Atomic force microscope and method for obtaining a sample stiffness value

Abstract

The present invention relates to an atomic force microscope (1) for obtaining stiffness values ​​of a sample (3), comprising: a cantilever (202) including a tip (203) for contacting a soft material sample (3); a light source (105) for generating light; and a focusing optical system (105a) configured to focus the light and guide a first beam (B1) along a first longitudinal axis (L1) onto the cantilever (202); a mirror (112) configured to receive a second beam (B2) reflected by the cantilever (202); and a detector (113) configured to detect the position of a third beam (B3) reflected by the mirror (112) and generate a deflection signal based on the detected position of the third beam (B3), the deflection signal indicating the deflection of the cantilever (202) along the first longitudinal axis (L1).

Description

Summary of the Invention

[0001] The above-mentioned objectives are achieved through the subject matter of the independent claims. Embodiments of the invention are defined in the dependent claims and described below.

[0002] A first aspect of the invention relates to an atomic force microscope for obtaining sample stiffness values ​​by force spectroscopy, comprising: a cantilever including a tip for contacting a soft material sample (particularly a biological tissue sample); a light source (particularly an optical fiber or laser) for generating light; focusing optics configured to focus the light and guide a first beam onto the cantilever along a first longitudinal axis; a mirror configured to receive a second beam reflected from the cantilever, the second beam being generated by reflection of the first beam onto the cantilever; a detector (particularly a photodiode) configured to detect the position of a third beam reflected from the mirror, the third beam being generated by reflection of the second beam onto the mirror, and configured to generate a deflection signal indicating deflection of the cantilever along the first longitudinal axis based on the detected position of the third beam, wherein the deflection signal is particularly useful for quantitatively determining the deflection value of the cantilever along the first longitudinal axis; a sample stage for receiving a sample; and an actuator (particularly a piezoelectric stacked actuator) configured to move the cantilever relative to the sample stage along the first longitudinal axis (particularly in the vertical direction).

[0003] An atomic force microscope includes at least one linear drive device configured to position a cantilever relative to the sample stage along a first longitudinal axis and / or along a second longitudinal axis perpendicular to the first longitudinal axis and / or along a third longitudinal axis perpendicular to the first and second longitudinal axes, wherein the at least one linear drive device includes a coarse linear motor (particularly having a spatial motor resolution of 100 nm or less) for coarse positioning of the cantilever relative to the sample stage and a fine linear motor (particularly having a spatial motor resolution of 1 nm or less) for fine positioning of the cantilever relative to the sample stage.

[0004] Specifically, the actuator has a spatial resolution of 1 nm or less along the first longitudinal axis, and / or the actuator has a travel range of 20 μm to 100 μm (e.g., 38 μm) along the first longitudinal axis.

[0005] Specifically, the position of the reflector ensures that the light reflected from the cantilever in the air and liquid reaches the detector at the same angle.

[0006] Specifically, the light generated by the light source is focused onto the top surface of the cantilever facing the light source. Preferably, the light is focused onto a portion of the top surface of the cantilever located at its free end. The free end of the cantilever can be formed by mechanical fastening the cantilever to the cantilever support disclosed herein, wherein the free end of the cantilever is the end portion of the cantilever away from its mechanical fastening to the cantilever support. The tip of the cantilever can be disposed on the back side of the cantilever facing away from its top surface; specifically, the tip protrudes from the back side portion located at the free end of the cantilever. The tip preferably faces the sample, such that the atomic forces between the tip and the sample cause bending of the cantilever, particularly the bending of the free end of the cantilever, which can be detected by changes in the deflection of light reflected from the cantilever, particularly changes in the deflection of light reflected from the top surface of the cantilever.

[0007] Specifically, the sample stage is configured to receive a sample holder for holding the sample, and more specifically, the sample stage includes a first slot for receiving the sample holder.

[0008] In some embodiments, the atomic force microscope includes a probe, particularly including a cantilever, a light source, a mirror, a detector, and / or an actuator, wherein the probe is connected to a mounting bracket via the at least one linear drive and the actuator, wherein more particularly, the actuator is directly connected to the mounting bracket, and the at least one linear drive connects the actuator and the probe (e.g., the probe frame).

[0009] In some embodiments, the coarse linear motor is a voice coil motor.

[0010] In some embodiments, the precision linear motor is a piezoelectric stick-slip motor.

[0011] In some embodiments, the coarse linear motor and the fine linear motor each include a stator and a rotor, the rotor being configured to move relative to the respective stator along a first longitudinal axis or a second longitudinal axis.

[0012] In some embodiments, the stator of the fine linear motor is connected to the stator of the coarse linear motor, wherein the at least one linear drive includes a coupling element (particularly including a magnet) configured to couple the rotor of the fine linear motor to the rotor of the coarse linear motor to couple the coarse and fine linear motors in a desired direction such that the rotor of the coarse linear motor can be moved relative to the stator of the coarse linear motor by moving the rotor of the fine linear motor relative to the stator of the fine linear motor.

[0013] In some embodiments, the coarse linear motor is configured to be deactivated, in particular de-energized, when the cantilever is moved relative to the sample stage by the fine linear motor, especially where the coarse and fine linear motors are coupled by a coupling element.

[0014] In some embodiments, the atomic force microscope includes a first linear drive mechanism configured to position the cantilever relative to the sample stage along a first longitudinal axis. The first linear drive mechanism includes a first coarse linear motor (particularly a voice coil motor) for coarse positioning of the cantilever relative to the sample stage along the first longitudinal axis, particularly having a spatial motor resolution of 100 nm or less, and a first fine linear motor (particularly a piezoelectric stick-slip motor) for fine positioning of the cantilever relative to the sample stage along the first longitudinal axis, particularly having a spatial motor resolution of 1 nm or less.

