High-speed atomic force microscope, preferably for dermatological measurements

Abstract

The invention relates to a high-speed atomic force microscope (AFM, 1) for measuring a relevant sample (S), such as a skin sample, the AFM having a sensor unit (SU, 10) comprising a light source (11) and a collimating lens (12), the collimating lens being mechanically displaced by a collimating lens actuator (12 '). An AFM cantilever having an AFM tip (14) is arranged relative to the photodetection beam (15) for providing a focus error signal (FES) indicative of mechanical engagement with the sample. The collimating lens (12) is arranged to be adjusted by a collimating lens actuator (12 ') along the beam path to compensate for at least thermal drift in the focus error signal (FES). According to the invention, the improved high-speed AFM can be obtained through the internal thermal drift compensation mechanism. The high-speed AFM of the embodiment of the invention can have a theoretical linear scanning speed which can reach 700000 um / s, which allows one-time imaging to be completed within about 3.4 seconds.

Description

Technical Field

[0001] This invention relates to a high-speed atomic force microscope (AFM) preferably used for dermatological measurements, and particularly to an AFM capable of non-invasively measuring skin morphology (e.g., human skin barrier dysfunction). The invention also relates to corresponding methods and computer programs for carrying out the invention. Background Technology

[0002] The integrity of the skin barrier is crucial for healthy skin; dysfunction of this barrier can lead to atopic dermatitis (AD), affecting over 790 million people. A durable, painless, non-invasive laboratory test, unlike biopsies, primarily for macroscopic detection, is preferred for AD assessment. Atomic force microscopy (AFM) is a powerful tool for studying nanomaterials and surfaces. This technique uses a tip (tip radius <8 nm) attached to a cantilever to probe the surface of various sample types, including biological specimens, polymers, and inorganic materials, and measures the force between the tip and the sample, thereby generating high-resolution images of the sample surface at the nanoscale or even atomic level.

[0003] Exfoliated keratinocytes can be imaged using amphoteric radiography (AFM). In recent years, AFM has been used to explore nanoscale anatomy, resulting in nanoobjects that serve as biomarkers. AFM is a suitable tool for non-invasively detecting any changes in the morphology of keratinocytes in both diseased and non-diseased skin. Furthermore, the nanoscale texture of keratinocytes in their natural state can be imaged without the vacuum environment or sample preparation required for scanning electron microscopy. However, traditional AFM is unsuitable for clinical testing due to its low throughput (5-10 minutes per image) and high cost, making it unsuitable for on-site skin measurements.

[0004] High-speed atomic force microscopy (HS-AFM) was developed to overcome these limitations and improve AFM imaging speed. HS-AFM can be achieved by increasing scanning speed, reducing cantilever beam damping, and increasing the cantilever beam resonant frequency. HS-AFM has been successfully applied to a wide range of materials and surfaces, including dynamic imaging of live cells, high-speed imaging of soft materials, and in-situ imaging of reaction processes. One of the key factors in improving AFM imaging speed is the development of faster scanning systems. Early AFM systems used slow piezoelectric scanning mechanisms, which limited imaging speed. In recent years, the development of high-speed piezoelectric scanners and resonant scanners has greatly improved AFM imaging speed. For example, high-speed piezoelectric scanners use high-frequency piezoelectric actuators to rapidly scan the tip of a sample surface.

[0005] Besides improving the scanning mechanism, reducing cantilever beam damping and increasing the cantilever beam resonant frequency are also important for achieving HS-AFM. Cantilever damping can be reduced by using a high-quality cantilever with a low-damping material (e.g., silicon nitride). The cantilever resonant frequency can be increased by using a smaller cantilever size or by using a material with a higher Young's modulus (e.g., silicon). Another crucial factor in achieving HS-AFM is the development of dedicated software for high-speed imaging. This software must be able to handle the large amounts of data generated by high-speed scanning and must be able to accurately track the tip's position in real time.

[0006] One of the key features of HS-AFM is its ability to perform dynamic imaging at high speeds. This allows for imaging of rapid processes, such as cell motility, with high temporal resolution. Furthermore, HS-AFM can be used to image soft materials such as polymers and gels, whose viscoelastic properties can be challenging for conventional AFM. Several imaging modes are commonly used in HS-AFM, including contact mode, intermittent contact mode, and tapping mode. In contact mode, the tip contacts the sample surface, and the deflection of the cantilever is measured as the tip scans across the surface.

[0007] However, traditional AFM has limitations in terms of speed, cost, and applicability to on-site skin measurements, making it unsuitable for clinical testing.

[0008] One technical problem is the drift of AFM components during measurement, especially thermal drift. In previous work, see E.-T. Hwu et al.'s Review of Scientific Instruments 83, 013703 (2012), due to signal drift issues in conventional DVD optical heads, anti-drift and auto-alignment mechanisms were applied to atomic force microscopy (AFM) based on an astigmatic detection system (ADS) for drift compensation and cantilever alignment. In previous settings, signal drift of several thousand nanometers was even measured. Utilizing the anti-drift and auto-alignment mechanism achievements since 2012, near-zero signal drift was achieved by activating the voice coil motor of the DVD optical head to compensate for signal drift.

[0009] WO2018109803 (Olympus Corp.) relates to an atomic force microscope equipped with an XY movable stage; an XY scanner for moving the XY movable stage in an XY scanning motion in a plane parallel to the XY plane; a Z scanner having a fixed end held by the XY movable stage, a free end holding a cantilever, and moving the cantilever in a Z scanning motion along a Z-axis perpendicular to the XY plane; and an optical displacement sensor for optically detecting the cantilever displacement. The optical displacement sensor includes a converging lens held by the XY movable stage. The converging lens converges the incident detection light, generating detection light with a focused spot, and irradiates the cantilever. The atomic force microscope also includes a Z-scan tracking system for suppressing movement of the irradiated area along the cantilever caused by the Z-scan. The optical detection is based on a split detector with a so-called position sensitive detector (PSD) (see [link to documentation]). Figure 1 (Figures 12 and 22) This dual-splitter detector can track changes in the position of the optical reflection spot on the cantilever, but it cannot effectively track any thermal drift in the AFM setup.

[0010] Other research groups have attempted to perform high-speed AFM in complex measurement settings with long waiting times for thermal equilibrium (e.g., several hours) without compensating for thermal results. Therefore, a high-speed AFM with effective thermal drift compensation is needed.

