Scattering-type scanning near-field optical microscope (SSNOM) system

Abstract

Scattering-type scanning near-field optical microscope (sSNOM) which comprises: a sample holder configured to hold a sample. The sample holder and the scanning probe tip are adjustable relative to each other in the x-, y-directions by means of a horizontal scanning device, wherein the horizontal scanning device is configured to move the sample located on the top side of the sample holder in the x- and y-directions relative to the scanning probe tip, wherein a plurality of frames is scanned subsequently, each frame representing the scan area of the sample at a specific time interval, and each frame comprising pixels arranged in lines and columns in the x- and y-directions, wherein for each pixel a measurement of phase and / or amplitude is made, and wherein the horizontal scanning device is configured to move the sample along the lines of each frame with a predetermined frequency.

Description

[0001] Scattering-type scanning near-field optical microscope (sSNOM) system

[0002] Description

[0003] The invention regards a scattering-type scanning near-field optical microscope system.

[0004] Scattering-type scanning near-field optical microscopy (sSNOM) is a technique that uses a sharp tip (such as an AFM tip) of an sSNOM probe to scatter light and measure optical properties of a sample at the nanoscale. The near-field probe is illuminated at its tip by focused light, e.g. in the infrared spectrum, to generate near-field light during tip-sample interaction. The optical near-field is typically measured by tip oscillation on the sample with tens of nanometer amplitude, wherein sSNOM measures the light scattered from regions directly under the AFM tip. By using an interferometer, the amplitude and / or phase of the scattered light can be measured, which relate to the local absorption and reflectivity of the sample. SSNOM can achieve a spatial resolution down to nanometer scale without diffraction limit.

[0005] The principles of an sSNOM scanning probe microscope have been described in document WO 2004 / 001764 A2.

[0006] E. Pfitzner et al.: “Infrared Scattering-Type Scanning Near-Field Optical Microscopy of Biomembranes in Water”, J. Phys. Chem. Lett. 2020, 11 , 8183-8188 discloses an sSNOM microscope in which the light from the laser passes through a solid immersion lens made of silicon and located below the sample before illuminating the microscope tip. Presently known sSNOM systems allow to scan a sample within one or several minutes. The result is a static image which does not allow to determine dynamic changes. However, when having a biological sample such as a protein, there is a desire to have a time resolution of the sSNOM microscope that allows the observe biological processes, such as dynamic structural changes of individual biological molecules.

[0007] It is an object of the invention to provide an sSNOM microscope that allows to make measurements of samples with an increased time resolution that allows to determine dynamic changes of the sample.

[0008] The invention provides for a scattering-type scanning near-field optical microscope (sSNOM) system with the features of claim 1. Embodiments of the invention are identified in the dependent claims.

[0009] Accordingly, aspects of the invention provide for a scattering-type scanning near-field optical microscope (sSNOM) system which comprises a sample holder configured to hold a sample and having a top side and a bottom side, a scanning probe having a tip, an IR- laser emitting infrared light, and means for directing the emitted infrared laser light through the sample holder onto the scanning probe tip, wherein the infrared light passes the sample holder from the bottom side to the top side, and wherein the scanning probe tip is configured to be located above the sample at a near-field distance such that a near-field is generated between the tip and the sample when the tip is illuminated with the infrared light. The sSNOM system further comprises an optical detection device, wherein the optical detection device comprises means for recovering phase and / or amplitude of the light scattered from the scanning probe tip and further scattered and modified by the sample, and the optical detection device further comprising a detector.

[0010] It is further provided that the sample holder and the scanning probe tip are adjustable relative to each other in the x-, y- and z-directions by means of a horizontal scanning device configured to move the sample holder in the x-, y- directions and by means of a vertical scanning device configured to move the scanning probe tip in the z-direction. The horizontal scanning device is configured to move the sample located on the top side of the sample holder in the x- and y-directions relative to the scanning probe tip, wherein a plurality of frames is scanned subsequently, each frame representing the scan area of the sample at a specific time interval, and each frame comprising pixels arranged in lines and columns in the x- and y-directions, wherein for each pixel a measurement of phase and / or amplitude is made, and wherein the horizontal scanning device is configured to move the sample along the lines of each frame with a predetermined frequency.

[0011] Aspects of the invention are thus based on the idea to provide for a fast scan rate that allows to make measurements of a sample with improved temporal resolution while at the same time providing a good spatial resolution. A faster scan rate is achieved in that a plurality of frames subsequent in time are scanned, wherein the frames, which are comprised of lines and columns in the x- and y- plane, are measured / scanned by moving the probe with the horizontal scanning device with a predetermined frequency, the frequency being defined as the number of lines that are scanned per second. The predetermined frequency is thus a frequency of the horizontal scanning device that identifies the speed with which the sample is moved by the horizontal scanning device in the x- and y-plane relative to the scanning probe tip.

[0012] For example, the horizontal scanning device may be configured to move the sample along the lines of each frame with at least a frequency that corresponds to scanning a line of 100 pixels with a frequency of 10 Hz (which means a scanning time of 1 / 10 s per line of 100 pixels). Assuming that the scanned frame comprises hundred lines (such that the frame comprises 100 x 100 pixels), the scanning time for the frame is 10 seconds or less. This provides for both a reasonable time resolution that allows to observe dynamic structural changes of a sample and a good spatial resolution.