[0015] In some embodiments, the probe is connected to a mounting bracket via a first linear drive and an actuator, wherein more specifically, the actuator is directly connected to the mounting bracket, and the first linear drive connects the actuator and the probe (e.g., the probe frame).

[0016] In some embodiments, the first coarse linear motor and the first fine linear motor each include a stator and a rotor, the rotor being configured to move relative to the respective stator along a first longitudinal axis.

[0017] In some embodiments, the stator of the first fine linear motor is connected to the stator of the first coarse linear motor, wherein the first linear drive includes a first coupling element (particularly including a magnet), wherein the first coupling element is configured to couple the rotor of the first fine linear motor to the rotor of the first coarse linear motor to couple the first coarse linear motor and the first fine linear motor in a desired direction such that the rotor of the first coarse linear motor can be moved relative to the stator of the first coarse linear motor by moving the rotor of the first fine linear motor relative to the stator of the first fine linear motor.

[0018] In some embodiments, the first coarse linear motor is configured to be deactivated (especially de-energized) when the cantilever is moved relative to the sample stage by the first fine linear motor, particularly wherein the first coarse linear motor and the first fine linear motor are coupled by a first coupling element.

[0019] In some embodiments, the atomic force microscope includes a second linear drive configured to position the cantilever relative to the sample stage along a second longitudinal axis perpendicular to the first longitudinal axis. The second linear drive includes a second coarse linear motor (particularly a voice coil motor) for coarsely positioning the cantilever relative to the sample stage along the second longitudinal axis, particularly having a spatial motor resolution of 100 nm or less, and a second fine linear motor (particularly a piezoelectric stick-slip motor) for finely positioning the cantilever relative to the sample stage along the second longitudinal axis, particularly having a spatial motor resolution of 1 nm or less.

[0020] In some embodiments, the probe is connected to the mounting bracket via a second linear drive and an actuator, wherein more specifically, the actuator is directly connected to the mounting bracket, and the second linear drive connects the actuator and the probe (e.g., the probe frame).

[0021] In some embodiments, the second coarse linear motor and the second fine linear motor each include a stator and a rotor, the rotor being configured to move relative to the respective stator along a second longitudinal axis.

[0022] In some embodiments, the stator of the second fine linear motor is connected to the stator of the second coarse linear motor, wherein the second linear drive includes a second coupling element (particularly including a magnet), wherein the second coupling element is configured to couple the rotor of the second fine linear motor to the rotor of the second coarse linear motor to couple the second coarse linear motor and the second fine linear motor in a desired direction such that the rotor of the second coarse linear motor can be moved relative to the stator of the second coarse linear motor by moving the rotor of the second fine linear motor relative to the stator of the second fine linear motor.

[0023] In some embodiments, the second coarse linear motor is configured to be deactivated, in particular de-energized, when the cantilever is moved relative to the sample stage by the second fine linear motor, and in particular, wherein the second coarse linear motor and the second fine linear motor are coupled by a second coupling element.

[0024] In some embodiments, the atomic force microscope includes at least one spring connecting the probe to a mounting bracket (in particular, connecting the probe to a first linear drive), wherein the at least one spring is configured to apply a force to the probe (including a cantilever, a light source, focusing optics, and a detector) along a first longitudinal axis, wherein the force remains constant when the spring is compressed or extended along the first longitudinal axis, and wherein the probe moves upward in particular when the coarse linear motor of the first linear drive is deactivated (in particular, de-energized).

[0025] In some embodiments, the atomic force microscope includes an alignment mechanism for aligning a first beam, comprising a support for holding a light source and focusing optics, a first carriage, and a first light source linear drive mechanism, particularly including a piezoelectric stick-slip motor configured to move the first carriage along a second longitudinal axis perpendicular to the first longitudinal axis, and a second carriage and a second light source drive mechanism, particularly including a piezoelectric stick-slip motor configured to move the second carriage along a third longitudinal axis perpendicular to the first and second longitudinal axes, wherein the support also includes a first plate and a second plate configured to hold the light source and focusing optics, and at least one adjusting screw connecting the first plate and the second plate, wherein the first plate can be tilted relative to the second plate by adjusting the adjusting screw to align the first beam.

[0026] In some embodiments, the atomic force microscope includes a frame, wherein a mirror is connected to the frame, and wherein the frame is pivotally connected to a mirror pivot. The atomic force microscope also includes a mirror drive configured to pivot the frame about the mirror pivot. In particular, the mirror drive is a linear drive, especially a piezoelectric stick-slip motor. The atomic force microscope includes a rod with a pin and a connecting rod with a slot. The mirror drive is configured to move the rod along a first longitudinal axis. The connecting rod is connected to the mirror pivot, and the pin of the rod is arranged in the slot of the connecting rod such that when the rod moves along the first longitudinal axis via the mirror drive, the frame pivots about the mirror pivot axis via the connecting rod.

[0027] In some embodiments, the atomic force microscope includes a detector holder, wherein the detector is connected to the detector holder, and wherein the atomic force microscope includes a detector linear drive, particularly a piezoelectric stick-slip motor, configured to move the detector holder along a second longitudinal axis perpendicular to the first longitudinal axis to adjust the position of the detector, wherein in particular the detector holder is tiltable to adjust the position of the detector.

[0028] In some embodiments, the atomic force microscope includes at least one position sensor (particularly a Hall sensor) configured to determine the position of the detector support along a second longitudinal axis.