[0011] Therefore, improved AFM would be advantageous, especially a more efficient and / or more reliable high-speed AFM for skin measurements.

[0012] Purpose of the invention

[0013] A further object of the present invention is to provide an alternative to the prior art.

[0014] In particular, it can be seen that the object of the present invention is to provide a high-speed AFM that solves the above-mentioned problems of the prior art by means of thermal drift compensation and / or by performing dermatological measurements in an efficient manner. Summary of the Invention

[0015] Therefore, in a first aspect of the invention, the above-mentioned objective and several other objectives are achieved by providing a high-speed atomic force microscope (AFM) for measuring relevant samples, the AFM comprising: Sensor unit, including A light source that can emit a beam of light. A collimating lens, arranged to receive and collimate the light beam in the optical path toward the relevant sample, is mechanically displaced by a corresponding collimating lens actuator. An objective lens that receives the light beam and focuses it onto an optical path toward the sample; the objective lens is mechanically displaced by a corresponding objective lens actuator. An AFM cantilever has an AFM tip arranged for mechanical engagement with a relevant sample, and the rear side of the AFM cantilever is arranged to optically reflect the light beam received from the objective lens in response to the mechanical engagement. A photoelectric detection beam is used to detect the reflected light from the AFM cantilever and provide a focus error signal (FES) indicating the mechanical engagement. A top scanning actuator, preferably a voice coil motor, is used for at least one-dimensional displacement of the sensor unit (SU) in the scanning direction (X) and displacement in the approach direction (Z) toward the relevant sample, and A sample stage is used to fix the relevant samples. The top scanning actuator and the sample stage are arranged to perform at least two-dimensional (XY) scanning of the sample surface using a sensor unit, wherein the collimating lens is arranged to be adjusted by the collimating lens actuator along the beam path in order to at least compensate for thermal drift in the focus error signal (FES).

[0016] This invention is particularly advantageous, but not uniquely advantageous, in achieving a more efficient and reliable high-speed AFM by providing a novel and advantageous internal thermal drift compensation mechanism. One embodiment of the high-speed AFM of this invention has a diameter of 1.4 × 1.4 × 3.8 mm. 3 Its full working range and theoretical linear scan speed of up to 700,000 µm / s allow a single imaging session to be completed in approximately 3.4 seconds.

[0017] For dermatological measurements, the atomic force microscope of this invention can measure skin morphology by imaging, for example, the nanotexture of skin keratinocytes in a constant-height scanning mode within seconds. Therefore, high-speed AFM may become the system recommended by the Atopic Dermatitis Care Quality of Care (ADQoC) initiative for the assessment of skin diseases (e.g., AD), prevention risk, and treatment effectiveness.

[0018] definition:

[0019] In the context of this invention, it should be understood that the term atomic force microscopy (AFM) is broadly interpreted to include various scanning probe microscopy (SPM) techniques with resolutions on the nanometer scale and fractions of those required. AFM was originally developed by Binnig, Quate, and Gerber in 1986, and it is assumed that those skilled in the art are familiar with the basic concepts and principles of AFM. For example, those skilled in the art may refer to NC Santos and FA Carvalho, "Atomic Force Microscopy - Methods and Protocols." pp. 1–369, 2019, the entire contents of which are incorporated herein by reference. Furthermore, the entire contents of the following three references are incorporated herein by reference: Ando, ​​T. and Uchihashi, T. (2015). High-speed atomic force microscopycoming of age. Nanotechnology, 26(43), 434001. DOI: 10.1088 / 0957-4484 / 26 / 43 / 434001, and Eghiaian, F., Rico, F., Colom, A., Casuso, I., & Scheuring, S. (2014). High-speed atomic force microscopy: Imaging and force spectroscopy. FEBSletters, 588(19), 3631-3638. (https: / / doi.org / 10.1016 / j.febslet.2014.06.028), and Ando, ​​T., Uchihashi, T., Kodera, N., Yamamoto, D., Taniguchi, M., Miyagi, A., & Yamashita, H. (2007). High-speed atomic force microscopy forobserving dynamic biomolecular processes. Journal of Molecular Recognition, 20(6), 448-458. (https: / / doi.org / 10.1002 / jmr.843), Furthermore, those skilled in the art can find more information about the principles of AFM, particularly information on the use of high-speed AFM for imaging biological materials and surfaces.

[0020] In the context of this invention, it should be understood that the term displacement is considered to include the meaning of relative displacement, that is, the first object can be displaced relative to the second object, which means that the first object can move and the second object can remain stationary, or vice versa, or both the first and second objects can move relative to each other.

[0021] In the context of this invention, it should be understood that thermal drift is caused by small thermal changes and / or fluctuations between the components of an AFM system and its surrounding environment. Therefore, thermal drift also includes changes in the characteristics, parameters, and / or operation of electronic and / or optical components used in the AFM system due to thermal differences and fluctuations. For example, aluminum has a linear coefficient of thermal expansion of 24 x 10⁻⁶. -6 The coefficient of linear thermal expansion (CTE) is K / m, so 1 cm of aluminum will expand by 240 nm when the temperature increases by 1 K. However, the CTE of some typical plastic materials is 10 times greater. Some internal heating sources in a typical AFM setup may include: laser diodes, sensors, photoelectric sensors, etc. Some external heating sources in a typical AFM setup may include: ambient / circular temperature, air conditions.

[0022] This invention is particularly applicable to skin measurements; therefore, AFM can be used to measure nanotexture patterns on the surface of corneal cells. Using this invention to identify nanoscale features and their association with skin diseases is an ideal choice. The first proven nanoscale feature may be a circular or other shaped nanotexture that is associated with the following non-limiting list of diseases related to skin barrier dysfunction: Atopic dermatitis (AD) as described above. Eczema (atopic dermatitis): This is a chronic inflammatory skin condition that causes redness, itching, and inflammation of the skin. Psoriasis: This is an autoimmune disease that causes skin cells to grow rapidly, resulting in thick, scaly patches on the skin. Acne: This is a common skin condition that causes pimples, blackheads, and whiteheads. Rosacea: This is a chronic inflammatory skin condition that causes redness, flushing, and small, pus-filled bumps on the face. Contact dermatitis: This is a skin condition that causes redness, itching, and inflammation when the skin comes into contact with irritants or allergens. Ichthyosis: This is a genetic disease that causes dry, scaly skin. Xerosis: This is a medical term used to describe dry skin that can cause itching, cracking, and peeling. Seborrheic dermatitis: This is a common skin condition that causes redness, scaly patches, and dandruff. Cutaneous lymphoma: This is a rare skin cancer that affects immune cells in the skin.

