Method of operating an AFM

Abstract

A method of operating an atomic force microscope, comprising a probe, the probe being moved forth and back during respective trace and retrace times of a scan line, the method comprising: a) during trace time, oscillating the probe, b) generating a z feedback signal to keep an amplitude of oscillation of the probe constant at a setpoint value, the z feedback signal being generated by a first feedback loop, c) during retrace time, placing the probe in a drift compensation state by changing the setpoint value to a different value so that the z feedback signal being generated by the first feedback loop causes the probe to move away from the sample and oscillate free, d) detecting an amplitude of free oscillation of the probe and adjusting with a second feedback loop its excitation signal to maintain the amplitude of free oscillation of the probe close to a set value.

Claims

1. A method of operating an atomic force microscope (AFM), comprising a probe displaceable relative to a sample along scan lines in an imaging x-y plane and along a z axis perpendicular to the x-y plane, the probe being moved forth and back during respective trace and retrace times of a scan line, and caused to oscillate by an excitation signal, the method comprising: a) setting a setpoint amplitude of a probe oscillation of the AFM at a value equal to A.sub.set and a parameter of free oscillation amplitude of the AFM at a value equal to A.sub.free, b) during trace time, placing the probe in an operating state in which the probe oscillates along the z axis and interacts with the sample, c) generating a z feedback signal to displace the probe relative to the sample along the z axis to keep an amplitude of oscillation of the probe constant at the value of the setpoint amplitude A.sub.set, the z feedback signal being generated by a first feedback loop at least on the basis of the value of the setpoint amplitude A.sub.set and having as input a measured amplitude value of oscillation of the probe, d) during retrace time, placing the probe in a drift compensation state by changing the value of the setpoint amplitude of the probe oscillation by sending a trigger signal to a processor, which generates a setpoint adjust signal, wherein the setpoint adjust signal is a voltage of an amplitude of probe oscillation chosen such that the setpoint amplitude of probe oscillation of the first feedback loop is changed from A.sub.set to a different value equal to A.sub.set+A.sub.mock, wherein A.sub.set+A.sub.mock is larger than A.sub.free, and wherein A.sub.mock is a mock amplitude value to be added to A.sub.set so that the z feedback signal being generated by the first feedback loop causes the probe to move away from the sample and oscillate free of interaction with the sample, e) detecting an amplitude of free oscillation of the probe and adjusting with a second feedback loop the excitation signal of the probe to maintain the amplitude of free oscillation of the probe close to the value of the parameter of free oscillation amplitude A.sub.free, f) returning the probe in interaction with the sample for a next scan line by restoring value of the setpoint amplitude of the probe oscillation to a value equal to A.sub.set for the generation of the z feedback signal by the first feedback loop while applying the adjusted excitation signal of previous step to the probe.

2. The method according to the claim 1, wherein the trigger signal for changing the value of the setpoint amplitude of the probe oscillation from A.sub.set to the value A.sub.set+A.sub.mock sent to the first feedback-loop at the beginning of the retrace time of the scan line is generated by converting a triangular x-scanning signal of the probe into a square wave by a differentiator.

3. The method according to claim 1, wherein A.sub.set is set to be between 90% of A.sub.free and A.sub.free.

4. The method according to claim 1, wherein the value of the parameter of free oscillation amplitude A.sub.free is set between 0.5 nm and 2 nm.

5. The method according to claim 1, in which A.sub.set is sent to the first feedback loop as a voltage input signal, the second feedback loop generating the setpoint amplitude adjust voltage signal that adds the mock value A.sub.mock to A.sub.set for changing the value of the setpoint amplitude from A.sub.set to A.sub.set+A.sub.mock.

6. The method according to claim 1, in which A.sub.set is sent to the first feedback loop as a voltage input signal, the second feedback loop generating the setpoint amplitude adjust voltage signal that substitute to A.sub.set the value A.sub.set+A.sub.mock for changing the value of the setpoint amplitude of the probe oscillation from A.sub.set to A.sub.set+A.sub.mock.

7. The method according to claim 1, the amplitude of free oscillation being detected at least ten times during of the retrace time of the scan line.

8. The method according to claim 7, an average value of the detected amplitude of free oscillation being calculated and compared to the set value A.sub.free for adjusting the amplitude of free oscillation.

