the first DC value a by the cosine wave signal cos (2πΔft) to obtain a · cos (2πΔft). The multiplier 53 multiplies the second DC value b by the sine wave signal sin (2πΔft) to obtain b · sin (2πΔft). The adder 55 calculates the sum of a · cos (2πΔft) and b · sin (2πΔft) and outputs the sum as an interference signal.

  With such a configuration, the extraction unit 25 removes the vibration components f1 and f2 from the lever vibration signal, and obtains an interference signal that is a vibration signal obtained by extracting only the component of the frequency Δf. The configuration of FIG. 6 extracts the interference signal from the lever vibration signal by reconstructing the first harmonic component of the Fourier series in the signal having the frequency Δf. In the present invention, such a filter is called a Fourier filter. Such a filter can extract an interference signal faster than a normal low-pass filter. The sine wave signal and cosine wave signal having the frequency Δf are signals supplied from the filter oscillator 41 and have almost no noise. Therefore, by using the Fourier filter, it is possible to extract an interference signal with high speed and low noise.

Amplitude detector
As already described, the amplitude detector 27 detects the amplitude of the interference signal, that is, the interference amplitude of the present invention. The amplitude detector 27 may be a circuit for obtaining (a ² + b ² ) ^1/2 (the square root of the sum of the two strips a and b in FIG. 6). However, more preferably, the circuit of the amplitude detection unit 27 is the highest within half a period after the highest point (peak) and lowest point (valley) (or lowest point and highest point) within one cycle of the interference signal are output. The difference between the point and the lowest point is output. Thereby, the amplitude detection can be speeded up.

  In order to realize the above high-speed detection, the amplitude detection unit 27 may include, for example, a phase shifter and a zero cross comparison circuit. The interference signal is delayed by 90 ° by the phase shifter. Then, the delayed signal is processed using a zero cross comparison circuit, and a pulse signal is generated at the zero cross point. This pulse signal is used as the sampling timing, and the interference signal is sampled to obtain the highest and lowest values. The difference between the highest and lowest points is output as the interference amplitude. This amplitude measurement technique is disclosed in Japanese Patent Application Laid-Open No. 2003-42931, and this document is incorporated herein by reference as a whole.

"Feedback controller"
FIG. 7 shows a preferred configuration of the feedback control unit 29 in the present embodiment. The feedback control unit 29 includes a subtraction circuit 61, a dynamic PID circuit 63, and a feedback gain adjustment circuit 65.

  The subtraction circuit 61 obtains a deviation signal by subtracting the interference amplitude from the set point. As described above, the set point is supplied from the computer 31. The deviation signal is input to the dynamic PID circuit 63 and the feedback gain adjustment circuit 65.

  The dynamic PID circuit 63 is a PID circuit whose gain can be changed, and amplifies the deviation signal input from the subtraction circuit 61. The feedback gain adjustment circuit 65 adjusts the gain of the dynamic PID circuit 63.

  The feedback gain adjustment unit 65 increases the feedback gain when the deviation signal is negative (when the interference amplitude is larger than the target value) compared to when the deviation signal is positive (when the interference amplitude is smaller than the target value). . Specifically, when the deviation signal is positive, the feedback gain is not adjusted, and therefore the feedback gain is fixed. When the deviation signal is negative, the feedback gain is increased according to the deviation signal. This adjustment increases the feedback gain in proportion to the deviation signal.

  The above-described feedback control provides the advantages described with reference to FIG. As shown in FIG. 4, when the probe is brought closer to the sample, the interference amplitude increases in the non-contact region, exceeds the set point, reaches an extreme value, and reaches the contact region. According to the present invention, the feedback gain when the interference amplitude is larger than the set point is larger than when the interference amplitude is smaller than the set point. As the contact amplitude approaches the set point beyond the interference amplitude, the feedback gain is increased and the control to pull the probe away from the sample is enhanced. Therefore, the non-contact state can be maintained as much as possible.

  In the present invention, the adjustment target is the feedback gain of the PID circuit. Thereby, the adjustment effect can be obtained instantaneously, which can also contribute to the maintenance of the non-contact region.

  In the present invention, when the interference amplitude is larger than the set point, the feedback gain increases in accordance with the deviation signal, more specifically in proportion to the deviation signal. According to this control, even if the interference signal exceeds the set point, the adjustment amount of the feedback gain can be reduced if the deviation signal is small. As the deviation signal increases, the adjustment amount can be gradually increased to enhance the adjustment effect. By the above control, it is possible to avoid a sudden change in the feedback gain when the deviation signal changes from positive to negative. Further, the feedback gain can be increased as the probe and the sample are closer to each other. In this way, smooth and stable control for gradually increasing the gain adjustment amount can be performed.