[0013] In an embodiment, the horizontal scanning device is configured to move the sample along the lines of each frame with a frequency that corresponds to scanning a line of 100 pixels with a frequency of 100 Hz (which means a scanning time of 1 / 100 s per line of 100 pixels). Assuming again that the scanned frame comprises hundred lines, the scanning time for the frame is 1 second only, which provides for a good time resolution.

[0014] In other embodiments, the scanning rate may be even much higher, e.g., up to 6 kHz per scanned line. Further, it is pointed out that, of course, if the number of pixels in a line may be more or less than 100 which represents an embodiment only . If the number of pixels in a is less than 100 , the frequency for scanning a line is increased accordingly, and if the number of pixels in a line is more than 100, the frequency is decreased accordingly.

[0015] One application of an improved scan rate lies in the measurement of a sample which is a living organic sample such as a biological molecule, or a polymer, in particular a large protein complex or polymer, which size is 5-100 nm height and more than 20 nm lateral size. The improved scan rate provides for a sequence of frame measurements that allows to record chemical composition changes of single biological molecules or of polymers. Accordingly, 4D-imaging is provided for, wherein the traditional 3D imaging is enhanced by a timeline.

[0016] Another advantage associated with the present invention lies in a highly compact design in which a sample is located at the top side of a sample holder which is illuminated with infrared light from the bottom, and wherein light scattered and modified by the sample is collected and provided to the optical detection device in the bottom direction.

[0017] The frames may in principle comprise any number of pixels, wherein the higher the number of pixels, the higher the spatial resolution. A desired resolution may be a resolution of about 20 nm. In embodiments, the frames comprise the same number of lines and columns. For example, the frames comprise 100x100, 128x128 or 256x256 or 512x512 pixels.

[0018] In an embodiment, the horizontal scanning device is coupled to or integrated with the sample holder. Accordingly, the scanner directly moves the sample holder and the sample attached to the sample holder surface.

[0019] In an embodiment, the horizontal scanning device comprises: an optical element having a flat top surface, the flat top surface being the top surface of the sample holder, wherein the optical element is transparent for infrared light and wherein the infrared light passes the optical element from the bottom to the top, a first piezoelectric actuator configured to move the optical element for scanning lines of each frame in the x-direction, a second piezoelectric actuator configured to move the optical element in the y- direction, wherein the first and second piezoelectric actuator are configured to scan a two- dimensional frame.

[0020] Accordingly, the first piezoelectric actuator is responsible for scanning the different pixels of a line of a frame (by moving the sample accordingly relative to the scanning probe tip). To this end, the voltage applied to the first piezoelectric actuator is increased (in steps or linearly) for scanning a line, each increase in voltage leading to the measurement of another pixel of the line. The second piezoelectric actuator is responsible for scanning different lines. To this end, the voltage applied to the second piezoelectric actuator is increased (in steps or linearly), wherein each increase is effected after a line is scanned by means of the first piezoelectric actuator.

[0021] In an embodiment, the optical element is a half sphere having a flat top surface, the flat top surface of the half sphere forming the top side of the sample holder. The half sphere optical element may be made of silicon or ZnSe. The half sphere optical element provides for a flat top surface on which the sample can be located.

[0022] In a further refinement of that embodiment, the first piezoelectric actuator and the second piezoelectric actuator are associated with a first and a second counter spring, wherein the first piezoelectric actuator is located in the x-direction at one end of the optical element and the first counter spring is located in the x-direction at the opposite end of the optical element, and wherein the second piezoelectric actuator is located in the y-direction at one end of the optical element and the second counter spring is located in the y-direction at the opposite end of the optical element. Accordingly, the optical element is arranged resiliently in the horizontal scanning device, this allowing the optical element and the sample arranged thereon to return quickly to the initial state after a line has been measured. Without the presence of counter springs, undesirable vibrations may prevent scanning. Further, the counter springs may provide a preload to the piezoelectric actuators. Also, passive damping is provided for.

[0023] To further improve such resilient arrangement and passive damping, it may be provided that the optical element is held by a resilient structure connecting the optical element (or a structure holding the optical element) and the first and second piezoelectric actuators and the first and second counter springs. The resilient structure may comprise resilient connecting elements, each connecting element connecting a piezoelectric actuator or a counter spring with the optical element.

[0024] In a further embodiment, the optical element is arranged in an octagonal structure which is located in an octagonal recess in the horizontal scanning device and is movable relative to the octagonal recess (which has a larger cross-section than the octagonal structure) dependent on the actuation status of the first and second piezoelectric actuators. The octagonal form of the structure and of the recess allow a relative movement both in the x- and y-directions. A silicone elastomer may fill the gap between the octagonal structure the and the octagonal recess as damping material to provide for passive damping. In a further embodiment, the vertical scanning device that is coupled to the scanning probe and thus the scanning probe tip is configured to modulate the scanning probe tip in the vertical direction with a frequency of at least 60 kHz. More particularly, the modulation frequency may be in the range between 60 kHz and 150 kHz, such as 100 kHz. The vertical modulation takes place at a defined distance to the sample such that the scanning probe tip is always at a distance to the sample and not contacting the sample.

[0025] In this respect, it is pointed out that, generally, the vertical z-coordinate of the sample is measured by the interference between the tip-scattered light and a reference beam in the optical detection device, such as a photoelastic modulator or Michelson different meter. The interference pattern depends on the optical phase of the scattered light, which is related to the height of the tip above the sample.