[0023] This invention is expected to enable large-scale data collection of skin samples with various medical conditions / diseases (i.e., one or more of the aforementioned medical conditions), and based on the data collection of characteristic textures and morphologies of these medical conditions / diseases, the invention can be applied to texture databases of these medical conditions / diseases. The vast amount of data in nanotexture databases can help associate skin medical conditions / diseases with nanotextures of different sizes / shapes / features, for example, C. Riethmüller, “Assessing the skin barrier via corneocyte morphometry,” Exp. Dermatol., vol. 27, no. 8, pp. 923–930, 2018, doi: 10.1111 / exd.13741. Furthermore, it is conceivable that artificial intelligence or machine learning can be applied to characterize texture databases and associate them with relevant medical conditions / diseases.

[0024] While this invention has been used to implement AFM contact mode imaging, those skilled in the art, upon fully understanding the general principles and teachings of the invention, will readily recognize that various embodiments and details of contact mode AFM can be combined with it. Therefore, some non-limiting contact AFM mode variations and embodiments may include: High resolution: Contact mode provides high-resolution images of the sample surface with high vertical and lateral resolution. High force sensitivity: The AFM tip applies a force to the sample surface, and the resulting cantilever deflection is used to determine the surface topography. The force sensitivity of the AFM tip is typically in the piconewton range to allow the measurement of very small forces. High wear: Contact mode AFM may cause wear and damage to both the AFM probe and the sample surface due to the direct contact between the AFM probe and the sample surface. Slow scan: Due to the large forces applied to the sample surface, contact mode AFM requires a slow scan rate. This slow scan rate may limit imaging speed and may also increase the risk of sample damage. Limited for soft materials: Contact mode AFM may not be suitable for imaging soft materials or delicate samples because the large force applied by the AFM probe may deform or damage the sample.

[0025] These are some key characteristics of contact mode AFM. While contact mode AFM can provide high-resolution images, it may not be suitable for all samples or applications. Other AFM modes, such as non-contact mode or tapping mode, can be used to minimize damage to the sample and improve imaging speed.

[0026] Furthermore, it is conceivable that, once the general principles and teachings of the invention are fully understood, those skilled in the art will readily recognize that other embodiments or modes besides the contact AFM can be combined with the invention. According to the invention, other modes besides the contact mode for operating the AFM may include these non-limiting modes: Non-contact mode: In this mode, the AFM tip approaches the sample surface but does not contact it. The tip then oscillates at a resonant frequency, and the amplitude of the oscillation is measured. The amplitude of the oscillation is related to the distance between the tip and the surface and is used to create a topographic map of the sample surface. Tapping Mode: In this mode, the AFM tip oscillates at a frequency slightly below its resonant frequency, gently tapping the sample surface. The amplitude of the tapping motion remains constant by adjusting the feedback signal. This mode causes less damage to the sample than the contact mode and is typically used for imaging soft or delicate samples. Dynamic Force Mode: In this mode, the AFM tip is brought close to the surface and oscillates at a frequency slightly higher than the surface's resonant frequency. The amplitude of the oscillation is measured and used to control the tip-sample distance. This mode is used to study the mechanical properties of materials. Phase imaging mode: In this mode, the phase difference between the tip oscillation and the sample surface is measured. This mode is used for imaging samples with different mechanical and electrical properties. Electrostatic force microscopy (EFM): In this mode, the AFM tip is used to measure the electric field above the sample surface. This mode is used to study the electrical properties of materials.

[0027] In an advantageous embodiment, compensation for thermal drift in at least the focus error signal (FES) can be performed in the control loop mechanism based on the focus error signal and the displacement (preferably linear displacement) of the collimating lens by the collimating lens actuator. As will be explained in the detailed description below, this will provide relatively high-quality high-speed imaging (e.g., a few seconds), effectively allowing for the quality of clinical use of the HS-AFM according to the invention for patient dermatological measurements. As explained in the detailed description below in conjunction with FIG2, in a preferred embodiment, the control loop mechanism may have an optimal sensing point, preferably an optimal sensing region, for the corresponding control loop curve.

[0028] Advantageously, the photodetector includes quadrant detectors arranged to sense a focus error signal (FES), for example, four photodetectors arranged in a 2x2 pattern, each photodetector having substantially the same size, such as a quadrilateral or square, or four detectors together forming a substantially circular detector, each photodetector having substantially the same size and forming approximately 90-degree circular portions of the circular detector. Furthermore, the quadrant detectors can be optically arranged relative to corresponding astigmatic lenses (ASLs) for sensing reflected light from the cantilever, as in typical setups known in optical recording techniques for detecting focus error signals (FES).

[0029] In a preferred embodiment, the AFM can mechanically connect the sample stage to a bottom scanning actuator, more preferably to a voice coil motor, the bottom scanning actuator being further arranged for at least one-dimensional displacement (Y), and optionally also for two-dimensional scanning (XY), to scan the surface of the relevant sample relative to the sensor unit (SU). Preferably, the bottom scanning can also be arranged for displacement between the relevant sample and the sensor unit (SU) in the proximity direction (Z), for example, for coarse displacement in the Z direction.

[0030] Advantageously, the AFM tip can be arranged together with the objective lens in the sensor unit (SU) for cross-correlated sample scanning to achieve a compact design of the AFM.

[0031] Generally, the AFM according to the present invention can be operated in contact mode, non-contact mode, tapping mode, dynamic force mode, phase imaging mode and / or as an electrostatic force microscope (EFM), as this will be easily understood by those skilled in the art of AFM once the general principles and teachings of the present invention are understood.

[0032] In an advantageous embodiment, the operating range of the top scan actuator and the optional bottom scan actuator can achieve scans of at least 0.5 mm, preferably 0.75 mm, and most preferably at least 1.0 mm in one or both directions within the plane in which the AFM scan is performed. These operating ranges advantageously enable dermatological measurements of patient skin samples in a clinical context and environment. Furthermore, these operating ranges advantageously enable additional surface measurements of nanostructures in the height (Z) direction, but at a macroscopic level in the scanning direction (XY).