  Here, the gain adjustment in the dynamic PID circuit 63 will be further described. In the PID circuit, gain is separately applied to the deviation signal in three systems of P (proportional), I (integral), and D (differential). After gain is applied, the P, I, and D components are added, and the added signal is output as a feedback signal.

  In the present embodiment, it is preferable to adjust the gains of P, I, and D separately. In order to avoid the contact state between the probe and the sample, it is preferable that the dynamic PID circuit 63 does not maintain the normal P, I, and D gain ratios. Normal gain distribution is applied when the deviation signal is positive (ie when contact is unlikely). In normal gain distribution, I (integration) occupies a large proportion. However, since I responds slowly, it is effective to slightly emphasize P (proportional) in an emergency. Therefore, when the deviation signal is negative, the feedback gain is suitably adjusted so that the feedback gain of P is greatly increased as compared with the feedback gain of I.

  Of the three systems P, I, and D in the PID circuit, it is appropriate to place the gain adjustment circuit section of I before the integrating circuit. This is because the integrated value (voltage corresponding to the height of the sample) itself is corrected when placed in the subsequent stage. The gain of D may be before or after the differentiation circuit. However, since it is difficult to increase the gain of the differentiation circuit to a high frequency, it is preferable to insert a gain circuit after the differentiation circuit so that the differentiation circuit does not need to handle a large voltage. As described above, gain adjustment is suitably performed before I for the integration circuit for I and after the differentiation circuit for D.

  If the D gain is adjusted before the differentiation circuit, the P, I, and D gains do not have to be adjusted separately. In this case, one variable gain circuit may be placed as a feedback gain adjustment circuit between the PID circuit and the subtraction circuit in the preceding stage.

  In the above description, the feedback control unit 29 includes the PID circuit. However, the feedback control unit 29 is not limited to this. For example, the feedback control unit 29 may be configured with a PI circuit. Also in this case, the feedback gain is suitably adjusted according to the deviation between the interference amplitude and the set point.

“Second Embodiment”
Next, another embodiment of the present invention will be described. In this embodiment, as described with reference to FIG. 4, feedback control using the bending of the cantilever is performed.

  As described with reference to FIG. 7 in the first embodiment, it is possible to maintain a non-contact state between the probe and the sample by adjusting the feedback gain. However, for example, when the unevenness of the sample is large, the probe and the sample may come into contact. In consideration of such a case, the present embodiment realizes feedback control that maintains the non-contact state as much as possible and can return to the non-contact state as soon as possible even if the contact state occurs.

  FIG. 8 shows the AFM of this embodiment. An AFM 71 in FIG. 8 includes a deflection detection unit 73 in addition to the configuration of the AFM 1 in FIG. Further, the feedback control unit 75 is different from the feedback control unit 29 of FIG. The deflection detection unit 73 detects the average deflection of the cantilever 3 from the lever vibration signal of the cantilever 3 and outputs the deflection to the feedback control unit 75.

  The feedback control unit 75 is supplied with the upper limit amplitude A1, the first upper limit deflection D1, and the second upper limit deflection D2 from the computer 31 together with the set point. The feedback control unit 75 performs feedback control using these set values and detected values such as interference amplitude and deflection.

  FIG. 9 shows the configuration of the deflection detection unit 73 and the feedback control unit 75. As illustrated, the deflection detection unit 73 includes a low-pass filter 81 and a high-pass filter 83. The lever vibration signal of the cantilever 3 is processed by the low-pass filter 81 to obtain a DC displacement. The DC displacement corresponds to the average amount of deflection. The deflection signal is further passed through a high pass filter 83 to remove low frequency noise and output to the feedback control unit 75.

  The feedback control unit 75 includes a subtraction circuit 61, a dynamic PID circuit 63, and a feedback gain adjustment circuit 65, similarly to the feedback control unit 29 of FIG. 5. The feedback control unit 75 further includes an amplitude adjustment unit 91 and a feedback signal adjustment unit 93.

  The amplitude adjustment unit 91 is a circuit that adjusts the interference amplitude detection signal before the subtraction circuit 61. The amplitude adjustment unit 91 includes comparators 101 and 103, an OR circuit 105, a switch circuit 107, and a multiplication circuit 109.

  The comparator 101 receives the interference amplitude and the upper limit amplitude A1 (FIG. 4). The comparator 101 compares these signals and outputs “1” to the OR circuit 105 when the interference amplitude is larger than the upper limit amplitude A1.