[0029] In some embodiments, the atomic force microscope includes at least one position sensor (particularly a Hall sensor) configured to determine the position of the first carriage of the alignment mechanism along a second longitudinal axis, and / or In some embodiments, the atomic force microscope includes at least one position sensor (particularly a Hall sensor) configured to determine the position of the second carriage of the alignment mechanism along a third longitudinal axis.

[0030] In some embodiments, the sample stage includes a second slot for receiving a cantilever support, particularly a cantilever chip support containing a cantilever, so that the cantilever support can be automatically mounted onto the atomic force microscope.

[0031] In some embodiments, the atomic force microscope further includes a third linear drive mechanism configured to position the sample stage relative to the cantilever along a third longitudinal axis (particularly extending horizontally, perpendicular to the first longitudinal axis and particularly perpendicular to the second longitudinal axis). The third linear drive mechanism includes, in particular, a third coarse linear motor (particularly a voice coil motor) for coarse positioning of the sample stage along the third longitudinal axis, with a spatial motor resolution of 100 nm or less, and a third fine linear motor (particularly including a piezoelectric stick-slip motor) for fine positioning of the sample stage along the third longitudinal axis, with a spatial motor resolution of 1 nm or less.

[0032] In some embodiments, the third coarse linear motor and the third fine linear motor each include a stator and a rotor, the rotor being configured to move relative to the respective stator along a second longitudinal axis.

[0033] In some embodiments, the stator of the third fine linear motor is connected to the stator of the third coarse linear motor, wherein the third linear drive includes a third coupling element (particularly including a magnet), wherein the third coupling element is configured to couple the rotor of the third fine linear motor to the rotor of the third coarse linear motor to couple the third coarse linear motor and the third fine linear motor in a desired direction such that the rotor of the third coarse linear motor can be moved relative to the stator of the third coarse linear motor by moving the rotor of the third fine linear motor relative to the stator of the third fine linear motor.

[0034] In some embodiments, the third coarse linear motor is configured to be deactivated (especially de-energized) when the sample stage is moved relative to the cantilever by the third fine linear motor, particularly wherein the third coarse linear motor and the third fine linear motor are coupled by a third coupling element.

[0035] In some embodiments, the atomic force microscope includes a first imaging system for imaging a sample (particularly a sample held by a sample holder) along a first longitudinal axis, particularly when the sample holder is accommodated by a sample stage, wherein the first imaging system is configured to acquire a full-focus image of the sample in the direction of the first longitudinal axis (particularly for acquiring the height profile of the sample), particularly with a resolution of 4 μm or less along the first longitudinal axis.

[0036] In some embodiments, the first imaging system is configured to acquire a depth-focused image or a z-stack.

[0037] In some embodiments, the first imaging system includes a microscope and a camera, wherein in particular the first imaging system is configured to obtain height profiles at a resolution of 1 μm or less along a second and / or third longitudinal axis.

[0038] In some embodiments, the atomic force microscope includes a second imaging system for imaging a sample (particularly a sample held by a sample holder) along a first longitudinal axis, particularly from above, especially when the sample holder is accommodated by a sample stage, wherein the second imaging system is configured to obtain an overview image of the complete sample.

[0039] In some embodiments, the atomic force microscope includes a third imaging system for imaging the cantilever (particularly the tip of the cantilever) along a first longitudinal axis (particularly from below), wherein the third imaging system is configured to obtain a full-focus image of the cantilever.

[0040] In some embodiments, the third imaging system has a fixed orientation relative to the second imaging system, so that the position of the cantilever on the sample can be determined based on the image obtained by the second imaging system.

[0041] Specifically, the first imaging system, the second imaging system, and / or the third imaging system are fixed to the mounting bracket such that the first, second, and / or third imaging systems comprise a defined position relative to the cantilever or probe.

[0042] In some embodiments, the atomic force microscope includes a vibration sensor.

[0043] Specifically, the atomic force microscope is placed on a vibration compensation stage, which includes a vibration sensor configured to sense vibrations and an actuator configured to compensate for the sensed vibrations.

[0044] In some embodiments, the atomic force microscope includes a control device configured to control the actuator and the at least one linear drive such that the cantilever and the sample stage move relative to each other along a first longitudinal axis, particularly such that the tip of the cantilever contacts the sample, wherein the control device is configured to receive a deflection signal from the detector.

[0045] In some embodiments, the control device includes multiple circuit boards arranged in a dice-like manner around at least a portion of the atomic force microscope (particularly the probe).

[0046] A second aspect of the invention relates to a method for obtaining sample stiffness values, particularly for samples from soft materials, and more particularly for biological tissue samples, using an atomic force microscope according to a first aspect of the invention, comprising the steps of: contacting the sample at a measurement point via the tip of a cantilever; in a loading step, moving the cantilever and the sample stage closer together parallel to a first longitudinal axis by an actuator, particularly automatically, such that the tip of the cantilever is pressed into the sample; in an unloading step, moving the cantilever and the sample stage away from each other parallel to the first longitudinal axis by an actuator, particularly automatically; determining multiple deflection values ​​of the cantilever from a deflection signal of a detector during the loading and / or unloading steps; and determining, particularly automatically, the stiffness value of the sample at the measurement point from the determined deflection values.

[0047] In some embodiments, the method further includes determining a plurality of force values ​​representing the forces exerted on the sample by the cantilever at corresponding positions along a first longitudinal axis, wherein the stiffness value of the sample is determined from at least one subset of the force values, particularly automatically, wherein force-position relationships and / or force-position curves are obtained in particular.

