Atomic force microscope and method for obtaining a stiffness value of a sample

Abstract

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

Description

[0001] Atomic force microscope and method for obtaining a stiffness value of a sample

[0002] Description

[0003] This objective is attained by the subject matter of the independent claims. Embodiments of the invention are specified in the dependent claims and described hereafter.

[0004] A first aspect of the invention relates to an atomic force microscope for obtaining a stiffness value of a sample by force spectroscopy comprising a cantilever comprising a tip for contacting a sample of soft material, particularly a biological tissue specimen, a light source, particularly an optical fiber or a laser, for generating light and focusing optics configured to focus the light and direct a first light beam along a first longitudinal axis onto the cantilever, a mirror configured to receive a second light beam reflected by the cantilever, the second light beam being generated by reflection of the first light beam by the cantilever, a detector, particularly a photodiode, configured to detect a position of a third light beam reflected by the mirror, generated by reflection of the second light beam on the mirror, and configured to generate a deflection signal indicating a deflection of the cantilever along the first longitudinal axis based on the detected position of the third light beam, wherein particularly the deflection signal can be used to quantitatively determine a deflection value of the cantilever along the first longitudinal axis, a sample stage for receiving the sample, and an actuator, particularly a piezo stack actuator, configured to move the cantilever relative to the sample stage along the first longitudinal axis, particularly in a vertical direction.

[0005] The atomic force microscope comprises at least one linear drive configured to position the cantilever relative to the sample stage along the first longitudinal axis and / or along a second longitudinal axis which is perpendicular to the first longitudinal axis, and / or along a third longitudinal axis which is perpendicular to the first longitudinal axis and the second longitudinal axis, wherein the at least one linear drive comprises a coarse linear motor for coarse positioning of the cantilever relative to the sample stage, particularly with a spatial motor resolution of 100 nm or less, and a fine linear motor for fine positioning of the cantilever relative to the sample stage, particularly with a spatial motor resolution of 1 nm or less.

[0006] In particular, the actuator comprises a spatial resolution of 1 nm or less along the first longitudinal axis and / or the actuator comprises a travel range of 20 pm to 100 pm, e.g. 38 pm, along the first longitudinal axis.

[0007] In particular, the mirror is positioned such that light reflected by the cantilever in air and in liquid arrives at the detector at the same angle.

[0008] Particularly, the light generated by the light source is focused onto a top surface of the cantilever facing the light source. Preferably, the light is focused onto a section of the top surface of the cantilever which is located at a free end of the cantilever. The free end of the cantilever can be formed resulting from a mechanical fastening of the cantilever on a cantilever holder disclosed herein, wherein the free end of the cantilever is an end section of the cantilever distant from its mechanical fastening to the cantilever holder. The tip of the cantilever can be arranged at a back surface of the cantilever facing away from its top surface, particularly wherein the tip is protruding away from a section of the back surface located at the free end of the cantilever. The tip preferably faces the sample, such that atomic forces between the tip and the sample cause a bending of the cantilever, particularly of the free end of the cantilever, which can be detected by variations of a deflection of light reflected from the cantilever, particularly from the top surface of the cantilever.

[0009] Particularly, the sample stage is configured to receive a specimen holder configured to hold the sample, wherein more particularly the sample stage comprises a first slot for receiving the specimen holder.

[0010] In certain embodiments, the atomic force microscope comprises a head, particularly comprising the cantilever, the light source, the mirror, the detector and / or the actuator, wherein the head 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 head, e.g. a frame of the head.

[0011] In certain embodiments, the coarse linear motor is a voice coil motor.

[0012] In certain embodiments, the fine linear motor is a piezoelectric stick-slip motor.

[0013] In certain embodiments, the coarse linear motor and the fine linear motor each comprise a stator and a runner configured to move along the first longitudinal axis or the second longitudinal axis relative to the respective stator.

[0014] In certain 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 comprises a coupling element, particularly comprising a magnet, wherein the coupling element is configured to couple the runner of the fine linear motor to the runner of the coarse linear motor, to couple the coarse linear motor and the fine linear motor in a desired orientation, such that the runner of the coarse linear motor can be moved relative to the stator of the coarse linear motor by moving the runner of the fine linear motor relative to the stator of the fine linear motor.

[0015] In certain embodiments, the coarse linear motor is configured to be inactivated, particularly deenergized, when the cantilever is moved relative to the sample stage by the fine linear motor, particularly wherein the coarse linear motor and the fine linear motor are coupled by the coupling element. In certain embodiments, the atomic force microscope comprises a first linear drive configured to position the cantilever relative to the sample stage along the first longitudinal axis, wherein the first linear drive comprises 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 with 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 with a spatial motor resolution of 1 nm or less.

[0016] In certain embodiments, the head is connected to the mounting bracket via the first linear drive and the actuator, wherein more particularly the actuator is directly connected to the mounting bracket and the first linear drive connects the actuator and the head, e.g. a frame of the head.