  The comparator 103 receives the detected value of the deflection and the first upper limit deflection D1 (FIG. 4). The comparator 103 compares these signals and outputs “1” to the OR circuit 105 when the detected value of the deflection is larger than the first upper limit deflection D1.

  The output of the OR circuit 105 changes according to the input from the comparators 101 and 103, and the switch circuit 107 switches according to the output of the OR circuit 105. The output of the switch circuit 107 is supplied to the multiplication circuit 109 as a gain of interference amplitude.

  When “0” is input from both the comparators 101 and 103, the output of the OR circuit 105 is also “0”, and the switch circuit 107 is switched to the “× 1” side. As a result, the gain “1” is output to the multiplication circuit 109. In the multiplication circuit 109, the interference amplitude detection signal is multiplied by the gain “1”, so that the interference amplitude detection signal is supplied to the subtraction circuit 61 without being adjusted.

  When “1” is input from at least one of the comparators 101 and 103, the output of the OR circuit 105 is “1”, and the switch circuit 107 is switched to the “× g” side. As a result, the gain “g (> 1)” is output to the multiplication circuit 109. In the multiplication circuit 109, the interference amplitude detection signal is multiplied by the gain “g”, and therefore the interference amplitude detection signal increases g times. The adjusted interference amplitude is supplied to the subtraction circuit 61.

  The feedback signal adjustment unit 93 is a circuit that adjusts a feedback signal (scanner control signal in the Z direction) at the subsequent stage of the dynamic PID circuit 63. The feedback signal adjustment unit 93 includes a comparator 111, a switch circuit 113, and an adder circuit 115.

  The comparator 111 receives the detected deflection value and the second upper limit deflection D2 (FIG. 4). The switch circuit 113 is switched according to the comparison result of the comparator 111. The output of the switch circuit 113 is supplied to the adder circuit 115 and added to the feedback signal generated by the dynamic PID circuit 63.

  When the detected value of deflection is smaller than the second upper limit deflection D2, the output of the comparator 111 is “0”, and the switch circuit 113 is switched to the ground side. The output of the switch circuit 113 becomes zero, and the adder circuit 115 outputs the feedback signal to the scanner 7 without adjusting it. More specifically, the feedback signal is output to the driver of the scanner 7.

  When the detected value of deflection is greater than the second upper limit deflection D2, the output of the comparator 111 is “1”, and the switch circuit 113 is switched to the separation set value side. The separation setting value is a voltage value set in advance, and is set to a value that allows the probe and the sample to be reliably and quickly separated. The separation set value may also be supplied from the computer 31 to the feedback control unit 75. Since the scanner 7 moves backward when the probe and the sample are separated from each other, the voltage of the separation set value is set to a large value on the negative (−) side.

  When the detected value of deflection is larger than the second upper limit deflection D2, the switch circuit 113 is switched to the separation setting value side, and the separation setting value is added to the feedback signal by the addition circuit 115, thereby adjusting the feedback signal. The The adding circuit 115 supplies the adjusted feedback signal to the scanner 7, and the scanner 7 separates the probe and the sample according to the feedback signal.

  The embodiment of FIG. 8 has been described above. The upper limit amplitude A1 is set to a value larger than the set point of feedback control, and the second upper limit deflection D2 is set to be larger than the first upper limit deflection D1 (see FIG. 4). These set values cause the AFM 71 to operate as follows.

  First, when the interference amplitude is a value near the set point, the upper limit amplitude A1 is not reached. In this region, the feedback gain is adjusted according to the deviation between the interference amplitude and the set point. When the deviation is negative, the feedback gain increases in accordance with the deviation, thereby maintaining a non-contact state.

  However, if the probe is likely to come into contact with the sample, such as when the unevenness of the sample is large, the interference amplitude increases away from the set point and exceeds the upper limit amplitude A1. Further, when the probe slightly touches the sample, the detected deflection value exceeds the first upper limit deflection D1. The amplitude adjusting unit 91 multiplies the detection signal of the interference amplitude by a gain g (> 1) when the interference amplitude exceeds the upper limit amplitude A1 or the detected deflection value exceeds the first upper limit deflection D1. In this way, when the probe is about to come into contact with the sample or is slightly in contact, the amplitude is separated from the probe by amplitude adjustment.

  It is assumed that the above control is not in time and the probe and the sample are in strong contact. In this case, the interference amplitude decreases, but the average amount of deflection increases. Therefore, in such a situation, it is advantageous to use the deflection amount for control. Therefore, the second upper limit deflection D2 (second stage deflection upper limit value) is set to the deflection value when the probe is reliably in contact with the sample even in consideration of noise or the like. When the detected deflection value exceeds the second upper limit deflection D2, the feedback signal adjustment unit 93 adjusts the feedback signal to a large value that can forcefully separate the probe and the sample. Thereby, when a probe and a sample have contacted, a non-contact state can be recovered more quickly.