[0026] To provide for the vertical modulation, according to an embodiment the vertical scanning device comprises a piezoelectric element with a resonance frequency of at least 60 kHz, in particular at least 100 kHz and the scanning probe comprises a cantilever to which the scanning probe tip is attached, wherein the cantilever also has a resonance frequency of at least 60 kilohertz.

[0027] In such embodiments, it may be provided that a z-coordinate of each pixel is determined by measuring the optical phase shift of the modulated signal at the resonance frequency of the cantilever in the optical detection device, as is in principle known to the person skilled in the art, such that a 3D measurement of the pixels and thus the sample is possible.

[0028] In an embodiment, the cantilever is further attached to a shaker imprinting a shaking movement onto the cantilever and the scanning probe tip, the shaker providing a tapping frequency in the range between 200 kHz and 1500 kHz in air. In an embodiment, the tapping frequency is around 1400 kHz in air and 200 kHz in water. At the same time, the oscillation amplitude of the cantilever may be more than 20 nm or more than 50 nm. Tapping mode is a type of an amplitude modulation technique in which vibrations of the probe are implemented for imaging. The shaker may be implemented by a further piezoelectric element.

[0029] Instead of implementing a tapping mode by a shaker, photothermal tapping may be implemented. Photothermal tapping includes the use of a power-modulated laser to excite the cantilever oscillation. The advantage of photothermal tapping compared to shaker tapping is that undesirable vibration is avoided when the cantilever oscillation and the Z scan frequency are within one order of magnitude.

[0030] In an embodiment, the system further comprises a lock-in measurement device which is configured to make a lock-in measurement of the sSNOM signal at a lock-in frequency which is the oscillation frequency of the cantilever. Accordingly, the signals of interest that are to be measured by the sSNOM which are the signals caused by the near-field coupling between the probe tip and the sample are measured with high precision at the lock-in frequency. In an embodiment, a lock-in amplifier uses the oscillation frequency of the cantilever from an AFM controller, wherein 2nd 3rd .. harmonics are extracted.

[0031] In a further embodiment, the area between the top side of the sample holder and the scanning probe including the scanning probe tip and possibly the cantilever of the scanning probe is immersed in a liquid. This allows to make sSNOM measurements on biological samples such as which are typically immersed in water. However, the frequencies referred to in the present application refer to the situation when the vibrating elements (such as the probe tip) are located in air and not in a liquid unless otherwise indicated. For example, the tapping frequency of around 1400 kHz mentioned above would be about 200 kHz in water.

[0032] When immersing the area between the top side of the sample holder and the scanning probe in a liquid, one further advantage of the embodiment in which the horizontal scanning device comprises an optical element lies in that the optical element prevents that the infrared light passes through the liquid such as water on its way to the sample, which is preferable as water absorbs infrared light. In other words, the infrared light coming from the bottom avoids passing through water.

[0033] In an embodiment, the wavelength of the infrared laser light lies in the region between 2 and 8 pm, in particular in a mid-infrared region between 3 and 6.5 pm.

[0034] In a further embodiment, the optical detection device comprises an interferometer such as a Michelson interferometer, wherein phase and / or amplitude for each pixel of each frame are measured using the interferometer.

[0035] In an alternative embodiment, the optical detection device comprises a photoelastic modulator, wherein phase and / or amplitude for each pixel of each frame are measured using the photoelastic modulator. A photoelastic modulator implements phase modulation instead of amplitude modulation as with a Michelson interferometer. Thereby, significantly less noise on the near-field signal is created. In a further embodiment, the system further comprises an AFM microscope, wherein the scanning probe is the probe of the AFM microscope. A signal for the AFM measurement is provided for by light reflected from the upper side of the scanning probe. In an embodiment, for each pixel a measurement of phase and / or amplitude is made both with the AFM microscope and with the sSNOM.

[0036] The invention will be explained in more detail on the basis of exemplary embodiments with reference to the accompanying drawings in which:

[0037] Fig. 1 is a schematic depiction of a scattering-type scanning near-field optical microscope (sSNOM) system in accordance with the present invention, wherein a sample is moved in the x-, y-directions by means of a horizontal scanning device and moved in the z-direction by means of a vertical scanning device;

[0038] Fig. 2 shows a frame that represents the scan area of a sample at a specific time interval, wherein the frame comprises pixels arranged in lines and columns in the x- and y-directions;

[0039] Fig. 3 is a more detailed embodiment of a scattering-type scanning near-field optical microscope (sSNOM) system in accordance with the present invention;

[0040] Fig. 4 is a top view of an embodiment of a horizontal scanning device that may be implemented in the systems of Figs. 1 and 3, the horizontal scanning device comprising an optical element and piezoelectric actuators and counter springs acting on the optical element;

[0041] Fig. 5 shows the horizontal scanning device of Fig. 4 at a moment when the optical element has been shifted in the x-direction by one of the piezoelectric actuators;

[0042] Fig. 6 is a schematic perspective view of an embodiment of a scanning probe which comprises a cantilever and a scanning probe tip;

[0043] Fig. 7 shows an embodiment of a scanning probe tip; Fig. 8 is a sectional view of the scanning probe of Fig. 6, wherein the scanning probe is coupled to a vertical scanning device;

[0044] Fig. 9 indicates the advantages of the scanning probe tip of Fig. 7;

[0045] Fig. 10 shows experimental results of an sSNOM microscope system in accordance with the present invention which include an AFM topography image, an sSNOM optical amplitude image and an sSNOM optical phase image; and

[0046] Fig. 11 shows an improvement of a Michelson interferometer reference arm.