[0017] In certain embodiments, the first coarse linear motor and the first fine linear motor each comprise a stator and a runner configured to move along the first longitudinal axis relative to the respective stator.

[0018] In certain 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 comprises a first coupling element, particularly comprising a magnet, wherein the first coupling element is configured to couple the runner of the first fine linear motor to the runner of the first coarse linear motor, to couple the first coarse linear motor and the first fine linear motor in a desired orientation, such that the runner of the first coarse linear motor can be moved relative to the stator of the first coarse linear motor by moving the runner of the first fine linear motor relative to the stator of the first fine linear motor.

[0019] In certain embodiments, the first coarse linear motor is configured to be inactivated, particularly 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 the first coupling element.

[0020] In certain embodiments, the atomic force microscope comprises a second linear drive configured to position the cantilever relative to the sample stage along a second longitudinal axis extending perpendicular to the first longitudinal axis, wherein the second linear drive comprises a second coarse linear motor, particularly a voice coil motor, for coarse positioning of the cantilever relative to the sample stage along the second longitudinal axis, particularly with a spatial motor resolution of 100 nm or less, and a second fine linear motor, particularly a piezoelectric stick-slip motor, for fine positioning of the cantilever relative to the sample stage along the second longitudinal axis, particularly with a spatial motor resolution of 1 nm or less.

[0021] In certain embodiments, the head is connected to the mounting bracket via the second linear drive and the actuator, wherein more particularly the actuator is directly connected to the mounting bracket and the second linear drive connects the actuator and the head, e.g. a frame of the head.

[0022] In certain embodiments, the second coarse linear motor and the second fine linear motor each comprise a stator and a runner configured to move along the second longitudinal axis relative to the respective stator.

[0023] In certain 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 comprises a second coupling element, particularly comprising a magnet, wherein the second coupling element is configured to couple the runner of the second fine linear motor to the runner of the second coarse linear motor, to couple the second coarse linear motor and the second fine linear motor in a desired orientation, such that the runner of the second coarse linear motor can be moved relative to the stator of the second coarse linear motor by moving the runner of the second fine linear motor relative to the stator of the second fine linear motor.

[0024] In certain embodiments, the second coarse linear motor is configured to be inactivated, particularly de-energized, when the cantilever is moved relative to the sample stage by the second fine linear motor, particularly wherein the second coarse linear motor and the second fine linear motor are coupled by the second coupling element.

[0025] In certain embodiments, the atomic force microscope comprises at least one spring connecting the head to the mounting bracket, particularly connecting the head to the first linear drive, wherein the at least one spring is configured to exert a force on the head, including the cantilever, the light source, the focusing optics and the detector, along the first longitudinal axis, particularly wherein the force remains constant when the spring is compressed or elongated along the first longitudinal axis, particularly resulting in an upward movement of the head when the coarse linear motor of the first linear drive is inactivated, particularly deenergized.

[0026] In certain embodiments, the atomic force microscope comprises an alignment mechanism for aligning the first light beam comprising a support for holding the light source and the focusing optics, a first carriage and a first light source linear drive, particularly comprising a piezo stickslip 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, particularly comprising a piezo stick-slip motor, configured to move the second carriage along a third longitudinal axis perpendicular to the first longitudinal axis and the second longitudinal axis, wherein particularly the support further comprises a first plate configured to hold the light source and the focusing optics, a second plate, and at least one set-screw connecting the first plate and the second plate, wherein the first plate is tiltable with respect to the second plate by adjusting the set-screw to align the first light beam. In certain embodiments, the atomic force microscope comprises a mirror frame, wherein the mirror is connected to the mirror frame, and wherein the mirror frame is pivotably connected to a mirror pivot axis, wherein the atomic force microscope further comprises a mirror drive configured to pivot the mirror frame about the mirror pivot axis, wherein particularly the mirror drive is a linear drive, particularly a piezo stick-slip motor, and wherein the atomic force microscope comprises a bar comprising a pin and a connecting lever comprising a slot, wherein the mirror drive is configured to move the bar along the first longitudinal axis, wherein the connecting lever is connected to the mirror pivot axis and the pin of the bar is arranged in the slot of the connecting lever, such that the mirror frame is pivoted around the mirror pivot axis by means of the connecting lever when the bar is moved along the first longitudinal axis by the mirror drive.

[0027] In certain embodiments, the atomic force microscope comprises a detector holder, wherein the detector is connected to the detector holder, and wherein the atomic force microscope comprises a detector linear drive, particularly a piezo 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 particularly the detector holder is tiltable to adjust the position of the detector.

[0028] In certain embodiments, the atomic force microscope comprises at least one position sensor, particularly a Hall sensor, configured to determine a rotational orientation of the mirror frame on the mirror pivot axis,

[0029] In certain embodiments, the atomic force microscope comprises at least one position sensor, particularly a Hall sensor, configured to determine a position of the detector holder along the second longitudinal axis,

[0030] In certain embodiments, the atomic force microscope comprises at least one position sensor, particularly a Hall sensor, configured to determine a position of the first carriage of the alignment mechanism along the second longitudinal axis, and / or

[0031] In certain embodiments, the atomic force microscope comprises at least one position sensor, particularly a Hall sensor, configured to determine a position of the second carriage of the alignment mechanism along the third longitudinal axis.