  The feedback signal adjustment unit 93 is arranged in parallel with the dynamic PID circuit 63. According to the present embodiment, when the probe and the sample are in strong contact and forced separation is necessary, the feedback signal can be immediately increased by the parallel configuration.

  Note that, within the scope of the present invention, either the interference signal or the feedback signal may be adjusted as long as the same effect can be obtained. The interference amplitude or feedback signal may be adjusted after generation as in the present embodiment, or may be adjusted during the generation process. Further, other signals may be adjusted within a range in which a similar effect can be obtained. For example, adjustment based on the upper limit amplitude A1 and the first upper limit deflection D1 may be performed on the feedback signal. Further, the adjustment based on the second upper limit deflection D2 may be performed on the amplitude signal. The upper limit amplitude A1 and the first upper limit deflection D1 may not be combined, that is, one of the upper limit amplitude A1 and the first upper limit deflection D1 may be used.

“Specific frequency components other than interference signals”
(Component of sample side frequency f1 or lever side frequency f2)
The present invention extracts a specific frequency component that changes according to the distance between the probe and the sample in a non-contact state from the lever vibration signal, and performs feedback control of the amplitude of the specific frequency component. The specific frequency component may be a signal other than the interference signal. As will be described below, the sample-side frequency component or the lever-side frequency component can be used as the specific frequency component.

  The cantilever vibrates at the sample-side frequency f1 and the lever-side frequency f2 before nonlinear interference occurs (that is, when the cantilever and the sample are not in proximity). That is, the lever vibration detection signal includes a component of the sample side frequency f1 and a component of the lever side frequency f2. When the probe approaches the sample surface in a non-contact manner, nonlinear interference occurs as described above, and a vibration with a frequency Δf (= | f1-f2 |) appears. In this observation, the inventor also examined the vibration at the sample side frequency f1 and the vibration at the lever side frequency f2. As a result, it was found that when the probe approaches the sample surface in a non-contact manner, the amplitude of the vibration of the component of the sample side frequency f1 decreases. Here, the amplitude of the component of the sample-side frequency f1 is simply referred to as f1 amplitude.

  FIG. 10 shows the measurement result of the f1 amplitude when the cantilever is brought close to the sample substrate in the arrangement of FIG. As shown in FIG. 10, when the cantilever approaches the sample substrate, the f1 amplitude decreases. Such a phenomenon occurs because a part of the vibration energy at the sample-side frequency f1 is converted into the vibration energy of the interference signal (frequency Δf = | f1-f2 |). That is, it is considered that the change in the f1 amplitude is also caused by ultrasonic interference.

  As described above, the signal of the component of the sample side frequency f1 is also a component that is converted according to the distance between the probe samples in a non-contact state, and corresponds to the specific frequency component of the present invention. Then, non-contact imaging of the sample shape is possible by feedback control using the f1 amplitude. Specifically, the feedback control target value is set to the f1 amplitude when the probe and the sample are close to each other in a non-contact state, and the feedback control is performed so that the detected value of the f1 amplitude matches the target value. As a result, non-contact imaging is performed.

  According to the measurement results made by the present inventors, the change in the f1 amplitude was more stable than the change in the interference amplitude (frequency | f1-f2 |) described above. In this respect, it is considered advantageous to use the component of the sample-side frequency f1 for feedback control.

"Improvement of feedback control (when using f1 amplitude)"
Further, as shown in FIG. 3, the interference signal has a biphasic property, that is, increases in the non-contact region and then decreases in the contact region. On the other hand, the f1 amplitude has a characteristic that continues to decrease even after the probe contacts the sample surface, that is, does not exhibit biphasic properties. Therefore, it is not known from the amplitude alone whether the probe has contacted the sample surface. Even in this case, the feedback control is preferably improved by applying the same principle as in the case of using the interference signal, and the non-contact state is suitably maintained.

  However, when the probe approaches the sample, the interference amplitude increases, but the f1 amplitude decreases. With this difference, the feedback control is also changed. Hereinafter, the improvement of the feedback control when the f1 amplitude is used will be described.

(1) Variable control of feedback gain The present invention increases the feedback gain when the f1 amplitude is smaller than the target value, compared to when the f1 amplitude is larger than the set point. Preferably, when the f1 amplitude is larger than the target value, the feedback gain may be fixed. When the f1 amplitude is smaller than the target value, the feedback gain is increased according to the difference between the f1 amplitude and the target value. The feedback gain may increase in proportion to the difference between the f1 amplitude and the target value.