[0033] Advantageously, the AFM according to the invention allows for linear scan speeds of at least 50,000 micrometers per second, preferably at least 100,000 micrometers per second, and most preferably at least 200,000 micrometers per second. In some embodiments, even very high speeds of 700,000 micrometers per second or higher can be achieved. An important factor is the resonance of the actuator, which must be appropriately controlled. Thus, if scanning at 128 x 128 pixels, 700,000 micrometers per second can be converted to at least 42 frames per second.

[0034] Advantageously, the AFM according to the present invention can be configured for dermatological measurements of relevant skin samples. Therefore, the HS-AFM of the present invention can be used to measure and study a non-limiting list of the various diseases and medical conditions described above.

[0035] In some specific embodiments, the AFM can be arranged to scan a sample surface under liquid conditions, wherein at least a portion of the relevant sample is immersed in the liquid, and wherein the AFM is capable of adjusting the optical path toward the sample focal point by shifting the collimating lens and / or by shifting the objective lens to at least partially compensate for the liquid conditions. Therefore, the collimating lens can compensate for liquid evaporation and / or thermal changes or other liquid changes, such as chemical or biochemical reactions in the liquid, and / or chemical or biochemical reactions between the sample and the liquid.

[0036] In a second aspect, the present invention relates to a method for operating HS-AFM and performing sample (S) measurements, preferably dermatological measurements of skin samples, the method comprising: Provide a sensor unit (SU), which includes: A light source that can emit a beam of light. A collimating lens, arranged to receive and collimate the light beam in the optical path toward the relevant sample, is mechanically displaced by a corresponding collimating lens actuator. An objective lens that receives the light beam and focuses it onto an optical path toward the sample; the objective lens is mechanically displaced by a corresponding objective lens actuator. An AFM cantilever has an AFM tip arranged for mechanical engagement with a relevant sample, and the rear side of the AFM cantilever is arranged to optically reflect the light beam received from the objective lens in response to the mechanical engagement. A photoelectric detection beam is used to detect the reflected light from the AFM cantilever and provide a focus error signal (FES) indicating the mechanical engagement. A top scanning actuator, preferably a voice coil motor, is provided for at least one-dimensional displacement of the sensor unit (SU) in the scanning direction (X) and displacement in the approach direction (Z) toward the relevant sample, and A sample stage is provided for fixing the relevant samples. The top scanning actuator and the sample stage are arranged relative to each other for scanning the surface of the sample (S) in at least two dimensions (XY) using a sensor unit (SU), wherein the collimating lens is adjusted by the collimating lens actuator along the beam path to at least compensate for thermal drift in the focusing error signal (FES).

[0037] This aspect of the invention is particularly advantageous, but not exclusively advantageous, because the invention can provide a method for operation and measurement using a high-speed AFM by providing an advantageous internal thermal drift compensation mechanism.

[0038] In a third aspect, the present invention relates to a computer program product suitable for enabling a computer system comprising at least one computer having a data storage device connected thereto to control an AFM according to the first aspect of the invention, such as a computer program product comprising instructions which, when executed by the computer, cause the computer to perform the method of the second aspect of the invention.

[0039] This aspect of the invention is particularly advantageous, but not exclusively advantageous, because when the computer program product is downloaded or uploaded to a computer system, the computer system can be enabled by the computer program product to perform the operation of the apparatus / system of the first aspect of the invention to realize the invention. Such a computer program product can be provided on any type of computer-readable medium or via a network.

[0040] Each aspect of the invention can be combined with any other aspect. These and other aspects of the invention will become apparent from the following description of the embodiments described herein. Attached Figure Description

[0041] The AFM according to the invention will now be described in more detail with reference to the accompanying drawings. The drawings illustrate one mode of implementing the invention and should not be construed as limiting other possible embodiments falling within the scope of the appended claims.

[0042] Figure 1 A schematic diagram of an embodiment of the AFM according to the present invention is shown.

[0043] Figure 2 shows a graph of the FES of AFM as a function of collimator adjustment.

[0044] Figure 3 Some representative AFM images with and without the present invention are shown.

[0045] Figure 4 This is a photograph of an AFM (Automatic Sensor Unit) with a top sensor unit and a lower sample stage according to the present invention.

[0046] Figure 5 A schematic diagram of another AFM embodiment according to the present invention is shown.

[0047] Figure 6 A schematic diagram of yet another AFM embodiment according to the present invention is shown, and

[0048] Figure 7 This is a schematic / detailed system diagram illustrating the operation of the method according to the present invention. Specific Implementation

[0049] Figure 1 A schematic diagram of an AFM embodiment according to the present invention is shown; namely, a high-speed atomic force microscope (AFM) 1 used to measure a relevant sample S.

[0050] Therefore, the sensor unit (SU) 10 includes a light source 11 capable of emitting a light beam 11' (e.g., a laser beam), a collimating lens 12, and an objective lens 13. The collimating lens 12 is arranged to receive and collimate the light beam in an optical path toward the relevant sample. The collimating lens is mechanically displaced by a corresponding collimating lens actuator 12' (schematically indicated by a thick arrow parallel to the optical path). The objective lens 13 is used to receive and focus the light beam in an optical path toward the sample S. The objective lens 13 can also be mechanically displaced, as schematically shown, by a corresponding objective lens actuator. Figure 1 As shown, in the optical path toward the sample S, the light 11' to the sample (and the reflected light 11'') can be redirected or reflected by one or more mirrors M.

[0051] Furthermore, an AFM cantilever 14' with an AFM tip 14 is arranged to mechanically engage with the associated sample S, and the rear side of the AFM cantilever is arranged to optically reflect the beam 11'' received from the objective lens 13 in response to the mechanical engagement. A corresponding photodetector beam 15 is arranged to detect the reflected light 11'' from the AFM cantilever 14' via a polarizing beam splitter PBS and an astigmatic lens ASL, and to provide a focusing error signal FES indicating the mechanical engagement (i.e., the displacement of the AFM tip 14 as a function of the surface structure of the sample S).