9. The method according to claim 1, wherein detection of the amplitude of free oscillation is performed for every scan line.

10. The method according to claim 1, the scanning being performed with different values for of setpoint amplitude A.sub.set at respective subregions of the sample.

11. The method according to claim 1, being applied for estimating kinetic parameters related to protein-protein interactions.

12. A method of operating an atomic force microscope (AFM), comprising a probe displaceable relative to a sample along scan lines in an imaging x-y plane and along a z axis perpendicular to the x-y plane, the probe being moved forth and back during respective trace and retrace times of a scan line, and caused to oscillate by an excitation signal, the method comprising a) setting a setpoint amplitude of a probe oscillation of the AFM at a value equal to A.sub.set and a parameter of free oscillation amplitude of the AFM at a value equal to A.sub.free, b) during trace time, placing the probe in an operating state in which the probe oscillates along the z axis and interacts with the sample, c) generating a z feedback signal to displace the probe relative to the sample along the z axis to keep an amplitude of oscillation of the probe constant at the value of the setpoint amplitude of the probe oscillation A.sub.set, the z feedback signal being generated by a first feedback loop at least on the basis of the value of the setpoint amplitude of the probe oscillation A.sub.set and having as input a measured amplitude value of oscillation of the probe, d) during retrace time, placing the probe in a drift compensation state by changing the value of the setpoint amplitude of the probe oscillation by sending a trigger signal to a processor, which generates a setpoint adjust signal, wherein the setpoint adjust signal is a voltage of an amplitude of probe oscillation chosen such that the setpoint amplitude of probe oscillation of the first feedback loop is changed from A.sub.set to a different value equal to A.sub.set+A.sub.mock which is larger than A.sub.free, wherein A.sub.mock is a mock value to be added to A.sub.set so that the z feedback signal being generated by the first feedback loop causes the probe to move away from the sample and oscillate free of interaction with the sample, e) detecting an amplitude of free oscillation of the probe and adjusting with a second feedback loop the excitation signal of the probe to maintain the amplitude of free oscillation of the probe close to the value of the parameter of free oscillation amplitude A.sub.free, f) returning the probe in interaction with the sample for a next scan line by restoring the value of the setpoint amplitude of the probe oscillation to a value equal to A.sub.set for the generation of the z feedback signal by the first feedback loop while applying the adjusted excitation signal of previous step to the probe, wherein A.sub.set+A.sub.mock is at least 5% larger than A.sub.free.

13. An atomic force microscope (AFM), comprising: a probe displaceable relative to a sample along scan lines in an imaging x-y plane and along a z axis perpendicular to the x-y plane, the probe being configured for being moved forth and back during respective trace and retrace times of a scan line, and caused to oscillate by an excitation signal, a detector for measuring an amplitude of oscillation of the probe, a first feedback loop configured for generating a z feedback signal to displace the probe relative to the sample along the z axis to keep an amplitude of oscillation of the probe constant at a value of the parameter of setpoint amplitude of the probe oscillation set to be equal to A.sub.set during the trace time, and a second feedback loop configured for changing, in the first feedback loop, the value of the setpoint amplitude of the probe oscillation by sending a trigger signal to a processor, which generates a setpoint adjust signal, wherein the setpoint adjust signal is a voltage of an amplitude of probe oscillation chosen such that the setpoint amplitude of probe oscillation changes A.sub.set to a different value A.sub.set+A.sub.mock during the retrace time, wherein A.sub.set+A.sub.mock is larger than A.sub.free, and wherein A.sub.mock is a mock amplitude value to be added to A.sub.set so that the first feedback loop causes the probe to move away from the sample and oscillate free of interaction with the sample, the second feedback loop being configured for adjusting the excitation signal of the probe to maintain the amplitude of free oscillation of the probe close to a set value equal to the value of the parameter of amplitude of free oscillation set to be equal to A.sub.free.

14. The atomic force microscope according to claim 13, comprising a differentiator configured for converting a triangular x-scanning signal of the probe into a square wave, and a processor configured for receiving the square wave as the trigger signal for sending the setpoint amplitude adjust signal to the first feedback loop.

15. The atomic force microscope according to claim 13, the second feedback loop being configured for restoring the value of the setpoint amplitude of the probe oscillation to A.sub.set in the first feedback loop after adjustment of the excitation signal of the probe.