[0047] Fig. 1 depicts schematically an embodiment of a scattering-type scanning near-field optical microscope system, referred to as “sSNOM” or “system” in the following.

[0048] Main components of the system are a sample holder 1 , a scanning probe 2, an infrared laser 3, means 41-44 for directing light emitted from the infrared laser through the sample holder 1 onto a tip 21 of the scanning probe 2, an optical detection device 5, a horizontal scanning device (horizontal scanner) 7 and a vertical scanning device (vertical scanner) 9.

[0049] More particularly, the sample holder 1 comprises a top side and a bottom side, wherein a sample 6 that is to be measured by the sSNOM is located on the top side of the sample holder 1. The sample 6 is depicted schematically and may be a living organic samples such as a protein. The system serves to detect changes of the sample such as conformation changes of specific chemicals with a time resolution that allows to observe such changes.

[0050] The scanning probe 2 is an AFM scanning probe of an atomic force microscope (AFM) 20 depicted schematically. The scanning probe 2 comprises a cantilever 22 and a tip 21 and is connected to the vertical scanner 9. The AFM microscope 20 generates images by scanning with the scanning probe 2 over the surface of the sample 6, wherein laser light is reflected from the back side of the scanning probe 2 and detected by a photodiode. As AFM microscopes are well known to the skilled person, it is refrained from providing more details about the AFM microscope 20.

[0051] In the system of Fig. 1 , the AFM microscope 20 is combined with an sSNOM microscope. The tip 21 of the scanning probe 2 is located above the sample 6 at a near-field distance such that a near-field coupling is generated between the tip 21 and the sample 6 when the tip 21 is illuminated with infrared light from the bottom. The tip 21 may have an apex with a radius in the range between 10 and 30 nm.

[0052] Infrared light 8 is emitted from an infrared laser 3 and directed through a beam splitter 41 , mirrors 42, 43 and an optical element 44 onto the scanning probe tip 21 , wherein the infrared light 8 illuminates the sample 6 and the tip 21 from the bottom, passing the sample holder 1 from the bottom side to the top side. The optical element 44 may comprise one or several lenses focusing the infrared light on the tip 21 of the scanning probe 2.

[0053] Infrared light 8 illuminating the tip 21 of the scanning probe 2 is scattered from the scanning probe tip 21 and further scattered and modified by the sample 6, which represents the basic physical interaction of an sSNOM as known to the skilled person. More particularly, the tip 21 acts as an antenna, enhancing the local electromagnetic field and interacting with the sample surface at a nanometer scale. The light scattered from the sample 6 in the bottom direction carries high resolution optical information and is collected and guided through elements 44, 43, 42 and 41 into a photoelastic modulator 52 which serves to recover phase and / or amplitude of the light scattered at the sample 6 together with beam splitter 41. The light is detected by a detector 51 after reflection at a parabolic mirror 53. The photoelastic modulator 52, the beam splitter 41 and the detector 51 together form an optical detection device 5. Alternatively, the optical detection device 5 may be formed by interferometric detection means.

[0054] A photoelastic modulator generally is an optical device that can change the polarization of a light source by using the photoelastic effect. The elastic modulator is used to modulate the polarization of the scattered light. This allows the detection of optical properties of the sample 6 with nanometer resolution. The photoelastic modulator also enhances the contrast and sensitivity of the sSNOM signal by filtering out the background light.

[0055] The photoelastic modulator 52 typically comprises a crystal (ZnSe for the mid-infrared) which is attached to a piezoelectric actuator. The refractive index of the unstrained crystal is isotropic. The crystal is designed such that the piezoelectric actuator excites an eigenmode of the crystal which has typically a frequency of tens to hundreds of kilohertz. Applying anisotropic strain to the crystal along one axis leads to a modulation of the refractive index along that axis but leaves the refractive index along the unstrained axis unaffected. If using linearly p-polarized light the phase will be retarded periodically. In an alternative embodiment, a Michelson interferometer is used instead of a photoelastic modulator.

[0056] As schematically depicted in Fig. 1 , the sample holder 1 and the scanning probe tip 21 are adjustable relative to each other in the horizontal x-, y-directions and in the vertical z- direction. To this end, the horizontal scanner 7 serves to move the sample holder in the x- and y-directions. The vertical scanner 9 serves to move the scanning probe tip 21 in the vertical direction. The vertical scanner 9 comprises a piezoelectric element that has a resonance frequency which determines the vertical movement of the scanning probe tip 21. The signal collected by the optical detection device 5 is collected at the oscillation frequency of the cantilever by a lock-in measurement, wherein scattered light from the tip 21 is modulated by device 5 and collected by detector 51.