  The gain variable control described above provides the same advantages as when using an interference signal. That is, when the f1 amplitude becomes larger than the set point, the probe and the sample can be separated earlier. Furthermore, smooth and stable control in which the gain adjustment amount is gradually increased can be performed.

(2) Control Using Cantilever Deflection When using interference amplitude, control utilizing bend was suitably performed. The deflection is also preferably used when using the f1 amplitude. That is, according to the present invention, the probe can be prevented from excessively contacting the sample by separating the cantilever from the sample when bending occurs. Specifically, the detection value of the f1 amplitude may be adjusted, and a feedback signal may be generated using the adjusted f1 amplitude. Further, the feedback signal may be adjusted during or after the generation of the feedback signal. Here, the feedback signal is a control signal for driving the actuator (scanner) so as to keep the f1 amplitude at the target value. Specific examples are given below.

(2.1) Lower limit amplitude A2, first upper limit deflection D1
As shown in FIG. 11, the lower limit amplitude A2 is set as the lower limit set value of the f1 amplitude. Further, the first upper limit deflection D1 is set as the upper limit set value for the first stage of deflection. The lower limit amplitude A2 corresponds to the threshold amplitude for amplitude adjustment of the present invention, and the first upper limit deflection D1 corresponds to the threshold deflection for amplitude adjustment of the present invention. As shown in the drawing, the first upper limit deflection D1 is set to a small value corresponding to immediately after the contact of the probe.

  According to the present invention, when the f1 amplitude becomes smaller than the lower limit amplitude A2 or the deflection becomes larger than the first upper limit deflection D1, the detection signal of the f1 amplitude is adjusted to decrease. Thereby, the decrease detection of the amplitude detection signal is performed immediately before or immediately after the contact of the probe. Therefore, it is possible to maintain the non-contact state between the probe and the sample, and even when the probe and the sample are in contact with each other, the non-contact state can be quickly restored.

  Here, the detection signal of the f1 amplitude is adjusted, but as described above, the feedback signal may be adjusted instead of the f1 amplitude.

(2.2) Second upper limit deflection D2
Further, as shown in FIG. 11, the second upper limit deflection D2 is set as the upper limit set value for the second stage of deflection. The second upper limit deflection D2 is set to be larger than the first upper limit deflection D1, and is set to be a deflection when the contact state is surely generated even when noise or the like is taken into consideration. The second upper limit deflection D2 corresponds to the threshold deflection for adjusting the feedback signal of the present invention. According to the present invention, when the deflection of the cantilever is larger than the second upper limit deflection D2, the feedback signal is adjusted to a value for forcibly separating the cantilever and the sample. Thereby, when a probe and a sample have contacted, a non-contact state can be recovered more quickly.

  Although the feedback signal is adjusted here, as described above, the detection signal having the f1 amplitude may be adjusted instead of the feedback signal.

  As described above, the bending is also preferably used in the feedback control of the f1 amplitude. In particular, the interference amplitude is biphasic, but the f1 amplitude is not biphasic. Therefore, the contact between the probe and the sample cannot be determined only from the f1 amplitude. However, according to the present invention, the above-described processing is performed by using the bending, the probe can be prevented from excessively contacting the sample, and the probe and the sample can be suitably returned to the non-contact state.

"Configuration of SPM using f1 amplitude"
Next, a specific example of the SPM configuration when using the f1 amplitude will be described.

  Here, the present invention is applied to the AFM as in the above-described embodiment. The configuration of this AFM may be basically the same as that of the above-described embodiment shown in FIG. However, in the above-described embodiment, the extraction unit 25 extracts the interference signal from the lever vibration signal, the amplitude detection unit 27 detects the interference amplitude, and the interference amplitude is used by the feedback control unit 29 for feedback control. In the present embodiment, the extraction unit 25 extracts the component of the sample side frequency f1 from the lever vibration signal, the amplitude detection unit 27 detects the amplitude of the component of the sample side frequency f1, that is, the f1 amplitude, and the feedback control unit 29. Performs feedback control using the f1 amplitude. Unlike the above-described embodiment, the feedback control set point is set to an appropriate f1 amplitude when the probe and the sample are in a non-contact state (FIG. 10). The set point is supplied from the computer 31.