[0048] In some embodiments, the resonant frequency of the cantilever is determined before it contacts the sample through the tip of the cantilever, wherein the spring constant of the cantilever is determined from the resonant frequency, particularly automatically.

[0049] In some embodiments, an image of the cantilever is obtained by a third imaging system, and at least one dimension of the cantilever (particularly the length of the cantilever along a second or third longitudinal axis and / or the width of the cantilever perpendicular to the length) is determined from the image, and the spring constant of the cantilever is determined based on the determined at least one dimension and the resonant frequency, wherein the temperature value is obtained, in particular, by a temperature sensor, and the spring constant of the cantilever is determined based on the determined at least one dimension, the resonant frequency, and the temperature value, particularly automatically.

[0050] In some embodiments, before contacting the sample by the tip of the cantilever, a reference sample with higher stiffness than the sample is contacted by the tip of the cantilever, and when the tip contacts the reference sample without indenting it, the cantilever moves a distance relative to the sample stage by an actuator, and wherein a deflection signal of the detector is obtained over the distance, and wherein the deflection sensitivity of the detector is determined from the distance and the deflection signal, particularly automatically.

[0051] In some embodiments, at least one image (particularly a full-focus image) of the sample is obtained by a first imaging system, and a surface map of the sample is determined from said at least one image, particularly automatically.

[0052] In some embodiments, a surface map is analyzed, and at least one measurement region of the sample is selected based on the analysis, wherein the measurement region includes a plurality of measurement points, and wherein stiffness values ​​are obtained at the measurement points of the measurement region, particularly automatically.

[0053] In some embodiments, at least one obstacle is identified from a surface view or overview image of the sample, and the cantilever moves away from the sample along a first longitudinal axis, particularly automatically, to avoid collisions between the cantilever and the obstacle.

[0054] In some embodiments, at least one image of the sample and the cantilever is obtained by a third imaging system, and the position of the cantilever tip relative to the sample is determined based on the image, particularly automatically.

[0055] In some embodiments, the sample stage is repeatedly moved in opposite directions along a third longitudinal axis by a third linear drive, particularly automatically. While the sample stage is repeatedly moved by the third linear drive, multiple images of the sample are sequentially acquired by a second imaging system, and the position of the sample on the sample stage (particularly on the sample holder) is automatically determined from the multiple images to determine whether the position of the sample on the sample stage remains constant during the movement of the sample stage, particularly automatically.

[0056] In some embodiments, vibration signals are obtained from vibration sensors, and in cases where the signal indicates the presence of vibration (particularly an amplitude or frequency above a predetermined threshold), the cantilever moves away from the sample along a first longitudinal axis, particularly automatically.

[0057] In this application, the first longitudinal direction is also referred to as the z-direction.

[0058] In this application, the second longitudinal direction is also referred to as the x-direction.

[0059] In this application, the third longitudinal direction is also referred to as the y-direction.

[0060] Regardless of whether alternatives to individual separable features are listed herein as "examples", it should be understood that these alternatives can be freely combined to form discrete embodiments of the invention disclosed herein. Attached Figure Description

[0061] Exemplary embodiments are described below with reference to the accompanying drawings. The drawings are attached to the claims and include text explaining the various features of the illustrated embodiments and aspects of the invention. Each individual feature shown in the drawings and / or mentioned in the accompanying text may (also separately) be incorporated into the claims relating to the first and / or second aspects of the invention.

[0062] Figure 1 An embodiment of an atomic force microscope according to the present invention is shown, wherein a cantilever support is arranged on the probe of the atomic force microscope; Figure 2 A cross-sectional view of the probe of an atomic force microscope according to an embodiment of the present invention is shown; Figure 3 An embodiment of an atomic force microscope according to the present invention is shown, wherein a tray for receiving a sample holder is arranged below the probe of the atomic force microscope; and Figure 4a , 4b The schematic depiction shows the optical path of light emitted from the light source and reflected from the cantilever and mirror to the detector. Figure 4a ), where the transparent body is arranged in Figure 4b In the cantilever bracket shown. Detailed Implementation

[0063] Figure 1An atomic force microscope 1 according to an embodiment of the present invention is shown. The atomic force microscope 1 includes a probe 100 movably connected to a mounting bracket 2 (not shown). Specifically, the probe 100 can be moved relative to the mounting bracket 2 via an actuator 140 of the atomic force microscope 1. For this purpose, the probe 100 can be connected to the mounting bracket 2 via the actuator 140. With the aid of the actuator 140, the probe 100 can be moved along a first longitudinal axis L1, according to… Figure 1 In the coordinate system shown, the first vertical axis L1 corresponds to the z-axis.

[0064] At the top of the upper portion 104a of the body 104 of the probe 100, the atomic force microscope 1 is provided with a light source 105. The light source 105 is configured to generate light for optically detecting the displacement of the cantilever 202, which is mounted on the cantilever chip 201 of the cantilever chip holder 310, which is mounted on the receiving unit 106 of the atomic force microscope 1. Alternatively, the cantilever 200 can be attached to the cantilever holder. The body 104 includes a mirror configured to receive light emitted from the light source and reflected from the cantilever, and a detector configured to detect the position of the light beam reflected from the mirror to the detector, such as... Figure 2 Further explanation is provided below.

[0065] The light source 105 may include focusing and collimating optics (such as lenses) and is connected to the body 104 via a cross slide 110 including a first slide 110a, a second slide 110b and a third slide 110c.