16. The atomic force microscope according to claim 13, the first feedback loop comprising a proportional-integral-derivative controller (PID controller).

17. The atomic force microscope according to claim 13, being configured for calculating an average value of the amplitude of free oscillation detected during the retrace time of a scan line, the adjustment of the amplitude of free oscillation being performed, for a scan line, at least on the basis of the comparison between the set value A.sub.free and the average value detected.

Description

(1) Preferred exemplary embodiments of the invention are illustrated in the accompanying drawings in which like reference numerals represent like parts throughout, and in which:

(2) FIG. 1 is a schematic representation of a portion of an AFM according to the present invention;

(3) FIG. 2 is a flow chart representing an embodiment of the invention;

(4) FIGS. 3a to 3c illustrate scan signals during free amplitude detection in a method according to the present invention;

(5) FIG. 4a is an image observed during retrace time of a scan line in a method according to the present invention and FIG. 4b shows a corresponding scan signal;

(6) FIGS. 5a to 5f are images during retrace time of a scan line in a method according to the present invention with different values of A.sub.set;

(7) FIG. 6 illustrates the variation of the amplitude of free oscillation when the second feedback loop is respectively on and off;

(8) FIG. 7a illustrates another example of the method of the invention and FIG. 7b shows images obtained in this example, and

(9) FIGS. 8 and 9 show results of the method illustrated in FIG. 7a.

(10) An AFM in accordance with the invention, as illustrated in FIG. 1, comprises a probe carried by a support (not shown) and comprising a cantilever 1 and a tip 2 attached to a free end of the cantilever 1.

(11) A sample (not shown) is mounted on a sample stage 8. An x-y-z drive 7 comprising a z-piezo, an x-piezo and a y-piezo (not shown) is used to displace the sample stage 8 with the sample in the x, y, and z directions with respect to the tip 2.

(12) The AFM is connected to a processor such as a personal computer, which serves, among other things, to generate scanning signals to the probe. The same computer may also be used for analysis of the scanning data and image formation on the basis of the scanning data.

(13) Although FIG. 1 shows the drive attached to the sample stage, the drive may also in a variant embodiment be attached to the probe, or independent drives may be attached to both, since it is the relative displacement of the sample and tip that needs to be controlled.

(14) In tapping mode, the cantilever 1 is driven to oscillate up and down along the z-axis, by acoustic waves, at or near its resonance frequency. This oscillation is commonly achieved with an excitation piezo 3 in the cantilever support, but other possibilities do not go beyond the scope of the invention. The excitation piezo 3 may be a piezoelectric element.

(15) The value of the amplitude of free oscillation A.sub.free when the probe is far away from the sample not to interact therewith is usually chosen to between less than 1 nm and 200 nm. When the sample consists of soft biological material, A.sub.free is preferably about 1 nm in order to avoid damaging the material.

(16) When the tip 2 is driven by the z-piezo close to the surface of the sample, this causes interaction between the sample and the tip 2. This interaction causes the amplitude of the cantilever's oscillation to change (usually decrease) as the tip 2 gets closer to the sample. This change of the amplitude of oscillation may be detected by measuring the deflection of the cantilever 1.

(17) As shown in FIG. 1, the cantilever-deflection measurement may be performed optically by a beam-deflection method. In such method, laser light from an emitter 4, for example a solid-state diode, is reflected off the back of the cantilever 1 and collected by a position sensitive detector (PSD) 5. The AFM comprises an amplitude detector 10 that receives the signal generated by the detector 5 and generates an amplitude signal transmitted to the first feedback loop. This signal is a voltage in the described embodiment.

(18) The amplitude detector also generates an excitation signal for oscillating the probe.

(19) When using the AFM to image the sample, the tip 2 is brought in interaction with the sample, and the sample is raster scanned along the x-y imaging plane.

(20) The amplitude signal delivered by the amplitude detector 10 is used as an input parameter in a first electronic feedback loop 11 that controls the height z of the probe above the sample. The first feedback loop 11 comprises for example a PID controller.

(21) As the probe scans over the sample, the first electronic feedback loop 11 adjusts the height z to maintain a user defined set cantilever oscillation amplitude, referred to as setpoint value A.sub.set.