[0052] In addition, a top scanning actuator 20, preferably a voice coil motor VCM, is provided for at least one-dimensional displacement of the sensor unit (SU) 10 in the scanning direction (X), optionally two-dimensional displacement, and displacement in the approach direction (Z) toward the sample S.

[0053] Furthermore, the sample stage 30 is arranged to fix the relevant sample S. As schematically illustrated in the figures, if the sample S is a skin sample, it can be positioned on top of a piece of adhesive tape between the sample stage and the sample itself. As schematically illustrated in the figures, the sample stage 30 is mechanically connected to a bottom scanning actuator, more preferably a voice coil motor (VCM), which is further arranged to displace at least one dimension (Y) relative to the sensor unit (SU) 10 to scan the surface of the relevant sample, preferably also displacing in the approach direction (i.e., the Z direction as schematically illustrated in the figures) between the sample S and the sensor unit 10.

[0054] Then, the top scanning actuator 20 and the sample stage 30 are functionally and / or structurally combined to perform a two-dimensional (XY) scan of the surface of the sample S using the sensor unit (SU) 10, in particular the collimating lens 12 is arranged to be adjusted by the collimating lens actuator 12 along the beam path in order to at least compensate for thermal drift in the focusing error signal (FES) detected by the photodetector 15.

[0055] Preferably, the photoelectric detection device 15 includes a two-dimensional detector arranged to provide a focus error signal (FES), preferably from a beam splitter ASL that splits the beam into three distinct sub-beams, such as... Figure 1 The upper right corner is schematically shown. Furthermore, the AFM according to the invention may include a controller (not shown for clarity) operatively connected to the sensor unit SU 10, the top scan actuator 20, and the sample stage 30 with a bottom scan actuator to control the AFM measurement and to create an AFM image of the sample S based on mechanical engagement detected in the focusing error signal (FES).

[0056] In some embodiments, the sensor unit may use one or more components from a CD, DVD, and Blu-ray data storage optical pickup unit (OPU), particularly a Blu-ray OPU, since the collimating lens can already be shifted within the Blu-ray OPU. Those skilled in the art will appreciate that CD, DVD, and Blu-ray data storage optical pickup units (OPUs) are used to read and write data on optical discs. An OPU incorporates a laser diode, a photodetector, and a lens system to focus a laser beam onto the optical disc and read data. A CD, DVD, or Blu-ray OPU can be used as a core optical element in an AFM setup; see EE te Hwu and A. Boisen, “Hacking CD / DVD / Blu-ray for Biosensing,” ACS Sens, vol. 3, no. 7, pp.1222–1232, 2018, doi: 10.1021 / acssensors.8b00340, where experienced readers can find more information on this subject, the entire contents of which are incorporated herein by reference. The OPU can be used to measure the position of the tip and collect data from the interaction between the tip 14 and the sample S. By using the OPU as the core optical element in AFM, the speed, accuracy, and / or resolution of AFM imaging can be improved. In particular, in one embodiment, the present invention has been implemented using a blue light optical pickup unit (OPU) specifically designed for high-speed dermatological AFM (HD-AFM) imaging of skin nanotextures.

[0057] Figure 1The HD-AFM diagram shows the AFM probe fixed to a moving part of a top voice coil motor (VCM) that carries an objective lens 13 for Z-axis and X-axis movement. Thus, the tip 14 and cantilever 14' together with the body chip BC form the AFM probe. The OPU, more generally SU 10, focuses a laser 11 (power: 0.42 mW, λ: 405 nm) onto the cantilever 14' of the AFM probe (Mikromasch CSC38) through the objective lens. The top VCM 20 carries the AFM probes 14 and 14', bringing the tip 14 close to the sample S (Z-axis) and scanning the probe in the X-axis direction. As schematically shown on the left, the top VCM can also perform tilting movements of the AFM probe. The bottom VCM moving part introduces the sample for Z'-axis adjustment and Y-axis scanning. As the tip 14 scans the sample surface in AFM mode at a constant height, the focused laser senses the nanoscale bending motion of the cantilever 14' via a focus error signal (FES) calculated by a quadrant detector in the photodetector 15, as is known in OPUs used for optical recording / reading. It is contemplated that one or more FES-related signals can be applied in the context of this invention. However, variations in the OPU laser diode and ambient temperature can cause thermal drift between one or more optical components, resulting in random drift of the FES. The newly developed HD-AFM uses an actuator or motor within the OPU to move the collimating lens 12, thereby fine-tuning the focus for drift compensation, preferably without substantially altering the distance between the AFM probe and the objective.

[0058] Figure 2A A graph showing the FES of the AFM as a function of collimator adjustment is presented. Therefore, motor 12' can precisely adjust the position of the collimating lens 12 and hold the FES at the most sensitive point. When the collimator adjustment motor steps (horizontal axis) to move the collimating lens position, the focus error signal (FES) (vertical axis) moves accordingly. Therefore, FES drift compensation can be achieved by driving the motor and holding the FES at the most sensitive point (dot) within the sensing area (the area with a basically linear slope indicated by the arrow).

[0059] Figure 2B (a) shows the relationship with Figure 2A Another similar graph of the FES signal, and corresponding AFM images obtained at different adjustment steps of the collimator motor. Therefore, during scanning of the one-dimensional grating sample, the microstepping motor embedded in the optical pickup unit (OPU) is adjusted to move in five-step increments. The current configuration allows the motor to be adjusted across 400 steps, covering the entire S-curve of the FES signal. This adjustment changes the position of the collimating lens, and consequently alters the focusing conditions of the cantilever. Figure 2BAs shown in (b), changes in image quality can be observed during the motor adjustment phase. The five-step incremental movement (from point 1 to point 12) causes the collimator to move, thus altering the cantilever's focusing conditions. Optimal imaging contrast is achieved when the focus error signal (SFE) aligns with the most sensitive point (position 7). Since most points are within the sensing area, sample features are still visible. It is noteworthy that the change in tilt distance is attributed to the VCM scanning mechanism, rather than as a result of the motor adjustment.

[0060] By using microstep settings of 1, 2, 4, 8, 16, 32, 64, 128, and 256, motor adjustment accuracy can be improved, achieving a maximum resolution of 102,400 (400 x 256) steps for precise adjustments to compensate for thermal drift.