[0057] The horizontal scanner 7 is configured to move the sample 6 in the x- and y-directions relative to the scanning probe tip 21. Thereby, a plurality of frames are scanned subsequently. Such frame 15 is schematically shown in Fig. 2. A frame 15 represents the area of the sample 6 which is scanned by the tip 21 in a specific time interval when the sample 6 is moved by the horizontal scanner 7, wherein the time interval is the time it takes to make measurements for all pixels 150 of a frame 15. More particularly, the frame 15 comprises lines 151 in the x-direction and columns 152 in the y-direction. In the depicted embodiment, the frame 15 comprises only 12 columns and 12 lines. This is only the case to simplify the drawing. In a realistic embodiment, the frame may have 100, 128, 256 or 512 lines and columns.

[0058] For each pixel 150, the measurement of phase and / or amplitude is made both with the AFM microscope 20 and with the sSNOM. After a pixel 150 has been measured, a subsequent pixel in the same line 151 is measured, and after all pixels of a line 151 have been measured, the pixels of a subsequent line are measured. The movement of the sample holder 1 and of the sample 6 through the horizontal scanner 7 is effected by means of two piezoelectric actuators, one acting in the x-direction and the other acting in the y- direction, as will be discussed in more detail with respect to Figs. 4 and 5. Movement of the sample holder 1 and of the sample 6 in pixel size steps can be achieved by changing the voltage of the respective piezoelectric actuators.

[0059] After a frame 15 is scanned / measured, a subsequent frame 15 is scanned / measured. The time it takes to scan a frame 15 determines if and to what extent it is possible to measure “in real time” the structural development of biological samples. To be able to observe conformational changes, it is necessary to be able to scan the lines 151 of each frame 15 with a frequency which allows to scan the complete frame 15 sufficiently fast to observe the conformational changes. It has been found that the ability to scan a line with 100 pixels of the frame 15 with a frequency of at least 10 Hz is required to have a time resolution that allows to observe structural changes of the sample - while keeping at the same time a reasonable spatial resolution. The scan area may, e.g., have a dimension of 4.5 x 4.5 pm.

[0060] The sSNOM of Fig. 1 is thus configured to allow scanning of each line 151 of each frame with a frequency of at least 10 Hz, preferably of at least 100 Hz. Of course, alternatively, each column could be scanned with such rate, wherein in such case the columns represent lines within the meaning of the present invention.

[0061] To achieve such scan rate, in an embodiment, the horizontal scanner 7 is designed in a particular manner as discussed with respect to Figs. 4 and 5.

[0062] Further, the components of the sSNOM are adapted for fast scanning. For example, the vertical scanner 9 has a frequency in the range between 60-100 kHz, this resulting in a scanning time of, e.g., 10 ps per pixel the z-direction. To implement the vertical scanner 9, a small piezoelectric stack may be implemented which comprises a high resonance frequency in the mentioned range. At the same time, the mass of the cantilever 22 of the scanning probe 2 is chosen to be small to ensure a small load and the 60-100 kHz resonance frequency of the piezoelectric stack.

[0063] The photoelastic modulator 52 implements a ZnSe crystal which is pushed for phase modulation. Using a photoelastic modulator and implementing phase modulation instead of amplitude modulation by a Michelson interferometer significantly reduces the noise of the near-field signal or a side band modulation.

[0064] Further improvement is achieved by the bottom illumination of the sample 6 and by combining an sSNOM and a High-speed AFM microscope 20.

[0065] It is further pointed out that the cantilever 22 of the scanning probe 2 is further attached to a shaker (not shown) which imprints a shaking movement onto the cantilever 22 and the scanning probe tip 21. The shaker provides a tapping frequency in the range between 200 and 1500 kHz in air. For example, the cantilever frequency is about 1400 kHz in air and 200 kHz in water. A short cantilever 22 is implemented to achieve such high frequency. The detector 51 implements a small detector chip which has a short rise time and a short exposure time to allow measurement of the signals for each pixel 150 of frame 15 in short time intervals in order to detect the signals with the same rate as the rate of scanning the lines 151 of each frame 15.

[0066] The wavelength of the infrared light 8 emitted by the IR laser 3 may be in the mid-infrared range, between 3 and 6.5 pm. For example, a laser may be used which emits in the range between 5.78 and 6.39 pm. Further, the detector 51 may be configured to detect light in the range between 2-14 pm, in particular in the range between 8 and 13 pm.

[0067] It is further pointed out that, in Fig. 1 , the horizontal scanner 7 comprises a half sphere 71 which comprises a flat top surface which is the surface of the sample holder. The half sphere 71 may be formed by Silicon (Si) as a highly refractive substrate. It allows for bottom illumination and enhances the numerical aperture.

[0068] Fig. 3 shows an embodiment of the general setup of Fig. 1 , showing additional details by way of example. In Fig. 3, the IR laser 3 and the optical detection device 5 are not shown. Infrared light 8 from the IR laser is guided through tilt mirror 41 , telecentric lenses 45, further mirror 43 (which is a 45 degrees mirror), lens 44 and half sphere 71 onto the scanning probe tip 21 of scanning probe 2. Lens 44 may be a focusing lens for the infrared light 8. It may be a ZnSe lens. It may also be a silver coated reflective lens. Also, lens 44 may be a lens system comprising a plurality of lenses.

[0069] The horizontal scanner 7 integrates the sample holder 1 in that it comprises the half sphere 71 on the flat top surface of which the sample 6 is located. The sample holder 1 and thus the horizontal scanner 7 comprises a top side 11 and a bottom side 12. The horizontal scanner 7 further comprises two piezoelectric actuators and two corresponding counter springs, wherein in Fig. 3 one such piezoelectric actuator 72 and the corresponding counter spring 74 is depicted. The horizontal scanner 7 will be explained with more detail with respect to Fig. 4.