(22) The first feedback loop 11 outputs a signal that controls the distance along the z axis between the probe support (not shown) and the sample stage 8.

(23) As the probe is in an operation state and the sample is scanned, height variations of the sample due to its topography will tend to change the amplitude of oscillation of the probe which is detected thanks to the detector 5. The first feedback loop 11 reacts to adjust the height of the probe support via the z-feedback signal sent to the z-piezoso that the deflection of the cantilever 1 representative of the oscillation amplitude is kept constant and equal to the setpoint value A.sub.set.

(24) Typically, A.sub.set is chosen to be between 95% and 99% of the amplitude of free oscillation of the probe A.sub.n.

(25) The AFM further comprises a second feedback loop 12.

(26) This second feedback loop 12 receives three input signals: (i) the excitation signal generated by the amplitude detector 10 for oscillating the probe, (ii) the same amplitude signal that the amplitude detector 10 sends to the first feedback loop 11 for controlling the distance along the z axis between the probe support and the sample stage 8, and (iii) the x-scanning signal that the computer 16 sends to the x-piezo to control the back and forth movement of the probe along the x-scan lines.

(27) The second feedback loop 12 sends two output signals out: (i) a setpoint adjust signal to the first feedback loop 11 for inducing the z-piezo retraction for detection of the amplitude of free oscillation, and (ii) a modified excitation voltage to the excitation piezo 3.

(28) The x-scanning signal is a triangular signal as shown in FIG. 3a and is converted into a square wave by a differentiator in the second feedback loop 12. This square wave is used as a trigger signal to detect the beginning of each retrace time.

(29) This trigger signal is sent to a processor which generates in turn a setpoint adjust signal sent by the second feedback loop to the first feedback loop 11. This signal is a voltage of an amplitude chosen such that the setpoint value of the first feedback loop is changed from A.sub.set to a value A.sub.set+A.sub.mock that is about 10% larger than A.sub.free.

(30) As a consequence, the PID controller of the first feedback loop drives away the tip from the sample and the probe is placed in a drift compensation state.

(31) During this period of lift-off, the actual amplitude of free oscillation of the probe is detected for example about 10 times and an accurate average is calculated by the processor. The averaged value is compared to the set value A.sub.free and an altered excitation voltage signal is sent by the second feedback loop 12 to the excitation piezo 3 to compensate for any amplitude drift so the free amplitude remains constant and equal to A.sub.free.

(32) Before the retrace time ends, the setpoint value of the imaging amplitude is restored to the original value A.sub.set, and the first feedback loop will automatically, via the z-feedback signal, bring the tip 2 back into interaction with the sample surface for next trace scan line.

(33) The drift compensation is preferably carried out for every x-scan line, meaning that in a classical AFM the force exerted by the probe on the sample would be adjusted between one and ten times per second and in HS-AFM several hundreds of times per second.

(34) In both cases, free amplitude drift occurs in the seconds to minutes range, and therefore the invention can be applied to any type of amplitude modulation AFM.

(35) The flow chart of FIG. 2 illustrates, for an x-scan line, the sequence of operations performed by the AFM in accordance with the invention.

(36) During image acquisition a triangular voltage signal as illustrated in FIG. 3a is sent to the x-piezo which leads to forth and back x scanning. In a method according to the invention, every time the x scanning turns around, i.e. the x-piezo voltage changes from increasing to decreasing values and the relative scan motion changes from left-right to right-left, a trigger signal is generated.

(37) When the second feedback loop 12 receives this trigger signal, it sends the setpoint adjust signal to the first feedback loop 11. As a consequence, the z-piezo immediately retracts the probe to result in complete separation of the tip 2 from the sample surface. This is illustrated in FIG. 3b showing the tip-sample distance.

(38) As illustrated in FIG. 3c, a short amplitude analysis window opens when the actual free amplitude is detected. Immediately after free amplitude detection, the second feedback loop reinitiates the first feedback loop with the imaging amplitude setpoint value A.sub.set, smaller than A.sub.free. The first feedback loop automatically drives the probe back into contact with the sample surface, before starting the next trace imaging fast scan line.

(39) As a consequence of the performed free amplitude detection, the second feedback loop adjusts the excitation power that is sent to cantilever excitation piezo 3 to keep the amplitude of free oscillation substantially constant to the set value A.sub.free.