[0032] In certain embodiments, the atomic force microscope comprises a temperature sensor, wherein particularly the temperature sensor is positioned at or close to the cantilever, such that the temperature of the cantilever can be determined from a temperature value measured by the temperature sensor. In certain embodiments, the sample stage comprises a second slot for receiving a cantilever holder, particularly a cantilever chip holder comprising a cantilever, such that the cantilever holder can be automatically mounted to the atomic force microscope.

[0033] In certain embodiments, the atomic force microscope further comprises a third linear drive configured to position the sample stage relative to the cantilever along a third longitudinal axis, particularly extending along a horizontal direction, perpendicular to the first longitudinal axis and particularly perpendicular to the second longitudinal axis, wherein particularly the third linear drive comprises a third coarse linear motor, particularly a voice coil motor, for coarse positioning of the sample stage along the third longitudinal axis, particularly with a spatial motor resolution of 100 nm or less, and a third fine linear motor, particularly comprising a piezoelectric stick-slip motor, for fine positioning of the sample stage along the third longitudinal axis, particularly with a spatial motor resolution of 1 nm or less.

[0034] In certain embodiments, the third coarse linear motor and the third fine linear motor each comprise a stator and a runner configured to move along the second longitudinal axis relative to the respective stator.

[0035] In certain 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 comprises a third coupling element, particularly comprising a magnet, wherein the third coupling element is configured to couple the runner of the third fine linear motor to the runner of the third coarse linear motor, to couple the third coarse linear motor and the third fine linear motor in a desired orientation, such that the runner of the third coarse linear motor can be moved relative to the stator of the third coarse linear motor by moving the runner of the third fine linear motor relative to the stator of the third fine linear motor.

[0036] In certain embodiments, the third coarse linear motor is configured to be inactivated, particularly 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 the third coupling element.

[0037] In certain embodiments, the atomic force microscope comprises a first imaging system for imaging the sample, particularly the sample held by the specimen holder, along the first longitudinal axis, particularly from above, particularly when the specimen holder is received by the sample stage, wherein the first imaging system is configured to obtain a full focus image, particularly for obtaining a height profile, of the sample in the direction of the first longitudinal axis, particularly with a resolution of 4 pm or less along the first longitudinal axis.

[0038] In certain embodiments, the first imaging system is configured to obtain a deep focusing image or a z-stack. In certain embodiments, the first imaging system comprises a microscope and a camera, wherein particularly the first imaging system is configured to obtain the height profile with a resolution of 1 pm or less along the second and / or the third longitudinal axis.

[0039] In certain embodiments, the atomic force microscope comprises a second imaging system for imaging the sample, particularly the sample held by the specimen holder, along the first longitudinal axis, particularly from above, particularly when the specimen holder is received by the sample stage, wherein the second imaging system is configured to obtain an overview image of the complete sample.

[0040] In certain embodiments, the atomic force microscope comprises a third imaging system for imaging the cantilever, particularly the tip of the cantilever, along the first longitudinal axis, particularly from below, wherein particularly the third imaging system is configured to obtain a full focus image of the cantilever.

[0041] In certain embodiments, the third imaging system comprises a fixed orientation with respect to the second imaging system, such that a position of the cantilever on the sample can be determined from an image obtained by the second imaging system.

[0042] In particular, the first imaging system, the second imaging system and / or the third imaging system are fixed to the mounting bracket, such that the first, the second and / or the third imaging system comprise a defined position relative to the cantilever or head.

[0043] In certain embodiments, the atomic force microscope comprises a vibration sensor.

[0044] In particular, the atomic force microscope rests on a vibration compensation table comprising the vibration sensor configured to sense vibrations and an actuator configured to compensate the sensed vibrations.

[0045] In certain embodiments, the atomic force microscope comprises a control device configured to control the actuator and the at least one linear drive, such that the cantilever and the sample stage are moved relative to each other along the first longitudinal axis, particularly such that the tip of the cantilever contacts the sample, wherein particularly the control device is configured to receive the deflection signal from the detector.

[0046] In certain embodiments, the control device comprises a plurality of circuit boards arranged in a dice-like fashion around at least a part of the atomic force microscope, particularly the head.

[0047] A second aspect of the invention relates to a method for obtaining a stiffness value of a sample, particularly a sample from a soft material, more particularly a biological tissue specimen, using the atomic force microscope according to the first aspect of the invention, comprising the following steps: contacting the sample by the tip of the cantilever at a measurement spot, moving the cantilever and the sample stage towards each other by the actuator parallel to the first longitudinal axis in a loading step, particularly automatically, such that the tip of the cantilever indents into the sample, moving the cantilever and the sample stage away from each other by the actuator parallel to the first longitudinal axis in an unloading step, particularly automatically, during the loading step and / or the unloading step, determining a plurality of deflection values of the cantilever from the deflection signal of the detector, determining a stiffness value of the sample at the measurement spot from the determined deflection values, particularly automatically.