[0070] A stepper approach motor 18 is used for a rough adjustment of the sample 6 with respect to the tip 21. Also, several micro positioners 171 , 172, 173 are provided.

[0071] The scanning probe tip 21 is connected to the vertical scanner 9. The AFM microscope 20 comprises a laser diode 201 , a focus lens 203 and a photodiode 202 such as a quadrant photodiode. The elements of the sSNOM and of the AFM microscope are held by a housing 30 which provides stability while the optical axis has an alignment in, e.g., 2.5 pm precision to place the tip 21 on the focal point of the infrared light.

[0072] It is pointed out that, in an embodiment, the area A between the top side 11 of the sample holder 1 including the top side of the optical element 71 and the scanning probe 2 including the scanning probe tip 21 and the cantilever 22 may be immersed in a liquid such as water (not shown). This may be sensible when having a biological probe 6 that needs to be in an aqueous solution. The cantilever 22 of course has a different resonance frequency when in an aqueous solution. As discussed, the resonance frequency of the cantilever 22 in water may be around 200 kHz.

[0073] When the area A is immersed in liquid, both the sSNOM and the AFM 20 are designed with a liquid system. In this respect, it is important to provide the cantilever 22 the possibility to be immersed in the liquid (such as water).

[0074] Fig. 4 shows an embodiment of the horizontal scanner ?. In its center the horizontal scanner 7 comprises an optical element 71 which has a flat top surface 710 on which the sample (not shown) is to be placed. The optical element 71 is transparent for infrared light, which passes the optical element for bottom illumination as discussed before. For example, the optical element 71 is made of Si (Silicon) or ZnSe (Zinc selenide). The optical element 71 may be formed as a half sphere as shown in Figs. 1 and 3.

[0075] The optical element 71 is arranged in an octagonal structure 78, which is centered in an octagonal recess 76 of horizontal scanner 7, wherein the octagonal structure 78 and the octagonal recess 76 are separated by a trench / gap 77 which allows it to move the octagonal structure 78 with the optical element 71 relative to the octagonal recess 76.

[0076] The horizontal scanner 7 further comprises at one side of the octagonal recess 76 a first piezoelectric actuator 72 which is configured to move the optical element 71 in the x- direction for scanning lines of each frame (see Fig. 2). There is further provided at another side of the octagonal recess 76 a second piezoelectric actuator 73 which is configured to move the optical element 71 in the y-direction. Together, the piezoelectric actuators 72, 73 are configured to scan a two-dimensional frame such as frame 15 of Fig. 2. To this end, the voltage applied to the actuators 72, 73 is raised (in steps or linearly). For example, the voltage applied to actuator 72 is raised linearly in each line, wherein all pixels in a line are passed. The respective z-value of the pixels is determined by the vertical scanner. After all pixels in one line have been scanned, the voltage applied to actuator 73 is changed to arrive at the next step in the y-direction, and subsequently the next line is scanned by raising again the voltage applied to the actuator 72.

[0077] The horizontal scanner 7 of Fig. 4 further comprises a first counter spring 74 and a second counter spring 75. The counter springs 74, 75 are arranged opposite the first and second actuators 72, 73, respectively. The counter springs 74, 75 allow to move the optical element 71 in the x- and y-directions by the actuators 72, 73 in a resilient and damped manner.

[0078] There is further provided a resilient structure 161-164 that couples the actuators 72, 73 and the springs 74, 75 with the octagonal structure 78 and thus with the optical element 71. The resilient structure consists of flexible and bendable connecting elements 161-164.

[0079] Fig. 5 shows the horizontal scanner 7 of Fig. 4 without depicting the actuators 72, 73 and the counter springs 74, 75. The situation is depicted when, by means of the actuator 72, the octagonal structure 78 and the optical element 71 has been moved in the x-direction (thereby compressing spring 74 of Fig. 4). The connecting elements 161-164 connect the octagonal structure 78 with the octagonal recess 76 in a flexible manner. Further, the recess 76 may be filled with an elastomer such as a silicone elastomer to provide for passive damping.

[0080] The piezoelectric actuators 72, 73 have a frequency of at least 10 Hz per line for a line that has 100 pixels, which means that a complete line 151 (consisting of 100 pixel) of a frame 15 (see Fig. 2) is scanned in at least 0.1 s. Accordingly, the scan rate is 0.001 s per pixel and a frame of 100 lines is scanned in 10 s. In an embodiment, a complete line of 100 pixel is scanned with a frequency of 100 Hz, which means that a frame with 100 x 100 pixel is scanned in 1 second.

[0081] The piezoelectric actuators 72, 73 are implemented as small piezoelectric stacks with high resonance frequency.

[0082] The connecting elements 161-164 may be made of titanium grade 5 and have a thickness between 0.1 mm and 0.5 mm, in particular 0.2 mm so that the connecting elements are flexible, rigid and allow to move the octagonal structure 78 with the optical element 71 with speed / high frequency. Fig. 6 shows an embodiment of a scanning probe 2 that comprises a cantilever 22 and a tip 21 . The cantilever 22 is attached to a vertical scanner 9, as can be seen in Fig. 8 which is a sectional view of the embodiment of Fig. 6. The vertical scanner 9 comprises a piezoelectric stack 91 with a resonance frequency in the range between 50 and 150 kHz. In an embodiment, the resonance frequency is 100 kHz. Optionally, there is provided a beam clamp to hold the cantilever 22. Adhesive may be used for fixation. The vertical scanner 9 moves cantilever 22. The cantilever 22 has a small mass such that the resonance frequency of the cantilever is the same or similar to the resonance frequency of the vertical scanner 9.