  In the above-described embodiment, the preferred configuration of the extraction unit 25 is the Fourier filter of FIG. Also in this embodiment, the extraction unit 25 is preferably configured by the Fourier filter of FIG. However, in the present embodiment, the filter oscillator 41 outputs a signal having the sample-side frequency f1. That is, the filter oscillator 41 outputs cos (2πf1t) to the multipliers 43 and 51, and outputs sin (2πf1t) to the multipliers 47 and 53. These signals cos (2πf1t) and sin (2πf1t) are used for the filter processing. Other configurations may be the same as those of the above-described embodiment.

  Further, in the above-described embodiment, the feedback control unit 29 has a configuration for performing dynamic control as shown in FIG. Also in this embodiment, the feedback control unit 29 may have the configuration of FIG.

  However, in the present embodiment, the f1 amplitude is input to the subtraction circuit 61 instead of the interference amplitude. Further, the set point is different from the above-described embodiment. Further, the subtraction circuit 61 subtracts the set point from the f1 amplitude, contrary to the above-described embodiment. This is because when the probe approaches the sample, the interference amplitude increases but the f1 amplitude decreases.

  Further, the feedback gain adjustment circuit 65 compares the feedback gain when the deviation signal is negative (when the f1 amplitude is smaller than the target value) as compared with when the deviation signal is positive (when the f1 amplitude is larger than the target value). Enlarge. Specifically, when the deviation signal is positive, the feedback gain is not adjusted, and therefore the feedback gain is fixed. When the deviation signal is negative, the feedback gain is increased according to the deviation signal. This adjustment increases the feedback gain in proportion to the deviation signal.

  By the above feedback control, the non-contact state can be maintained as much as possible as in the above-described embodiment. Further, smooth and stable control for gradually increasing the gain adjustment amount can be performed.

"Configuration of SPM using f1 amplitude"
(When using cantilever deflection)
Next, the configuration of the SPM when using cantilever deflection will be described.

  Again, the SPM is an AFM as in the previous embodiment. The configuration of this AFM may be basically the same as that of the above-described embodiment shown in FIGS. However, the extraction unit 25 extracts the component of the sample-side frequency f1 from the lever vibration signal, the amplitude detection unit 27 detects the f1 amplitude, and the feedback control unit 75 processes the f1 amplitude instead of the interference amplitude (this point is As already explained).

  Further, the comparator 101 of the amplitude adjusting unit 91 compares the f1 amplitude with the lower limit amplitude A2 (FIG. 11). Then, the comparator 101 outputs “1” to the OR circuit 105 when the f1 amplitude is smaller than the lower limit amplitude A2. A gain g smaller than 1 is supplied from the switch circuit 107 to the multiplication circuit 109 (g <1).

  Therefore, in the present embodiment, the amplitude adjustment unit 91 applies the gain g (<<) to the detection signal of the f1 amplitude when the f1 amplitude falls below the lower limit amplitude A1 or the detected deflection value exceeds the first upper limit deflection D1. Multiply 1) to reduce the detection signal. In this way, when the probe is about to come into contact with the sample or is slightly in contact, the amplitude is separated from the probe by amplitude adjustment.

  In addition, also in this embodiment, the subtraction circuit 61, the dynamic PID circuit 63, and the feedback gain adjustment circuit 65 are changed so as to be adapted to the processing of the f1 amplitude. This point has already been explained.

“Use of the component of lever side frequency f2”
Finally, a configuration using the component of the lever side frequency f2 will be described. When the probe and the sample approach in a non-contact state, not only the amplitude of the component of the sample side frequency f1, but also the amplitude of the component of the lever side frequency f2 decreases. The cause of such a phenomenon is considered similarly to the component of the sample-side frequency f1. That is, it is considered that a part of the vibration energy of the lever side frequency f2 is converted into the vibration energy of the interference signal (frequency Δf = | f1-f2 |). And it can be said that the amplitude change of the component of the lever side frequency f2 is also caused by the ultrasonic interference.

  As described above, the amplitude of the component of the lever side frequency f2 also decreases as the probe sample distance decreases. That is, the signal of the component of the lever side frequency f2 also corresponds to the specific frequency component of the present invention. Therefore, the above feedback control and imaging may be similarly performed using the component of the lever side frequency f2 instead of the component of the sample side frequency f1. That is, the extraction unit 25 may extract the component of the lever side frequency f2 from the lever vibration signal, the amplitude detection unit 27 may detect the amplitude of the component of the lever side frequency f2, and the feedback control unit 29 may detect the lever side frequency f2. The feedback control may be performed using the amplitude of the component. Improvements in feedback control may be made as well. However, in the measurement result of the present inventor, the amplitude of the component of the sample side frequency f1 changes more significantly according to the probe sample distance than the amplitude of the component of the lever side frequency f2. Therefore, the shape measurement can be performed more accurately by using the component of the sample side frequency f1.