[0048] In certain embodiments, the method further comprises determining a plurality of force values indicating the force of the cantilever onto the sample at a corresponding position along the first longitudinal axis, wherein the stiffness value of the sample is determined, particularly automatically, from at least a subset of the force values, wherein particularly a force-position relationship and / or a force-position curve is obtained.

[0049] In certain embodiments, prior to contacting the sample by the tip of the cantilever, a resonance frequency of the cantilever is determined, wherein a spring constant of the cantilever is determined, particularly automatically, from the resonance frequency.

[0050] In certain embodiments, an image of the cantilever is obtained by the third imaging system, and wherein at least one dimension of the cantilever, particularly a length of the cantilever along the second longitudinal axis or the third longitudinal axis and / or a width of the cantilever perpendicular to the length, are determined from the image, and wherein the spring constant of the cantilever is determined based on the determined at least one dimension and the resonance frequency, wherein particularly a temperature value is obtained by the temperature sensor, wherein the spring constant of the cantilever is determined, particularly automatically, based on the determined at least one dimension, the resonance frequency and the temperature value.

[0051] In certain embodiments, prior to contacting the sample by the tip of the cantilever, a reference sample having a higher stiffness compared to the sample is contacted by the tip of the cantilever, and the cantilever is moved relative to the sample stage by the actuator over a moving distance while the tip contacts the reference sample without indentation of the reference sample, and wherein the deflection signal of the detector is obtained over the moving distance, and wherein a deflection sensitivity of the detector is determined, particularly automatically, from the moving distance and the deflection signal.

[0052] In certain embodiments, at least one image, particularly a full focus image, of the sample is obtained by the first imaging system and a surface map of the sample is determined, particularly automatically, from the at least one image.

[0053] In certain embodiments, the surface map is analyzed, and wherein at least one measurement region of the sample is selected based on the analysis, wherein the measurement region comprises a plurality of measurement spots, and wherein stiffness values are obtained, particularly automatically, at the measurement spots of the measurement region.

[0054] In certain embodiments, at least one obstacle is identified from the surface map or the overview image of the sample, and wherein the cantilever is moved away from the sample, particularly automatically, along the first longitudinal axis to avoid collision of the cantilever with the obstacle.

[0055] In certain embodiments, at least one image of the sample and the cantilever is obtained by the third imaging system, and wherein a position of the tip of the cantilever relative to the sample is determined, particularly automatically, based on the image.

[0056] In certain embodiments, the sample stage is repeatedly moved in opposite directions along the third longitudinal axis by means of the third linear drive, particularly automatically, a plurality of images of the sample are sequentially obtained by the second imaging system while the sample stage is being repeatedly moved by the third linear drive, and it is determined from the plurality of images, particularly automatically, whether the position of the sample on the sample stage, particularly on the specimen holder, has remained constant during the movement of the sample stage.

[0057] In certain embodiments, a vibration signal is obtained from the vibration sensor, and wherein the cantilever is moved away from the sample along the first longitudinal axis, particularly automatically, in case the signal indicates the presence of vibrations, particularly having an amplitude or a frequency above a predefined threshold.

[0058] In the present application, the first longitudinal direction is also referred to as z-direction.

[0059] In the present application, the second longitudinal direction is also referred to as x-direction.

[0060] In the present application, the third longitudinal direction is also referred to as y-direction.

[0061] Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

[0062] Exemplary embodiments are described below in conjunction with the Figures. The Figures are appended to the claims and are accompanied by text explaining individual features of the shown embodiments and aspects of the present invention. Each individual feature shown in the Figures and / or mentioned in the text of the Figures may be incorporated (also in an isolated fashion) into a claim relating to the first aspect and / or the second aspect according to the present invention. Fig. 1 shows an embodiment of an atomic force microscope according to the invention, wherein a cantilever holder is arranged on the head of the atomic force microscope;

[0063] Fig. 2 shows a cut view through the head of an atomic force microscope according to an embodiment of the invention;

[0064] Fig. 3 shows an embodiment of an atomic force microscope according to the invention, with a tray for receiving a specimen holder arranged below the head of the atomic force microscope; and

[0065] Figs. 4a, b schematically depict an optical path of light emitted from a light source and reflected from the cantilever and a mirror towards a detector (Fig. 4a), wherein a transparent body is arranged in the cantilever holder shown in Fig. 4b.

[0066] Fig. 1 shows an embodiment of an atomic force microscope 1 according to the invention. The atomic force microscope 1 comprises a head 100 which is movably connected to a mounting 2 (not shown). Particularly, the head 100 is movable with respect to the mounting 2 by an actuator 140 of the atomic force microscope 1. To this end, the head 100 can be connected to the mounting 2 via the actuator 140. By means of the actuator 140, the head 100 can be moved along a first longitudinal axis L1 , which corresponds to the z-axis according to the coordinate system indicated in Fig. 1.