[0061] Figure 3 Representative AFM images (i.e., HS-AFM thermal drift compensation test and skin nanotexture measurement results) with and without the present invention are shown. One image (area: 17 × 17 µm) 2 The measurement time (512×512 pixels) was 3.4 seconds. These figures show: (a) without collimator adjustment, (b) with collimator adjustment, and (c) keratinocyte morphology of healthy skin keratinocyte samples.

[0062] therefore, Figure 3 HD-AFM measurements of DVD data tracks and skin keratinocyte samples are shown. Due to limitations in the controller's buffer circuitry, the scan speed was limited to 150 lines / second (tip-sample velocity: 5100 µm / s). Figure 3 As shown in (a), without adjusting the collimator, the FES drifts outside the sensing area and cannot measure cantilever bending. After adjusting the collimator, the FES remains within the sensing area and a topographic image of the DVD data track is obtained. Figure 3 (b) The top VCM oscillates at the start of the sampling scan, causing instability in the top 36 rows. Figure 3 (c) shows the morphology of a healthy skin keratinocyte sample. Instability was observed, but the image stabilized after row 28. As shown in Figure 2, the nanoscale texture on the keratinocytes can be clearly seen when measured at the sensitive point of the FES.

[0063] Figure 4 This is a photograph of an AFM with a top sensor unit and a lower sample stage according to the present invention. Figure 4 HD-AFM images containing two identical blue light-emitting OPUs are shown. Coarse AFM position adjustment and nanometer-level resolution scanning were performed along the X, Y, and Z, Z' axes at working distances of 1400 µm and 1900 µm, respectively. The complete image is 1.4 × 1.4 × 3.8 mm. 3The increased operating range eliminates the need for external tip-sample proximity and sample coarse-tuning mechanisms, while simultaneously improving throughput for measuring different points on skin keratinocyte samples. Four 16-bit digital-to-analog converter channels drive the actuation axes at resolutions of 21 nm (X, Y) and 29 nm (Z, Z'). The first and second resonant frequencies of the top and bottom VCMs are 48 Hz and >20 kHz, respectively. When HD-AFM is scanned at 20 kHz, the VCM attenuates by -38 dB across its entire operating range, still achieving a sufficient AFM scan range of 17.5 µm. The theoretical linear scan speed reaches 700,000 µm / s, a significant improvement of three orders of magnitude faster than conventional high-speed AFM.

[0064] Therefore, this invention is an HD-AFM with an internal thermal drift compensation mechanism. The tip-scanning HD-AFM can image nanotextures on skin keratinocytes in a constant height scanning mode within 3.4 seconds. Further improvements are needed to enhance imaging stability. The HD-AFM has the potential to become a system recommended by the Global Atopic Dermatitis Care Quality (ADQoC) initiative for the assessment of skin diseases such as AD, prevention risk, and treatment effectiveness.

[0065] Figure 5 It shows the relationship with Figure 1 The illustrated embodiment is similar to another AFM embodiment according to the present invention. Therefore, as... Figure 5 As schematically illustrated, the AFM according to this embodiment is arranged to scan the sample surface under liquid conditions, wherein at least a portion of the relevant sample is immersed in liquid L. The liquid can be water or other liquids, particularly liquids that preserve / maintain certain biologically relevant conditions of the sample S of biological interest during sample scanning. The AFM can then adjust the focal point of the optical path toward the sample S by moving the collimating lens 12 and / or the objective lens 13 to at least partially compensate for these liquid conditions. Thus, the collimating lens can compensate for liquid evaporation and / or thermal changes, or other liquid changes, such as reactions occurring in the sample S and / or the liquid L above the sample.

[0066] Figure 6 A schematic diagram of yet another AFM embodiment according to the present invention is shown. This embodiment is similar to... Figure 1 and Figure 5 The AFM is schematically shown, but in this embodiment, an additional beam of light (e.g., a red laser 9) is applied to find the approximate location of the AFM tip 14, i.e., sample ranging. Figure 6 (a) Part shows the internal dual blue light optical hardware design, in which a voice coil motor (VCM) performs X-axis and Z-axis motion. Figure 6(b) shows an exploded view, where the top VCM carries a pre-aligned AFM tip and objective for Z-axis coarse approach and X-axis coarse adjustment, as well as high-speed scanning, and the bottom VCM carries a skin band for Y-axis coarse adjustment and high-speed scanning. Such a compact embodiment of the optics and VCM is anticipated to allow the entire AFM system and its optomechanical components to be handheld in size, making it particularly suitable for clinical applications of skin imaging. For example, this handheld miniature HS-AFM could be used for imaging the nanotexture of skin keratinocytes. The digitized keratinocyte data can be further analyzed for assessment of the severity of skin conditions.

[0067] Figure 7 This is a schematic / detailed system diagram illustrating the operation of the method according to the invention. Therefore, the invention relates to a method for operating HS-AFM and measuring sample S (preferably dermatological measurements of skin samples), see [link to relevant documentation]. Figure 1 , Figure 5 and Figure 6 The method includes: S1 provides a sensor unit (SU) 10, which includes: Light source 11, which is capable of emitting a light beam, Collimating lens 12, which is arranged to receive and collimate the light beam in the optical path toward the relevant sample, see [link to relevant document]. Figure 1 The collimating lens is mechanically displaced by a corresponding collimating lens actuator. Objective lens 13 receives the light beam and focuses it onto an optical path toward the sample; the objective lens is mechanically displaced by a corresponding objective lens actuator. An AFM cantilever having an AFM tip 14 arranged for mechanical engagement with an associated sample, and a rear side of the AFM cantilever arranged to optically reflect the light beam received from the objective lens in response to the mechanical engagement. Photodetector beam 15 is used to detect the reflected light from the AFM cantilever and provide a focus error signal (FES) indicating the mechanical engagement. S2 provides a top scanning actuator 20, preferably a voice coil motor, for at least one-dimensional displacement of the sensor unit (SU) in the scanning direction (X) and displacement in the approach direction (Z) toward the relevant sample, and S3 provides a sample stage (30) for fixing the relevant sample. S4 In this configuration, the top scanning actuator and the sample stage are arranged relative to each other to perform at least a two-dimensional (XY) scan of the surface of the sample (S) using the sensor unit (SU) 10, see [link to relevant documentation]. Figure 1 , Figure 5 and Figure 6,as well as S5 In this case, the collimating lens 12 is adjusted along the beam path by the collimating lens actuator to at least compensate for thermal drift in the focus error signal (FES), see Figure 2.