[0066] The first carriage 110a of the cross slide 110 can be configured according to... Figure 1 The coordinate system shown is moved in the y-direction, and its resolution is approximately 10 μm. This movement is achieved by a first light source linear actuator 111a, which includes a piezoelectric drive, specifically a piezoelectric LEGS actuator, a piezoelectric friction actuator, or a piezoelectric stick-slip motor.

[0067] The second carriage 110b of the cross slide 110 can be configured according to... Figure 1 The coordinate system shown moves in the x-direction, particularly with a resolution of approximately 10 μm, via a second light source linear actuator 111b, which includes a piezoelectric actuator, specifically a piezoelectric LEGS or a piezoelectric friction actuator or a piezoelectric stick-slip motor or a piezoelectric stick-slip motor.

[0068] The third carriage 110c of the cross slide can be configured according to... Figure 1The coordinate system shown moves in the z-direction, particularly with a resolution of approximately 10 μm, via a second light source linear actuator, which includes a piezoelectric actuator 111c, particularly a piezoelectric LEGS or a piezoelectric friction actuator or a piezoelectric stick-slip motor or a piezoelectric stick-slip motor.

[0069] By moving the light source 105 relative to the probe 100 (especially relative to the cantilever 202) using the cross slide 110 and its carriages 110a, 110b, 110c, the position of the spot of the first beam B1 emitted from the light source 105 along the first longitudinal axis L1 toward the cantilever 202 can be aligned with and focused on the cantilever 202. This allows a strong signal indicating the deflection of the cantilever 202 to be obtained during measurement, thereby improving the measurement accuracy of the atomic force microscope 1.

[0070] Figure 2 A cross-sectional view of a probe 100 of an atomic force microscope 1 according to an embodiment of the present invention is shown. This cross-sectional view highlights components arranged inside the probe 100, such as the optical path and focusing optics 105a for focusing light onto the cantilever 202. From the cantilever 202, light can be reflected to a mirror 112 mounted on a frame 114, which is configured to reflect light to a detector 113, which detects the reflected light indicating the deflection of the cantilever 202. In this embodiment, the detector 113 is a four-quadrant photodiode mounted on a detector support 116. The probe 100 includes a detector linear actuator 116a, particularly a piezoelectric motor, configured to move the detector support 116 and the detector 113 along the x-direction, perpendicular to the x-axis. Figure 2 The z and y directions are shown in the diagram. For this purpose, the detector linear actuator 116a is connected to the detector holder 116 via a connecting rod extending in the z direction. The translational movement of the connecting rod 117 caused by the detector linear actuator 116 can thus be transferred to the detector holder 116. The detector holder 116 can be tilted by three adjusting screws 116b to adjust the direction of the detector holder 116 relative to the incident light beam reflected from the reflector 112. A magnet-based position sensor 126a is mounted on the photodetector holder 116 so that the position of the photodetector holder 116 carrying the photodetector 113 can be determined.

[0071] according to Figure 2In the coordinate system shown, the frame 114 is rotatable about axis 114a, which extends along the x-direction and is perpendicular to the y and z directions. A mirror actuator 114b (particularly a piezoelectric motor) forms a clamping actuator 1222, configured to move the device 1224 along a first longitudinal axis L1. By moving the device 1224, the clamping element 122 for clamping the cantilever chip 310 to the head 100 of the atomic force microscope 1 can be transferred between a receiving position and a clamping position, in which the cantilever chip 310 can be arranged in the receiving unit 106, and in the clamping position, the cantilever chip 310 is fixed to the receiving unit 106 by the clamping element 122.

[0072] The shaft 114a of the frame 114 is mechanically connected to the connecting rod 121, which in turn is mechanically connected to the push rod 119. The connecting rod 121 includes a groove 121a, and the push rod includes a pin 120 disposed in the groove. Therefore, when the mirror actuator 114b moves the device 1224 in the z-direction, the pin 120 moves in the groove 121, while the mirror 112 pivots on the frame 114. Thus, the mirror actuator 114b is configured to both engage the clamping element 122 and pivot the mirror 112.

[0073] Figure 3 An atomic force microscope 1 according to an embodiment of the present invention is shown, wherein a tray 300 for receiving a sample holder 430 is disposed below the probe 100 of the atomic force microscope 1.

[0074] The probe 100 is schematically depicted as being mounted on a first linear actuator 101, which is used to linearly move the probe 100 relative to the tray 300 along the vertical z-direction. The first linear actuator 101 includes a coarse-adjustment actuator 101a for coarse-adjustment movement of the probe 100 along the z-direction and a fine-adjustment actuator 101b for fine-adjustment movement of the probe 100 along the z-direction. The coarse-adjustment actuator 101a may include or be composed of a voice coil motor. The fine-adjustment actuator 101b may include or be composed of a piezoelectric motor.

[0075] The atomic force microscope according to this embodiment includes a constant-force magnetic spring 130 that applies an upward force to the probe 100 along the z-direction (i.e., away from the tray 300) to counteract the gravity of the probe 100. The magnetic spring 130 is configured such that when the first linear actuator 101 (particularly the coarse adjustment actuator 101a and / or fine adjustment actuator 101b of the first linear actuator 101) is de-energized, the probe 100 moves upward along the z-direction, causing the cantilever 202 to move away from the sample. This represents a safety measure as it prevents the cantilever 202 from colliding with the sample in the event of a power outage of the first linear actuator 101.