[0067] On top of an upper part 104a of a main body 104 of the head 100, the atomic force microscope 1 comprises a light source 105. The light source 105 is configured to generate light used to optically detect a displacement of a cantilever 202 attached to cantilever chip 201 of a cantilever chip holder 310 arranged at a receiving unit 106 of the atomic force microscope 1. Alternatively, the cantilever 200 can be attached to a cantilever holder. The main body 104 frames a mirror configured to receive light emitted from the light source and reflected from the cantilever as well as a detector configured to detect a position of a light beam reflected from the mirror toward the detector, as explained further in Fig. 2.

[0068] The light source 105 may comprise focusing and collimating optics, such as lenses, and is connected to the main body 104 via a cross-table 110 comprising a first carriage 110a, a second carriage 110b and a third carriage 110c.

[0069] The first carriage 110a of the cross-table 110 is movable in the y-direction according to the coordinate system indicated in Fig.1 , particularly with a resolution of about 10 pm, by a first light source linear drive 111a comprising a piezo electric drive, particularly a piezo LEGS or Piezo Friction drive or piezo stick slip motor.

[0070] The second carriage 110b of the cross-table 110 is movable in the x-direction according to the coordinate system indicated in Fig. 1 , particularly with a resolution of about 10 pm, by a second light source linear drive 111 b comprising a piezo drive, particularly a piezo LEGS or Piezo Friction drive or piezo stick slip motor or a piezo stick slip motor.

[0071] The third carriage 110c of the cross-table is movable in the z-direction according to the coordinate system indicated in Fig. 1 , particularly with a resolution of about 10 pm, by a second light source linear drive comprising a piezo drivel 11c, particularly a piezo LEGS or Piezo Friction drive or piezo stick slip motor or a piezo stick slip motor.

[0072] By moving the light source 105 with respect to the head 100, particularly with respect to the cantilever 202, by means of said cross-table 110 and its carriages 110a, 110b, 110c, a spot position of a first light beam B1 emitted from the light source 105 along the first longitudinal axis L1 toward the cantilever 202 can be aligned with and focused onto the cantilever 202, which allows for a strong signal indicative of the deflection of the cantilever 202 during a measurement and thus increases the measurement accuracy of the atomic force microscope 1.

[0073] Fig. 2 shows a cut view through the head 100 of an atomic force microscope 1 according to an embodiment of the invention. The cut view highlights components arranged inside the head 100, such as light and focusing optics 105a for focusing light onto the cantilever 202. From the cantilever 202, light can be reflected towards a mirror 112 mounted on a mirror frame 114 that is configured to reflect the light towards a detector 113, where the reflected light indicative for the deflection of the cantilever 202 is detected. The detector 113 in this embodiment is a 4- quadrant photodiode arranged on a detector holder 116. The head 100 comprises a detector linear drive 116a, particularly a piezo-electric motor, configured to move the detector holder 116 with the detector 113 along the x-direction, perpendicularly to the z- and the y-direction indicated in Fig. 2. To this end, the detector linear drive 116a is connected to the detector holder 116 via a connecting rod extending 1 in the z-direction. A translation movement of the connecting rod 117 caused by the detector linear drive 116 can thereby be transferred onto the detector holder 116. The detector holder 116 can be tilted by three set-screws 116b for adjusting the orientation of the detector holder 116 with respect to the incoming light beam reflected from the mirror 112. A magnet-based position sensor 126a is arranged on the photodetector holder 116, such that the position of the photodetector holder 116 with the photodetector 113 can be determined. The mirror frame 114 is rotatable around an axis 114a extending in the x-direction, perpendicularly to the y- and the z-direction according to the coordinate system indicated in Fig. 2. A mirror drive 114b, particularly a piezo-electric motor, forms a clamping drive 1222 configured to move a means 1224 along the first longitudinal axis L1. By moving the means 1224, clamping elements 122 for clamping the cantilever chip 310 to the head 100 of the atomic force microscope 1 can be transferred between a receiving position in which the cantilever chip 310 can be arranged in the receiving unit 106 and a clamping position in which the cantilever chip 310 is secured at the receiving unit 106 by the clamping elements 122.

[0074] The axis 114a of the mirror frame 114 is mechanically connected to a connecting lever 121 , which in turn is mechanically connected to a push bar 119. The connecting lever 121 comprises a slot 121a and the push bar comprises a pin 120 arranged in the slot. As such, when the mirror drive 114b moves said means 1224 along the z-direction, the pin 120 moves in the slot 121 under a pivot movement of the mirror 112 on the mirror frame 114. The mirror drive 114b is thus configured to both effect the engagement of the clamping elements 122 as well as the pivoting of the mirror 112.

[0075] Fig. 3 shows an embodiment of an atomic force microscope 1 according to the invention, with a tray 300 for receiving a specimen holder 430 arranged below the head 100 of the atomic force microscope 1.