  The preferred embodiments of the present invention have been described above. However, the present invention is not limited to the above-described embodiments, and it goes without saying that those skilled in the art can modify the above-described embodiments within the scope of the present invention.

  As described above, the scanning probe microscope according to the present invention is useful for observing biomolecules and the like.

It is a figure which shows SPM using the conventional ultrasonic wave. It is a figure which shows the principle of SPM which concerns on embodiment of this invention. It is a figure which shows the dependence with respect to the distance between a probe and a sample of the interference amplitude and average deflection amount of a cantilever. It is a figure explaining suitable feedback control using interference amplitude and average deflection. It is a figure which shows the structure of AFM of this Embodiment. It is a figure which shows the circuit which extracts an interference signal from a lever vibration signal. It is a figure which shows the structure of a feedback control part. It is a figure which shows the structure of AFM of another embodiment. It is a figure which shows the structure of the bending detection part in the embodiment of FIG. 8, and a feedback control part. It is a figure which shows the dependence with respect to the distance between a probe and a sample of the sample side frequency component amplitude and average deflection amount in cantilever vibration. It is a figure explaining suitable feedback control using sample side frequency ingredient amplitude and average deflection.

Explanation of symbols

1,71 Atomic force microscope (AFM)
3 Cantilever 5 Sample stage 7 Scanner 9 Lever side piezoelectric body 11 Sample side piezoelectric body 13 Sample substrate 15 Oscillator 21 Laser unit 23 Sensor 25 Extraction unit 27 Amplitude detection unit 29 Feedback control unit 31 Computer 33 Monitor 61 Subtraction circuit 63 Dynamic PID circuit 65 Feedback gain adjustment circuit 73 Deflection detection unit 75 Feedback control unit 91 Amplitude adjustment unit 93 Feedback signal adjustment unit f1 Sample side frequency f2 Lever side frequency

Claims (24)