[0076] The first linear actuator 101 is disposed between the probe 100 and the second linear actuator 102, for linear movement of the probe 100 relative to the tray 300 in the x-direction (perpendicular to the y and z directions). The second linear actuator 102 includes a coarse adjustment actuator 102a for driving the probe 100 to perform coarse adjustment movement in the x-direction and a fine adjustment actuator 102b for driving the probe 100 to perform fine adjustment movement in the x-direction. The coarse adjustment actuator 102a may include or be composed of a voice coil motor. The fine adjustment actuator 102b may include or be composed of a piezoelectric motor.

[0077] Therefore, with the aid of the first and second linear actuators 101 and 102, the probe 100 can move relative to the tray 300 in two orthogonal directions.

[0078] Furthermore, the atomic force microscope 1 according to this embodiment includes a third linear actuator 103 for linearly moving the tray 300 relative to the probe 100 along the y-direction. The third linear actuator 103 includes a coarse adjustment actuator 103a for coarsely adjusting the tray 300 along the y-direction and a fine adjustment actuator 103b for finely adjusting the tray 300 along the y-direction. The coarse adjustment actuator 103a may include or be composed of a voice coil motor. The fine adjustment actuator 103b may include or be composed of a piezoelectric motor.

[0079] Therefore, the probe 100 and the tray 300 can be moved relative to each other in three orthogonal directions using the first, second and third linear actuators 101, 102, 103, which allows for precise and flexible positioning of the sample relative to the cantilever 202 mounted on the probe 100.

[0080] The atomic force microscope 1 according to this embodiment also includes a first imaging system 450 disposed on the first linear actuator 101. The first imaging system 450 is configured to image a sample placed on a sample holder 430 from above along the z-direction. The first imaging system 450 is specifically configured to acquire a depth map of the sample, particularly a 3D reconstructed or topographic depth-focused image, particularly a true-color image with all pixels in focus. For example, the z-resolution (i.e., the optical resolution in the z-direction) can be approximately 4 μm, and the xy-resolution (i.e., the optical resolution along the x and y directions) can be approximately 1 μm.

[0081] like Figure 3 As further seen, a second imaging system 460 is disposed on the probe 100. The second imaging system 460 is configured to capture an overview image of the sample disposed in the sample holder 430. Furthermore, the first imaging system 450 may be configured to read barcodes on the sample holder and / or the cantilever support (particularly the cantilever chip holder) along the z-direction from above (i.e., from the angle of the probe 100).

[0082] Figure 3 Two shuttle units 650 disposed on the tray 300 are also shown. The shuttle units 650 are movable between the tray 300 and the drawer unit 620. The shuttle units 650 are configured to receive the sample holder 430 and / or cantilever holder, particularly the cantilever chip holder 310, with samples.

[0083] A third imaging system 440 is disposed on the tray 300. The third imaging system 440 is an optical microscope, such as a bottom-light microscope, configured to image the cantilever 202 from below (i.e., from the angle of the tray 300). The third imaging system 440 can be configured to determine the dimensions of the cantilever 202, particularly its length, width, and / or height, in order to determine the spring constant of the cantilever 202. Additionally, the position of the tip of the cantilever 202 can be determined using the third imaging system 440. Therefore, the third imaging system 440 can be used to automatically align a light beam to the tip of the cantilever 202 in the x, y, and z directions. The third imaging system 440 can be configured to acquire depth maps and / or depth-focused images of the cantilever 202 to automatically determine the position of the tip in the x, y, and z directions.

[0084] The atomic force microscope 1 according to this embodiment also includes a controller 410. The controller 410 may be made of a system-on-a-module (SoM), which consists of an FPGA (Field Programmable Gate Array) for computation (particularly for computation on a timescale of about 5 ns), a real-time Linux running on an ARM processor (for computation, particularly for computation on a timescale of about 50 μs), and a device controller (particularly a Linux operating system for device automation and / or user input).

[0085] The inputs of the controller 410 may come from and / or the outputs of the controller 410 may be directed to one or more of the following: the detector 113 (particularly via a preamplifier and analog-to-digital converter), the first, second, and third imaging systems 450, 460, and 440, the magnetic positioning sensor 126a, an ambient temperature sensor, an ambient air pressure sensor, an ambient air humidity sensor, a voltage sensor, a sample temperature sensor, a vibration compensation stage provided with the atomic force microscope 1, the first, second, and third linear actuators 101, 102, and 103 (particularly their coarse adjustment actuators 101a, 102a, and 103a and / or their fine adjustment actuators 101b, 102b, and 103c), a position controller for operating the first linear actuator, the second, and / or the third linear actuators 101, 102, and 103, the detector linear actuator 116a, the mirror actuator 114b (for pivoting the mirror 112 and for engaging the clamping element 122), a light source controller for controlling the light source 105, and an SLED controller.

[0086] like Figure 3 As further seen, the optical target 402 is disposed on the tray 300 below the probe 100. When no cantilever support (specifically the cantilever chip holder 310) is mounted on the probe 100, the atomic force microscope 1 is configured to automatically determine and calibrate the position of the probe 100 in the x and y directions. For positioning and calibration, a light beam emitted from the light source 105 passes through the optical path and focusing optics 105a of the probe 100 and illuminates the optical target 402. The light reflected from the optical target 402 returns to the optical path and focusing optics 105a, which is connected to a beam splitter of an optical fiber configured to calibrate the position of the probe 100 relative to the tray 300 based on the light reflected from the optical target 402 along the x and y directions.