[0076] The head 100 is schematically depicted arranged on a first linear drive 101 for linear movement of the head 100 relative to the tray 300 along the vertical z-direction. The first linear drive 101 comprises a coarse drive 101a for coarse movement of the head 100 along the z-direction and a fine drive 101b for fine movement of the head 100 along the z-direction. The coarse drive 101a can comprise or consist of a voice coil motor. The fine drive 101 b can comprise or consist of a piezo motor.

[0077] The atomic force microscope according to the present embodiment comprises a constant force magnetic springs 130 which exerts an upward force along the z-direction, i.e. away from the tray 300, on the head 100 that counteracts the weight of the head 100. The magnetic spring 130 is configured such that the head 100 moves upward along the z-direction when the first linear drive 101 , particularly the coarse drive 101a and / or the fine drive 101 b of the first linear drive 101 , is de-energized to move the cantilever 202 away from the sample. This represents a safety measure as it can prevent a collision of the cantilever 202 with the sample in case the first linear drive 101 is de-energized.

[0078] The first linear drive 101 is arranged between the head 100 and a second linear drive 102 for linear movement of the head 100 relative to the tray 300 along the x-direction, perpendicular to the y- and the z-direction. The second linear drive 102 comprises a coarse drive 102a for coarse movement of the head 100 along the x-direction and a fine drive 102b for fine movement of the head 100 along the x-direction. The coarse drive 102a can comprise or consist of a voice coil motor. The fine drive 102b can comprise or consist of a piezo motor.

[0079] As such, the head 100 can be moved with respect to the tray 300 along two orthogonal directions by means of the first and second linear drive 101 ,102.

[0080] Moreover, the atomic force microscope 1 according to the present embodiment comprises a third linear drive 103 for linear movement of the tray 300 relative to the head 100 along the y- direction. The third linear drive 103 comprises a coarse drive 103a for coarse movement of the tray 300 along the y-direction and a fine drive 103b for fine movement of the tray 300 along the y-direction. The coarse drive 103a can comprise or consist of a voice coil motor. The fine drive 103b can comprise or consist of a piezo motor.

[0081] Thus, the head 100 and the tray 300 can be moved with respect to each other along three orthogonal directions using the first, second and third linear drive 101 ,102,103, which allows for precise and flexible positioning of the sample with respect to the cantilever 202 arranged on the head 100.

[0082] The atomic force microscope 1 according to the present embodiment further comprises a first imaging system 450 arranged on the first linear drive 101. The first imaging system 450 is configured to image a sample arranged on the specimen holder 430 along the z-direction from above. The first imaging system 450 is in particular configured to obtain a depth map, particularly a 3D reconstruction or topography deep focus image, particularly a true color image with all pixels in focus, of the sample. For example, a z-resolution, i.e. the optical resolution in the z-direction, can be about 4 pm and an xy-resolution, i.e. an optical resolution along the x- and the y-direction, can be about 1 pm.

[0083] As can further be seen in Fig. 3, a second imaging system 460 is arranged on the head 100. The second imaging system 460 is configured to capture an overview image of a sample arranged in the specimen holder 430. Moreover, the first imaging system 450 can be arranged and configured to read a barcode on the specimen holder, and / or on the cantilever holder, particularly the cantilever chip holder along the z-direction from above, i.e. from the perspective of the head 100.

[0084] Fig. 3 also shows two shuttle units 650 arranged on the tray 300. The shuttle units 650 can be transferred between the tray 300 and a drawer unit 620 The shuttle units 650 are configured to receive said specimen holder 430 with a sample and / or a cantilever holder, particularly a cantilever chip holder 310. A third imaging system 440 is arranged on the tray 300. The third imaging system 440 is an optical microscope, for example a bottom light microscope, configured to image the cantilever 202 from below, i.e. from the perspective of the tray 300. The third imaging system 440 can be configured to determine the dimensions of the cantilever 202, particularly a length, a width and / or a height of the cantilever 202, in order to determine a spring constant of the cantilever 202. Additionally, the position of the tip of the cantilever 202 can be determined by means of the third imaging system 440. As such, the third imaging system 440 can be used for automated alignment of the light beam on the tip of the cantilever 202 in x-, y- and z-direction. The third imaging system 440 can be configured to obtain a depth map and / or deep focus image of the cantilever 202 to automatically determine the location of the tip in x-, y- and z- direction.

[0085] The atomic force microscope 1 according to the present embodiment also comprises a controller 410. The controller 410 can be made from a SoM (system on module) consisting of an FPGA (field programmable gate array) for calculations, particularly for calculations on a timescale of about 5 ns, a Real Time Linux running on ARM processors for calculations, particularly for calculations on a timescale of about 50 ps, and a device controller, particularly a Linux operating system for device automation and / or user input.