[0083] Fig. 7 is an example embodiment of a scanning probe with a cantilever 22 and a tip 21. The material of the cantilever may be Si or Si3N4. The tip 21 may of or coated with platinum Pt. The length of the cantilever 22 may be 160 pm or less, such as 60 pm. The width of the cantilever 22 is 10 pm or less. The thickness of the cantilever 22 is 1.5 pm or less. The resonance frequency is 1 ,400 kHz or more in air (equivalent to about 200 kHz or more in water). The oscillation amplitude in z-direction may be 40 nm or more.

[0084] The scanning probe of Fig. 7 is optimized for fast sSNOM and has been experimentally evaluated as indicated in Fig. 9. Fig. 9 indicates frequency f on the x-axis and phase ph on the y-axis. Two different tips have been measured, a state of the art tip (graph 901) and a fast scanning cantilever tip in accordance with Figure 7 (graph 902). The measurement was in water, and during the measurement the tip and the sample were engaged with the tapping mode at the resonance frequency: as mentioned before, the scanning probe tip is coupled to a vertical scanner which comprises a piezoelectric element. Further, the cantilever is attached to a shaker imprinting a shaking movement onto the cantilever and the scanning probe tip, the shaker providing the tapping frequency. While the tip and the sample are engaged with the tapping mode at the resonance frequency, the response of the cantilever to actuation of the vertical scanner was measured at each frequency. Phase decay identifies the phase difference between the input phase into the piezoelectric element of the vertical scanner and the output phase of the cantilever, wherein ideally this phase difference is zero. A phase decay starts at about 250 Hz for the state of the art tip, graph 901 , and only at about 8 kHz for the arrow-formed tip of Fig. 7, graph 902, this indicating the advantages of the tip of Fig. 7. Respective oscillation amplitudes of the cantilever for optimal near-field signal or high-speed AFM are above 50 nm or ca 2 nm, respectively. The tip of Fig. 7 has a mechanical bandwidth more than an order of magnitude higher than the conventional sSNOM lever in water. Fig. 10 shows experimental results of the sSNOM microscope system in accordance with the present invention. The results include an AFM topography image (left-hand side), an sSNOM optical amplitude image (middle) and an sSNOM optical phase image (right hand side). Horizontal scanning was implemented with a frequency of 10 Hz (10 lines per second). The wave number of the infrared light was 1665 cm-1. The experiment was conducted with the sample and the scanning probe in water. The sSNOM optical amplitude image and the sSNOM optical phase image provide additional information.

[0085] Fig. 11 shows an improvement of a Michelson interferometer. As mentioned before, a Michelson interferometer may be used as an alternative to a photoelastic modulator to implement the optical detection device 5 of Fig. 1. Fig. 11 illustrates an improvement in the reference arm of a Michelson interferometer. As known to the skilled person, in a Michelson interferometer, a reference beam reflects off a mirror in the reference arm, while a sample beam reflects off a mirror in the sample arm, wherein the beams are then recombined at the beam splitter. The reference arm is crucial because it provides a stable, known path length for one of the beams.

[0086] In the left drawing a) of Fig. 11 , a conventional reference arm is depicted, which comprises a piezoelectric stack 111 and a mirror 112. Piezoelectric stacks are used because they can make very fine adjustments to the position of the mirror in the reference arm, allowing for accurate phase shifts and interference pattern adjustments. The reference arm may oscillate laterally with the frequency of 350 Hz or more which is unwanted. The right drawing b) of Fig. 11 shows an improvement with a faster Michelson reference arm which may be implemented with a Michelson interferometer used with the present invention. Here, the size of the mirror has been reduced. For example, a small gold coated mirror 113 is provided for. Further, the piezo stack 112 is preloaded with a force p. This improves the mechanical stability and overall performance. The piezo stack 112 may now move vertically (arrow 114) without lateral oscillation up to 1200 Hz estimated from near-field signals.

[0087] It should be understood that the above description is intended for illustrative purposes only and is not intended to limit the scope of the present disclosure in any way. Also, those skilled in the art will appreciate that other aspects of the disclosure can be obtained from a study of the drawings, the disclosure and the appended claims. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Various features of the various embodiments disclosed herein can be combined in different combinations to create new embodiments within the scope of the present disclosure. In particular, the disclosure extends to and includes all combinations and sub-combinations of one or more features described herein. Any ranges given herein include any and all specific values within the range and any and all sub-ranges within the given range.