[0068] In short, the present invention relates to a high-speed atomic force microscope (AFM) 1, see [link to relevant documentation]. Figure 1 The high-speed atomic force microscope (AFM) 1 is used to measure a relevant sample (S) (e.g., a skin sample). The AFM has a sensor unit (SU) 10 including a light source 11 and a collimating lens 12, which is mechanically displaced by a collimating lens actuator 12'. An AFM cantilever with an AFM tip 14 is arranged relative to a photodetector beam 15 to provide a focusing error signal (FES) indicating mechanical engagement with the sample S. The collimating lens 12 is arranged to be adjusted along the beam path by the collimating lens actuator 12' to at least compensate for thermal drift in the focusing error signal. This invention facilitates improved high-speed AFM through this internal thermal drift compensation mechanism. One embodiment of the high-speed AFM of the invention can have a theoretical linear scan speed of up to 700,000 µm / s, allowing an imaging operation to be completed in approximately 3.4 seconds.

[0069] This invention can be implemented through hardware, software, firmware, or any combination thereof. This invention, or certain features thereof, can also be implemented as software running on one or more data processors and / or digital signal processors.

[0070] The various elements of this invention can be implemented physically, functionally, and logically in any suitable manner, such as in a single unit, multiple units, or as part of a separate functional unit. This invention can be implemented in a single unit or physically and functionally distributed among different units and processors.

[0071] Although the invention has been described in conjunction with specific embodiments, it should not be construed as being limited in any way to the examples presented. The scope of the invention will be interpreted in accordance with the appended claims. In the context of the claims, the terms "comprising" or "comprises" do not exclude other possible elements or steps. Furthermore, references such as "a" or "an" should not be construed as excluding a plurality. The use of reference numerals in the claims relating to elements shown in the drawings should also not be construed as limiting the scope of the invention. Moreover, the various features mentioned in different claims can be advantageously combined together, and the mention of these features in different claims does not preclude the possibility and advantage of combining features. Some other embodiments: E1. A high-speed atomic force microscope (AFM, 1) for measuring a relevant sample (S), said AFM comprising: The sensor unit (SU, 10) includes: Light source (11), which is capable of emitting a light beam, A collimating lens (12) is arranged to receive and collimate the light beam in the optical path toward the relevant sample, the collimating lens being mechanically displaced by a corresponding collimating lens actuator. Objective lens (13), which receives the light beam and focuses it onto the optical path toward the sample, is mechanically displaced by a corresponding objective lens actuator. An AFM cantilever having an AFM tip (14) arranged for mechanical engagement with a relevant sample, and a rear side of the AFM cantilever arranged to optically reflect the light beam received from the objective lens in response to said mechanical engagement. A photoelectric detection beam (15) is used to detect the reflected light from the AFM cantilever and provide a focus error signal (FES) indicating the mechanical engagement. A top scanning actuator (20), preferably a voice coil motor, is used for at least one-dimensional displacement of the sensor unit (SU) in the scanning direction (X) and displacement in the approach direction (Z) toward the relevant sample, and A sample stage (30) is used to fix the relevant sample. The top scanning actuator and sample stage are arranged to perform at least two-dimensional (XY) scanning of the surface of the sample (S) using a sensor unit (SU, 10), wherein the collimating lens (12) is arranged to be adjusted by the collimating lens actuator along the beam path to at least compensate for thermal drift in the focusing error signal (FES). 2. According to the AFM of Embodiment 1, thermal drift compensation in the focus error signal (FES) is performed in the control loop mechanism based on the focus error signal and the displacement of the collimating lens by the collimating lens actuator, preferably a linear displacement. 3. The AFM according to Example 2, wherein the AFM operates at or near the optimal sensing point of the corresponding control loop curve. 4. The AFM according to any one of Examples 1-3, wherein the sample stage is mechanically connected to a bottom scanning actuator, more preferably to a voice coil motor, the bottom scanning actuator being further arranged for at least one-dimensional displacement (Y) to scan the surface of the relevant sample relative to the sensor unit (SU). 5. The AFM according to Example 4, wherein the bottom scanning actuator is further arranged to displace in the approach direction (Z) between the relevant sample and the sensor unit (SU). 6. The AFM according to any one of the foregoing embodiments, wherein the AFM tip is arranged to scan the relevant sample together with the objective lens in the sensor unit (SU). 7. The AFM according to any one of Examples 1-6, wherein the AFM is capable of operating in contact mode, non-contact mode, tapping mode, dynamic force mode, phase imaging mode and / or as an electrostatic force microscope (EFM). 8. The AFM according to Embodiment 1 and Option Embodiment 4, wherein the operating range of the top scan actuator and the optional bottom scan actuator is capable of scanning at least 0.5 mm, preferably 0.75 mm, and most preferably at least 1.0 mm in one or both directions within the plane in which the AFM scan is performed. 9. The AFM according to any one of the foregoing embodiments, wherein the linear scan rate is at least 50,000 micrometers / second, preferably at least 100,000 micrometers / second, and most preferably at least 200,000 micrometers / second. 10. The AFM according to any one of the foregoing embodiments, wherein the AFM is arranged for dermatological measurements of relevant skin samples. 11. The AFM according to any one of the foregoing embodiments, wherein the AFM is arranged to scan a sample surface under liquid conditions, wherein at least a portion of the relevant sample is immersed in the liquid, and the AFM is capable of adjusting the focal point of the optical path toward the sample by displacing the collimating lens and / or displacing the objective lens to at least partially compensate for the liquid conditions. 12. A method for operating HS-AFM and measuring a sample (S), preferably a skin sample, for dermatological measurements, the method comprising: - Provide a sensor unit (SU, 10), which includes: Light source (11), which is capable of emitting a light beam, A collimating lens (12) is arranged to receive and collimate the light beam in the optical path toward the relevant sample, the collimating lens being mechanically displaced by a corresponding collimating lens actuator. Objective lens (13), which receives the light beam and focuses it onto the optical path toward the sample, is mechanically displaced by a corresponding objective lens actuator. An AFM cantilever having an AFM tip (14) arranged for mechanical engagement with a relevant sample, and a rear side of the AFM cantilever arranged for optical reflection of the light beam received from the objective lens in response to said mechanical engagement. A photoelectric detection beam (15) is used to detect the reflected light from the AFM cantilever and provide a focus error signal (FES) indicating the mechanical engagement. A top scanning actuator (20), preferably a voice coil motor, is provided for at least one-dimensional displacement of the sensor unit (SU) in the scanning direction (X) and displacement in the approach direction (Z) toward the relevant sample, and A sample stage (30) is provided for fixing the relevant sample. The top scanning actuator and the sample stage are arranged to perform at least two-dimensional (XY) scanning of the surface of the sample (S) using a sensor unit (SU, 10), wherein the collimating lens (12) is adjusted by the collimating lens actuator along the beam path to at least compensate for thermal drift in the focusing error signal (FES). 13. A computer program product suitable for enabling a computer system including at least one computer having a data storage device connected thereto to control the AFM according to claim 12.