[0086] Inputs to the controller 410 can be from and / or outputs from the controller 410 can be to one or more of the following: the detector 113, particularly via a pre-amplifier and an analog-digital- converter, the first, second and third imaging system 450,460,440, the magnetic positioning sensor 126a, an environment temperature sensor, an ambient air pressure sensor, an ambient air humidity sensor, voltage sensors, a sample temperature sensor, a vibration compensation table on which the atomic force microscope 1 is arranged, the first, second and third linear drive 101 ,102,103, particularly their coarse drives 101 a, 102a, 103a and / or their fine drives 101 b, 102b, 103c, a position controller for operating the first linear drive, second and / or third linear drive 101 ,102,103, the detector linear drive 116a, the mirror drive 114b, for pivoting the mirror 112 and for engaging the clamping elements 122, a light source controller for controlling the light source 105, a SLED controller.

[0087] As can further be seen in Fig. 3, an optical target 402 is arranged on the tray 300 below the head 100. The atomic force microscope 1 is configured to automatically determine and calibrate the position of the head 100 in x-direction and in y-direction when no cantilever holder, particularly cantilever chip holder 310 is mounted on the head 100. For the determination and calibration, the light beam emitted from the light source 105 passes through the light and focusing optics 105a of the head 100, and hits the optical target 402. The light reflected from the optical target 402 goes back towards the light and focusing optics 105a, which has a beam splitter connected to an optical fiber that is configured to calibrate the position of the head 100 with respect to the tray 300 along the x- and the y-direction based on the light reflected from the optical target 402.

[0088] Fig. 4a schematically depicts an optical path of light emitted from said light source 105 and detected by said detector 113. The light is emitted from the light source 105 as a first light beam B1 towards the cantilever 202 held by the cantilever holder 310. From the cantilever 202, particularly from a top surface of the cantilever 202 facing the light source 105, the light is reflected as a second light beam B2 towards the mirror 112, which can be rotated by means of said mirror drive 114b, cf. Fig. 2. From the mirror 112, the light is finally reflected as a third light beam B3 towards the detector 113, which may be a 4-quadrant photodiode. The optical path can be changed by moving the light source 105 with respect to the cantilever 202 using the cross table 110 with its first, second and third light source linear drives 111 a, 111b, 111c along the x-y- and the z-direction, respectively. Additionally, the photodetector 113 can be moved along the x-direction, i.e., outside the drawing plane shown in Fig. 4a, by means of said detector linear drive 116a. The deflection of the cantilever 202 is indicative of the distance between the tip 203 of the cantilever and the sample 3 arranged on the sample stage 300.

[0089] Fig. 4a also schematically indicates a first linear drive 101 with a coarse linear motor 101a and a fine linear motor 101 b for moving the cantilever 202 held by the cantilever holder 310 along the first longitudinal axis L1 as well as a second linear drive 102 with a coarse linear motor 102a and a fine linear motor 102b for moving the cantilever 202 held by the cantilever holder 310 along the second longitudinal axis L2. Additionally, the sample stage 300 can be moved by a third linear drive 103 comprising a coarse linear motor 103a and a fine linear motor 103b along the third longitudinal axis L3.

[0090] Fig. 4b shows an embodiment, wherein the cantilever holder 310 is configured to receive a transparent body 320. The transparent body 320 is formed from an optically clear material, such as a glass material. In particular, the material of the transparent body comprises a refractive index which is greater than the refractive index of air. In certain embodiments, the material of the transparent body comprises a refractive index which is greater than the refractive index of the material of the holder. For example, the transparent body comprises a refractive index of 1.6 to 2.2, more particularly about 1.9.

[0091] In case a transparent body 320 is provided, the light beam used to determine the deflection of the free end of the cantilever 202 travels through the transparent body 320 and onto the cantilever 202 and the light beam reflected from the cantilever 202 travels towards the mirror 112 through the transparent body 320. At the interfaces of the transparent body 320, the light is refracted. The transparent body 320 improves the stability of an assembly formed by the transparent body 320 and the cantilever holder 310, such that the cantilever holder 310 can be made longer in the z-direction, which simplifies the handling of the cantilever holder 310. In addition, in case the material of the transparent body comprises a higher refractive index than air, this allows the optics of the atomic force microscope 1 to be built more compactly, since the refraction of the light beam at the transparent body 320 allows to refract the light beam towards the mirror 112, whereas in the case without the transparent body 320 the light has to be guided from the cantilever 202 to the mirror entirely due to the reflection of the cantilever 202, which requires a rather bulky shape of the cantilever holder 310, as can be understood from comparing the cantilever holders 310 of Fig. 4a and 4b.