[0087] Figure 4aThe optical path of light emitted from the light source 105 and detected by the detector 113 is schematically depicted. Light is emitted from the light source 105, forming a first beam B1, directed toward the cantilever 202 fixed by the cantilever bracket 310. From the cantilever 202, particularly from the top surface of the cantilever 202 facing the light source 105, the light is reflected to form a second beam B2, directed toward the reflector 112, which can be rotated by means of the reflector driver 114b. See [reference needed]. Figure 2 From the reflector 112, light is ultimately reflected to form a third beam B3, which is then reflected to the detector 113, which may be a four-quadrant photodiode. The position of the light source 105 relative to the cantilever 202 can be changed by moving the cross slide 110 and its first, second, and third light source linear drivers 111a, 111b, and 111c along the x, y, and z directions, respectively, thereby altering the optical path. Furthermore, the photodetector 113 can be moved along the x-direction by the detector linear driver 116a, even if it exceeds... Figure 4a The drawing plane is shown. The deflection of the cantilever 202 reflects the distance between the cantilever tip 203 and the sample 3 placed on the sample stage 300.

[0088] Figure 4a The diagram also schematically indicates a first linear actuator 101 having a coarse linear motor 101a and a fine linear motor 101b for moving the cantilever 202, which is fixed by the cantilever bracket 310, along the first longitudinal axis L1; and a second linear actuator 102 having a coarse linear motor 102a and a fine linear motor 102b for moving the cantilever 202, which is fixed by the cantilever bracket 310, along the second longitudinal axis L2. Additionally, the sample stage 300 can be moved along the third longitudinal axis L3 by a third linear actuator 103 including a coarse linear motor 103a and a fine linear motor 103b.

[0089] Figure 4b An embodiment is shown in which the cantilever bracket 310 is configured to receive a transparent body 320. The transparent body 320 is formed of an optically transparent material (such as glass). Specifically, the material of the transparent body has a refractive index greater than that of air. In some embodiments, the refractive index of the transparent body material is greater than that of the bracket material. For example, the refractive index of the transparent body is between 1.6 and 2.2, more particularly about 1.9.

[0090] With the transparent body 320 provided, the light beam used to determine the deflection of the free end of the cantilever 202 passes through the transparent body 320 and reaches the cantilever 202, and the light beam reflected from the cantilever 202 passes through the transparent body 320 and is directed towards the reflector 112. At the interface of the transparent body 320, the light is refracted. The transparent body 320 improves the stability of the assembly formed by the transparent body 320 and the cantilever support 310, allowing the cantilever support 310 to be made longer in the z-direction, which simplifies the handling of the cantilever support 310. Furthermore, if the refractive index of the transparent body 320 is higher than that of air, the optical system of the atomic force microscope 1 can be made more compact, because the refraction of the light beam at the transparent body 320 can refract the light beam to the reflecting mirror 112. Without the transparent body 320, the light must rely entirely on the reflection of the cantilever 202 to be guided from the cantilever 202 to the reflecting mirror, which requires the cantilever support 310 to have a rather large shape, as can be understood from the comparison of the cantilever support 310 in Figures 4a and 4b.

Claims

1. An atomic force microscope (1) for obtaining the stiffness value of a sample (3), comprising: a. A cantilever (202) including a tip (203) for contacting a soft material sample (3); b. A light source (105) for generating light; and a focusing optics (105a) configured to focus the light and guide the first beam (B1) onto the cantilever (202) along the first longitudinal axis (L1); c. A reflector (112) configured to receive a second beam (B2) reflected by the cantilever (202); d. Detector (113), configured to detect the position of the third beam (B3) reflected by the mirror (112), and generate a deflection signal indicating that the cantilever (202) has deflected along the first longitudinal axis (L1) based on the detected position of the third beam (B3); e. Sample stage (300) for accommodating sample (3); f. An actuator (140) configured to move the cantilever (202) relative to the sample stage (300) along a first longitudinal axis (L1); The atomic force microscope (1) includes at least one linear drive device (101, 102, 103) configured to position the cantilever (202) relative to the sample stage (300) along a first longitudinal axis (L1) and / or along a second longitudinal axis (L2) perpendicular to the first longitudinal axis (L1), and / or along a third longitudinal axis (L3) perpendicular to the first longitudinal axis (L1) and the second longitudinal axis (L2), wherein the at least one linear drive device (101, 102, 103) includes a coarse linear motor (101a, 102a, 103a) for coarse positioning of the cantilever (202) relative to the sample stage (300) and a fine linear motor (101b, 102b, 103b) for precise positioning of the cantilever (202) relative to the sample (3).

2. The atomic force microscope (1) according to claim 1, wherein the atomic force microscope (1) includes a probe (100), particularly the probe (100) includes a cantilever (202), a light source (105), a mirror (112), a detector (113) and / or an actuator (140), wherein the probe (100) is connected to a mounting bracket (2) via the at least one linear drive device (101, 102, 103) and the actuator (140), wherein more particularly, the actuator (140) is directly connected to the mounting bracket (2), and the at least one linear drive device (101, 102, 103) connects the actuator (140) and the probe (100).

3. The atomic force microscope (1) according to claim 1 or 2, wherein the coarse linear motor (101a, 102a, 103a) is a voice coil motor.

4. The atomic force microscope (1) according to any of the preceding claims, wherein the precision linear motors (101b, 102b, 103b) are piezoelectric stick-slip motors.

5. The atomic force microscope (1) according to any of the preceding claims, wherein the coarse linear motor (101a, 102a, 103a) and the fine linear motor (101b, 102b, 103b) each include a stator and a rotor, the rotor being configured to move relative to the corresponding stator along a first longitudinal axis (L1), a second longitudinal axis (L2) or a third longitudinal axis (L3).