Claims

CLAIMS1. A scattering-type scanning near-field optical microscope (sSNOM) system which comprises: a sample holder (1) having a top side (11) and a bottom side (12), wherein the sample holder top side (11) is configured to hold a sample (6), a scanning probe (2) having a tip (21), an IR-laser (3) emitting infrared light (8), means (41-44) for directing the emitted infrared light (8) through the sample holder (1) onto the scanning probe tip (21), wherein the infrared light passes the sample holder (1) from the bottom side (12) to the top side (11), and wherein the scanning probe tip (21) is configured to be located above the sample (6) at a near-field distance such that a near-field is generated between the tip (11) and the sample (6) when the tip (21) is illuminated with the infrared light (8); and an optical detection device (5), the optical detection device (5) comprising means (52) for recovering phase and / or amplitude of the light scattered from the scanning probe tip (21) and further scattered and modified by the sample (6), and the optical detection device (5) further comprising a detector (51), wherein the sample holder (1) and the scanning probe tip (21) are adjustable relative to each other in the x-, y- and z-directions by means of a horizontal scanning device (7) configured to move the sample holder (1) in the x-, y- directions and by means of a vertical scanning device (9) configured to move the scanning probe tip (21) in the z-direction, wherein the horizontal scanning device (7) is configured to move the sample (6) located on the top side (11) of the sample holder (1) in the x- and y-directions relative to the scanning probe tip (21), wherein a plurality of frames (15) is scanned subsequently, each frame (15) representing the scan area of the sample at a specific time interval, and each frame (15) comprising pixels (150) arranged in lines (151) and columns (152) in the x- and y-directions, wherein for each pixel(150) a measurement of phase and / or amplitude is made, and wherein the horizontal scanning device (7) is configured to move the sample (6) along the lines(151) of each frame (15) with a predetermined frequency.

2. The system of claim 1 , characterized in that the horizontal scanning device (7) is configured to move the sample (6) along the lines (151) of each frame (15) with at leasta frequency that corresponds to scanning a line of 100 pixels with a frequency of 10 Hz.

3. The system of claim 1 , characterized in that the horizontal scanning device (7) is coupled to or integrated with the sample holder (1).

4. The system of claim 2 or 3, characterized in that the horizontal scanning device (7) comprises: an optical element (71) having a flat top surface (710), the flat top surface (710) being the top surface (11) of the sample holder (1), wherein the optical element (71) is transparent for infrared light and wherein the infrared light passes the optical element (71) from the bottom to the top, a first piezoelectric actuator (72) configured to move the optical element (71) for scanning lines (151) of each frame (15) in the x-direction, a second piezoelectric actuator (73) configured to move the optical element (71) in the y-direction,- wherein the first and second piezoelectric actuators (72, 73) are configured to scan a two-dimensional frame (15).

5. The system of claim 4, characterized in that the optical element (71) is a half sphere having a flat top surface (710), the flat top surface (710) forming the top side (11) of the sample holder (1) on which the sample (8) is located.

6. The system of claim 4 or 5, characterized in that the first piezoelectric actuator (72) and the second piezoelectric actuator (73) are associated with a first and second counter spring (74, 75), wherein the first piezoelectric actuator (72) is located in the x- direction at one end of the optical element (71) and the first counter spring (74) is located in the x-direction at the opposite end of the optical element (71), and wherein the second piezoelectric actuator (73) is located in the y-direction at one end of the optical element (71) and the second counter spring (75) is located in the y-direction at the opposite end of the optical element (71).

7. The system of claim 6, characterized in that the optical element (71) is held by a resilient structure (161-164) connecting the optical element (71) with the first and second piezoelectric actuators (72, 73) and the first and second counter springs (74, 75).

8. The system of claim 6, characterized in that the optical element (71) is arranged in an octagonal structure (78) which is located in an octagonal recess (76) in the horizontal scanning device (7) and is movable relative to the octagonal recess (76) dependent on the actuation status of the first and second piezoelectric actuators (72, 73).

9. The system of any of the preceding claims, characterized in that the vertical scanning device (9) is configured to modulate the scanning probe tip (21) in the vertical direction with a frequency of at least 60 kHz.

10. The system of claim 9, characterized in that the vertical scanning device (9) comprises a piezoelectric element (91) with a resonance frequency of at least 60 kHz, and characterized in that the scanning probe (2) comprises a cantilever (22) to which the scanning probe tip (21) is attached.11 . The system of claim 10, characterized in that the cantilever (22) is further attached to a shaker imprinting a shaking movement onto the cantilever (22) and the scanning probe tip (21), the shaker providing a tapping frequency in the range between 200 kilohertz and 1500 kilohertz.

12. The system of any of claims 10 to 11 , characterized in that the system further comprises a lock-in measurement device configured to make a lock-in measurement at a lock-in frequency which is the oscillation frequency of the cantilever (22).

13. The system of any of the preceding claims, characterized in that an area (A) between the top side (11) of the sample holder (1) and the scanning probe (2) including the scanning probe tip (21) is immersed in a liquid.

14. The system of any of the preceding claims, characterized in that the wavelength of the infrared light (8) lies in the region between 2 and 8 pm.

15. The system of any of the preceding claims, characterized in that the optical detection device (52) comprises an interferometer.

16. The system of any of claims 1 to 14, characterized in that the optical detection device (52) comprises a photoelastic modulator.

17. The system of any of the preceding claims, characterized in that the system further comprises an AFM microscope (20), the scanning probe (2) being the probe of the AFM microscope (20).

18. The system of claim 17, characterized in that for each pixel (150) a measurement of phase and / or amplitude is made both with the AFM microscope (20) and with the sSNOM.

19. The system of any of the preceding claims, characterized in that the system is configured to measure in real time a sample (6) which is a living organic sample.

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