Claims

1. A high-speed atomic force microscope (AFM, 1) for measuring a relevant sample (S), the AFM comprising: a sensor unit (SU, 10) comprising: a light source (11) capable of emitting a light beam, a collimating lens (12) arranged to receive and collimate the light beam on an optical path towards the relevant sample, the collimating lens being mechanically displaceable by a respective collimating lens actuator, an objective lens (13) receiving the light beam and focusing the light beam on an optical path towards the sample, the objective lens being mechanically displaceable by a respective objective lens actuator, an AFM cantilever having an AFM tip (14) arranged to mechanically engage with the relevant sample, a backside of the AFM cantilever being arranged to optically reflect the light beam received from the objective lens in response to the mechanical engagement, and a photodetected light beam (15) for detecting the reflected light from the AFM cantilever and providing a focus error signal (FES) indicative of the mechanical engagement, a top scanning actuator (20), preferably a voice coil motor, for at least one-dimensional displacement of the sensor unit (SU) in a scanning direction (X) and in a proximity direction (Z) towards the relevant sample, and a sample stage (30) for fixing the relevant sample, wherein the top scanning actuator and the sample stage are arranged to perform at least two-dimensional (X-Y) scanning of a surface of the sample (S) using the sensor unit (SU, 10), wherein the collimating lens (12) is arranged to be adjusted by the collimating lens actuator along the light beam path to at least compensate for thermal drift in the focus error signal (FES), wherein compensation of thermal drift in the focus error signal (FES) is performed in a control loop mechanism based on the focus error signal and the displacement, preferably linear displacement, of the collimating lens by the collimating lens actuator.

2. The AFM of claim 1, wherein, The photodetected device (15) comprises a quadrant detector (A, B, C, D) arranged to sense the focus error signal (FES).

3. The AFM of claim 2, wherein, The quadrant detector is optically arranged with respect to an astigmatism lens (ASL) for sensing the reflected light from the cantilever.

4. The AFM of claim 1, wherein, The AFM is operated at or near an optimum sensing point of a respective control loop curve.

5. The AFM of any one of claims 1-4, wherein, The sample stage is mechanically connected to a bottom scanning actuator, more preferably to a voice coil motor, the bottom scanning actuator being further arranged for at least one-dimensional displacement (Y) of the surface of the relevant sample relative to the sensor unit (SU).

6. The AFM of claim 5, wherein, The bottom scanning actuator is further arranged to displace in the proximity direction (Z) between the relevant sample and the sensor unit (SU).

7. The AFM of any of the preceding claims, wherein, The AFM tip is arranged to scan on the relevant sample together with the objective lens in the sensor unit (SU).

8. The AFM of any one of claims 1-7, wherein, The AFM is capable of operating in contact mode, non-contact mode, tapping mode, dynamic force mode, phase imaging mode and / or as an electrostatic force microscope (EFM).

9. The AFM of claim 1 and optional claim 5, wherein, The working range of the top scanning actuator and optionally the bottom scanning actuator is capable of scanning at least 0.5 mm, preferably 0.75 mm, most preferably at least 1.0 mm in one or both directions within the plane of performing the AFM scan.

10. The AFM of any of the preceding claims, wherein, The linear scan speed is at least 50.000 micrometer / sec, preferably at least 100.000 micrometer / sec, most preferably at least 200.000 micrometer / sec.

11. The AFM of any of the preceding claims, wherein, The AFM is arranged for dermatological measurements of a related skin sample (S).

12. The AFM of any one of the preceding claims, wherein, The AFM is arranged for scanning a sample surface under liquid conditions, wherein at least a portion of the related sample is immersed in a liquid (L), the AFM is capable of adjusting the focus of the optical path towards the sample by displacing the collimating lens and / or displacing the objective lens to at least partially compensate for the liquid conditions.

13. A method for operating an HS-AFM (1) and performing measurements of a sample (S), preferably dermatological measurements of a skin sample, the method comprising: providing a sensor unit (SU, 10) comprising: a light source (11) capable of emitting a light beam, a collimating lens (12) arranged to receive and collimate the light beam on an optical path towards the related sample, the collimating lens being mechanically displaceable by a respective collimating lens actuator, an objective lens (13) receiving the light beam and focusing it on an optical path towards the sample, the objective lens being mechanically displaceable by a respective objective lens actuator, an AFM cantilever having an AFM tip (14) arranged to mechanically engage with the related sample, a back side of the AFM cantilever being arranged to optically reflect the light beam received from the objective lens in response to the mechanical engagement, and a photodetected light beam (15) for detecting the reflected light from the AFM cantilever and providing a focus error signal (FES) indicative of the mechanical engagement, providing a top scanning actuator (20), preferably a voice coil motor, for at least one-dimensional displacement of the sensor unit (SU) in a scanning direction (X) and displacement in a proximity direction (Z) towards the related sample, and providing a sample stage (30) for fixing the related sample, wherein the top scanning actuator and the sample stage are arranged to each other for at least two-dimensional (X-Y) scanning of a surface of the sample (S) using the sensor unit (SU, 10), wherein the collimating lens (12) is adjusted by the collimating lens actuator along the light beam path to at least compensate for thermal drift in the focus error signal (FES), wherein compensation of thermal drift in the focus error signal (FES) is performed in a control loop mechanism based on the focus error signal and displacement, preferably linear displacement, of the collimating lens by the collimating lens actuator.

14. A computer program product adapted to enable a computer system comprising at least one computer having data storage means connected thereto to control an AFM according to claim 13.

Images