6. The atomic force microscope (1) according to claim 5, wherein the stator of the fine linear motors (101b, 102b, 103b) is connected to the stator of the coarse linear motors (101a, 102a, 103a), wherein the at least one linear drive device (101, 102, 103) includes a coupling element, particularly the coupling element including a magnet, wherein the coupling element is configured to couple the rotor of the fine linear motors (101b, 102b, 103b) to the rotor of the coarse linear motors (101a, 102a, 103a) to connect the coarse linear motors (101a, 102a, 103a) and the fine linear motors (101b, 102b, 103b). Coupled in the desired orientation, the rotor of the coarse linear motor (101a, 102a, 103a) can be moved relative to the stator of the fine linear motor (101b, 102b, 103b) by moving the rotor of the fine linear motor (101b, 102b, 103b) relative to the stator of the coarse linear motor (101a, 102a, 103a).

7. The atomic force microscope (1) according to any of the preceding claims, wherein, when the precision linear motors (101b, 102b, 103b) move the cantilever (202) relative to the sample stage (300), the ordinary precision linear motors (101a, 102a, 103a) are configured to be in a deactivated state, particularly a power-off state; particularly wherein, Precision linear motors (101b, 102b, 103b) and ordinary precision linear motors (101a, 102a, 103a) are coupled through coupling elements.

8. The atomic force microscope (1) according to any of the preceding claims, wherein the atomic force microscope includes a first linear drive device (101) configured to position the cantilever (202) relative to the sample stage (300) along a first longitudinal axis (L1), wherein the first linear drive device (101) includes a first coarse linear motor (101a), particularly a voice coil motor, for coarsely positioning the cantilever (202) relative to the sample stage (300) along the first longitudinal axis (L1), and a first fine linear motor (101b), particularly a piezoelectric stick-slip motor, for finely positioning the cantilever (202) relative to the sample stage (300) along the first longitudinal axis (L1).

9. The atomic force microscope (1) according to any one of the preceding claims, wherein the atomic force microscope (1) includes a second linear drive device (102) configured to position the cantilever (202) relative to the sample stage (300) along a second longitudinal axis (L2) perpendicular to the first longitudinal axis (L1), wherein the second linear drive device (102) includes a second coarse linear motor (102a), particularly a voice coil motor, for coarsely positioning the cantilever (202) relative to the sample stage (300) along the second longitudinal axis (L2), and a second fine linear motor (102b), particularly a piezoelectric stick-slip motor, for finely positioning the cantilever (202) relative to the sample stage (300) along the second longitudinal axis (L2).

10. The atomic force microscope (1) according to any of the preceding claims, wherein the atomic force microscope (1) further comprises a third linear drive (103) configured to position the sample stage (300) relative to the cantilever (202) along a third longitudinal axis (L3) perpendicular to the first longitudinal axis (L1) and particularly perpendicular to the second longitudinal axis (L2), wherein the third linear drive (103) comprises a third coarse linear motor (103a), particularly a voice coil motor, for coarsely positioning the sample stage (300) along the third longitudinal axis (L3), and a third fine linear motor, particularly a piezoelectric stick-slip motor, for finely positioning the sample stage (300) along the third longitudinal axis (L3).

11. The atomic force microscope (1) according to any of the preceding claims, wherein the atomic force microscope (1) includes a control device configured to control the actuator (140) and the at least one linear drive device (101, 102, 103) such that the cantilever (202) and the sample stage (300) move relative to each other along a first longitudinal axis (L1), in particular such that the tip (203) of the cantilever (202) contacts the sample (3), wherein the control device is particularly configured to receive a deflection signal from the detector (113).

12. A method for obtaining stiffness values ​​of a sample (3), particularly from a sample (3) of a soft material, and more particularly from a biological tissue sample, using an atomic force microscope (1) according to any one of claims 1 to 11, wherein the method comprises the following steps: a. At the measurement point, the sample (3) is contacted through the tip (203) of the cantilever (202); b. In the loading step, the cantilever (202) and the sample stage (300) are moved toward each other parallel to the first longitudinal axis (L1) by the actuator (140), and in particular automatically, the tip (203) of the cantilever (202) is pressed into the sample (3); c. During the unloading step, the cantilever (202) and the sample stage (300) are moved in opposite directions parallel to the first longitudinal axis (L1) by the actuator (140), particularly automatically; d. During the loading and / or unloading steps, determine multiple deflection values ​​of the cantilever (202) from the deflection signal of the detector (113); e. Based on the determined deflection value, specifically, the stiffness value of the sample (3) at the measurement point is automatically determined.

13. The method of claim 12, wherein the method further comprises: Determine multiple force values, which represent the forces exerted by the cantilever (202) on the sample (3) at corresponding positions along the first longitudinal axis (L1); The stiffness value of sample (3) is determined based on at least one subset of force values, in particular automatically, wherein force-position relationships and / or force-position curves are obtained.

14. The method according to claim 12 or 13, wherein the resonant frequency of the cantilever (202) is determined before contacting the sample (3) by the tip (203) of the cantilever (203), wherein the spring constant of the cantilever (202) is determined from the resonant frequency, in particular automatically.

15. The method according to any one of claims 12 to 14, wherein, before contacting the sample (3) by the tip (203) of the cantilever (202), a reference sample having higher stiffness than the sample (3) is contacted by the tip (203) of the cantilever (202), and the cantilever (202) is moved a distance relative to the sample stage (300) by an actuator (140), while the tip (203) contacts the reference sample without indenting the reference sample, and wherein a deflection signal of the detector (113) is obtained over the distance moved, and wherein the deflection sensitivity of the detector is determined, in particular automatically, based on the distance moved and the deflection signal.

Images