Claims

Claims1. An atomic force microscope (1) for obtaining a stiffness value of a sample (3) comprising a. a cantilever (202) comprising a tip (203) for contacting a sample (3) of soft material, b. a light source (105) for generating light and focusing optics (105a) configured to focus the light and direct a first light beam (B1) along a first longitudinal axis (L1) onto the cantilever (202), c. a mirror (112) configured to receive a second light beam (B2) reflected by the cantilever (202), d. a detector (113) configured to detect a position of a third light (B3) beam reflected by the mirror (112) and generate a deflection signal indicating a deflection of the cantilever (202) along the first longitudinal axis (L1) based on the detected position of the third light beam (B3), e. a sample stage (300) for receiving the sample (3), f. an actuator (140) configured to move the cantilever (202) relative to the sample stage (300) along the first longitudinal axis (L1), wherein the atomic force microscope (1) comprises at least one linear drive (101 , 102, 103) configured to position the cantilever (202) relative to the sample stage (300) along the first longitudinal axis (L1) and / or along a second longitudinal axis (L2) which is perpendicular to the first longitudinal axis (L1), and / or along a third longitudinal axis (L3) which is perpendicular to the first longitudinal axis (L1) and the second longitudinal axis (L2), wherein the at least one linear drive (101 , 102, 103) comprises 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 (101 b, 102b, 103b) for fine 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) comprises a head (100), particularly comprising the cantilever (202), the light source (105), the mirror (112), the detector (113) and / or the actuator (140), wherein the head (100) is connected to a mounting bracket (2) via the at least one linear drive (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 (101 , 102, 103) connects the actuator (140) and the head (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 one of the preceding claims, wherein the fine linear motor (101 b, 102b, 103b) is a piezoelectric stick-slip motor.

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

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

7. The atomic force microscope (1) according to one of the preceding claims, wherein the coarse linear motor (101a, 102a, 103a) is configured to be inactivated, particularly deenergized, when the cantilever (202) is moved relative to the sample stage (300) by the fine linear motor (101 b, 102b, 103b), particularly wherein the coarse linear motor (101a, 102a, 103a) and the fine linear motor (101 b, 102b, 103b) are coupled by the coupling element.

8. The atomic force microscope (1) according to one of the preceding claims, wherein the atomic force microscope comprises a first linear drive (101) configured to position the cantilever (202) relative to the sample stage (300) along the first longitudinal axis (L1), wherein the first linear drive (101) comprises a first coarse linear motor (101a), particularly a voice coil motor, for coarse positioning of 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 fine positioning of the cantilever (202) relative to the sample stage (300) along the first longitudinal axis (L1).

9. The atomic force microscope (1) according to one of the preceding claims, wherein the atomic force microscope (1) comprises a second linear drive (102) configured toposition the cantilever (202) relative to the sample stage (300) along a second longitudinal axis (L2) extending perpendicular to the first longitudinal axis (L1), wherein the second linear drive (102) comprises a second coarse linear motor (102a), particularly a voice coil motor, for coarse positioning of 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 fine positioning of the cantilever (202) relative to the sample stage (300) along the second longitudinal axis (L2).

10. The atomic force microscope (1) according to one 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 particularly the third linear drive (103) comprises a third coarse linear motor (103a), particularly a voice coil motor, for coarse positioning of the sample stage (300) along the third longitudinal axis (L3) and a third fine linear motor, particularly comprising a piezoelectric stick-slip motor, for fine positioning of the sample stage (300) along the third longitudinal axis (L3).

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

12. A method for obtaining a stiffness value of a sample (3), particularly a sample (3) from a soft material, more particularly a biological tissue specimen, using the atomic force microscope (1) according to one of the claims 1 to 11 , wherein the method comprises the following steps: a. contacting the sample (3) by the tip (203) of the cantilever (202) at a measurement spot, b. moving the cantilever (202) and the sample stage (300) towards each other by the actuator (140) parallel to the first longitudinal axis (L1) in a loading step, particularly automatically, such that the tip (203) of the cantilever (202) indents into the sample (3),c. moving the cantilever (202) and the sample stage (300) away from each other by the actuator (140) parallel to the first longitudinal axis (L1) in an unloading step, particularly automatically, d. during the loading step and / or the unloading step, determining a plurality of deflection values of the cantilever (202) from the deflection signal of the detector (113), e. determining a stiffness value of the sample (3) at the measurement spot from the determined deflection values, particularly automatically.

13. The method according to claim 12, wherein the method further comprises determining a plurality of force values indicating the force of the cantilever (202) onto the sample (3) at a corresponding position along the first longitudinal axis (L1), wherein the stiffness value of the sample (3) is determined, particularly automatically, from at least a subset of the force values, wherein particularly a force-position relationship and / or a force-position curve is obtained.

14. The method according to claim 12 or 13, wherein prior to contacting the sample (3) by the tip (203) of the cantilever (203), a resonance frequency of the cantilever (202) is determined, wherein a spring constant of the cantilever (202) is determined, particularly automatically, from the resonance frequency.

15. The method according to one of the claims 12 to 14, wherein prior to contacting the sample (3) by the tip (203) of the cantilever (202), a reference sample having a higher stiffness compared to the sample (3) is contacted by the tip (203) of the cantilever (202), and the cantilever (202) is moved relative to the sample stage (300) by the actuator (140) over a moving distance while the tip (203) contacts the reference sample without indentation of the reference sample, and wherein the deflection signal of the detector (113) is obtained over the moving distance, and wherein a deflection sensitivity of the detector is determined, particularly automatically, from the moving distance and the deflection signal.

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