A cantilever with a probe,
A scanner for relative scanning of the cantilever and the sample;
A sample-side ultrasonic wave emitting unit that emits ultrasonic waves of a sample-side frequency to the sample;
A lever side ultrasonic wave emitting unit for emitting ultrasonic waves of a lever side frequency different from the sample side frequency to the cantilever;
A sensor for detecting a lever vibration signal representing the vibration of the cantilever;
An extraction unit that extracts a specific frequency component that changes according to the distance between the probe and the sample in a non-contact state from the lever vibration signal;
An amplitude detector for detecting the amplitude of the specific frequency component;
A feedback control unit that performs feedback control of the scanner so that the amplitude of the specific frequency component matches a predetermined target value;
The scanning probe microscope according to claim 1, wherein the target value of the feedback control is set to an amplitude of the specific frequency component when the probe and the sample are close to each other in a non-contact state.   The scanning probe microscope according to claim 1, wherein an absolute value of a difference between the sample side frequency and the lever side frequency is set to a resonance frequency of the cantilever.   The scanning probe microscope according to claim 1, wherein the lever side frequency is set smaller than the sample side frequency. The extraction unit extracts an interference signal that is a frequency component of an absolute value of a difference between the sample side frequency and the lever side frequency as the signal of the specific frequency component,
The amplitude detector detects an interference amplitude which is an amplitude of the interference signal;
The scanning probe microscope according to claim 1, wherein the target value of the feedback control is set to the interference amplitude when the probe and the sample are close to each other in a non-contact state.   5. The scanning type according to claim 4, wherein the feedback control unit increases a feedback gain when the interference amplitude is larger than the target value as compared to when the interference amplitude is smaller than the target value. Probe microscope.   6. The feedback control unit according to claim 5, further comprising a feedback gain adjustment unit that increases the feedback gain according to a difference between the interference amplitude and the target value when the interference amplitude is larger than the target value. A scanning probe microscope as described in 1. above.   A bend detection unit that detects the bend of the cantilever from the lever vibration signal, and the feedback control unit is configured to perform a process of separating the cantilever and the sample based on the bend. The scanning probe microscope according to claim 4.   The feedback control unit includes an amplitude adjustment unit that increases a detection signal of the interference amplitude detected by the amplitude detection unit based on the deflection of the cantilever, and the feedback control unit is controlled by the amplitude adjustment unit. The scanning probe microscope according to claim 7, wherein the feedback control is performed using the adjusted interference amplitude.   The amplitude adjustment unit is configured to reduce the interference amplitude when at least one of the deflection is larger than a predetermined amplitude adjustment threshold deflection and when the interference amplitude is larger than a predetermined amplitude adjustment threshold amplitude. The scanning probe microscope according to claim 8, wherein the detection signal is adjusted.   The feedback control unit includes a feedback signal adjustment unit that adjusts a feedback signal generated by the feedback control unit so that the cantilever and the sample are separated based on the deflection of the cantilever. 8. A scanning probe microscope according to 7.   The feedback signal adjustment unit adjusts the feedback signal to a value for forcibly separating the cantilever and the sample when the deflection is larger than a threshold deflection for a predetermined feedback signal adjustment. 10. A scanning probe microscope according to 10. The extraction unit extracts the signal of the sample side frequency component or the lever side frequency component as the signal of the specific frequency component,
The amplitude detector detects the amplitude of the sample side frequency component or the lever side frequency component,
The target value of the feedback control is set to an amplitude of the sample-side frequency component or the lever-side frequency component when the probe and the sample are close to each other in a non-contact state. Item 2. A scanning probe microscope according to Item 1.   The feedback control unit is configured such that the amplitude of the sample-side frequency component or the lever-side frequency component is smaller than the target value compared to when the amplitude of the sample-side frequency component or the lever-side frequency component is larger than the target value. The scanning probe microscope according to claim 12, wherein a feedback gain at the time is increased.   When the amplitude of the sample-side frequency component or the lever-side frequency component is smaller than the target value, the feedback control unit responds to a difference between the amplitude of the sample-side frequency component or the lever-side frequency component and the target value. The scanning probe microscope according to claim 13, further comprising a feedback gain adjustment unit that increases the feedback gain.   A bend detection unit that detects the bend of the cantilever from the lever vibration signal, and the feedback control unit is configured to perform a process of separating the cantilever and the sample based on the bend. The scanning probe microscope according to claim 12.   The feedback control unit includes an amplitude adjustment unit that reduces the amplitude detection signal detected by the amplitude detection unit based on the deflection of the cantilever, and the feedback control unit is adjusted by the amplitude adjustment unit. The scanning probe microscope according to claim 15, wherein the feedback control is performed using the measured amplitude.   The amplitude adjustment unit detects the amplitude detection signal at least one of when the deflection is larger than a predetermined amplitude adjustment threshold deflection and when the amplitude is smaller than a predetermined amplitude adjustment threshold amplitude. The scanning probe microscope according to claim 16, wherein the scanning probe microscope is adjusted.   The feedback control unit includes a feedback signal adjusting unit that adjusts a feedback signal generated by the feedback control unit so that the cantilever and the sample are separated from each other based on the bending of the cantilever. 15. A scanning probe microscope according to 15.   The feedback signal adjustment unit adjusts the feedback signal to a value for forcibly separating the cantilever and the sample when the deflection is larger than a threshold deflection for a predetermined feedback signal adjustment. The scanning probe microscope according to 18. A sample stage attached to the scanner;
The scanning probe microscope according to claim 1, wherein an ultrasonic wave generation source of the sample-side ultrasonic wave emitting unit is disposed between the sample stage and a substrate that holds the sample.   The scanning probe microscope according to claim 1, wherein the extraction unit includes a Fourier filter that reconstructs a first harmonic component of a Fourier series in the signal of the specific frequency component from the lever vibration signal.   The Fourier filter multiplies the lever vibration signal by a cosine wave signal having a frequency of the specific frequency component, performs integration for one period, obtains a first DC value, and obtains a sine wave having the same frequency as the cosine wave signal. A signal obtained by multiplying the lever vibration signal by integration for one period to obtain a second DC value, and a signal obtained by multiplying the first DC value by the cosine wave signal and the second DC value by the sine wave signal. The scanning probe microscope according to claim 21, wherein a sum with a signal multiplied by is obtained. Place the sample close to the cantilever with the probe,
Firing ultrasonic waves of the sample side frequency against the sample,
Firing an ultrasonic wave of a lever side frequency different from the sample side frequency to the cantilever,
Detecting a lever vibration signal representing the vibration of the cantilever;
From the lever vibration signal, extract a specific frequency component that changes according to the distance between the probe and the sample in a non-contact state,
Detecting the amplitude of the specific frequency component;
Feedback control of the distance between the cantilever and the sample is performed so that the amplitude of the specific frequency component matches a predetermined target value, and the target value of the feedback control is determined when the probe and the sample are not in contact with each other. A sample observation method using a scanning probe microscope, wherein the amplitude is set to the amplitude of the specific frequency component when close to each other.   The signal of the specific frequency component is an interference signal that is a frequency component of an absolute value of a difference between the sample side frequency and the lever side frequency, a signal of the sample side frequency component, or a signal of the lever side frequency component. The sample observation method according to claim 23.