EXAMPLES

(40) Material and Methods:

(41) High-Speed Atomic Force Microscopy (HS-AFM)

(42) All images in the following examples were taken by amplitude modulation mode HS-AFM from the company RIBM, Japan. 8 m short cantilevers Ultra-Short Cantilevers (USC) from the company NanoWorld, Switzerland with nominal spring constant k of 0.15 N/m, resonance frequency f(r) of 0.6 MHz and a quality factor Q of about 1.5 in buffer, were used. The HS-AFM fluid cell was connected to a buffer control system constituted by a constant pressure and flow pump from the company Harvard Instruments, USA connected over two silicon tubes to the fluid cell.

Example 1

(43) The imaging conditions areas follows: the value of free amplitude A.sub.free is set to 1 nm, the setpoint value of the imaging amplitude A.sub.set is 90% of A.sub.free, i.e. 0.9 nm. The frame size is 200200 pixels and the scan speed is 1 frame per second. A.sub.set+A.sub.mock is adjusted to 110% of A.sub.free, leading to complete separation of tip and sample, here about 4.5 nm. Such distance is far enough from the surface to detect accurately the actual free amplitude, that is around 1 nm and close enough to the surface to reach contact before beginning of the next trace imaging scan line.

(44) The retrace image illustrated in FIG. 4a shows a vertical bright line on the right edge. This line corresponds to the lift-off regime during which the amplitude of free oscillation is detected.

(45) As illustrated in FIG. 4b, when the probe moves along an x-scan line (from right to left in the illustrated figure), the tip-sample distance reaches a peak.

(46) Tests are performed with A.sub.set chosen to 90%, 92%, 94%, 96% 98% or 99%. The results of the tests, illustrated respectively in FIGS. 5a to 5f, show that at A.sub.set=98% of A.sub.free, the return of the probe back to the sample is clearly visible but the tip gets readily back into contact to the sample surface before the next trace scan. Only when A.sub.set=0.99 A.sub.free, the return might eventually reach the next trace scan line, as can be seen in FIG. 5f.

(47) However, typical amplitude modulation AFM is performed at about A.sub.set=0.95 A.sub.free, which is sufficiently for getting the tip readily back into contact to the sample surface before the next trace scan.

Example 2

(48) A read out of the amplitude of free oscillation for 90 minutes is performed with the second feedback loop active (period I)-off (period ID-active (period III)-off (period IV). It can be seen in FIG. 6 that during periods I and III when the second feedback loop was active, the amplitude of free oscillation is maintained substantially constant, while free amplitude changes were detected during periods II and IV when the second feedback loop was switched off.

(49) In this example, at around 40 minutes, the amplitude of free oscillation A.sub.free is raised from 1 nm to 1.8 nm within only 3 minutes. When the setpoint value of the imaging amplitude A.sub.set is kept constant at 0.95 nm, this free amplitude change corresponds to a force increase from about 55 pN to about 730 pN. The above mentioned average force is estimated following equation eq.1, where the cantilever spring constant k=0.15 N/m and the quality factor Q=1.5 in liquid.

(50) $\begin{array}{cc}.Math.F.Math.{\frac{{\mathrm{kA}}_{\mathrm{free}}}{2Q}\left[1-{\left(\frac{A_{\mathrm{set}}}{A_{\mathrm{free}}}\right)}^2\right]}^{1/2}& \left(\mathrm{eq}.1\right)\end{array}$

(51) A force of about 730 pN is excessive and damaging for fragile biological samples like proteins.

(52) Detecting by eye changes in the image appearance and readjusting the setpoint force during imaging based on experience is undesirable. The operator's evaluation of the image derivations is a highly subjective parameter and implies that the operator knows what a good representation of the object under study was. Also, it is not exactly the same to scan at A.sub.free of 1 nm with A.sub.set of 0.95 nm or to scan at A.sub.free of 1.8 nm trying to adjust A.sub.set manually to reach similar image quality.

Example 3

(53) In addition to free amplitude control for long-duration force-controlled imaging stabilization, the second feedback loop can equally be used to adjust the setpoint value of imaging amplitude A.sub.set together with A.sub.free in subregions of images.

(54) To illustrate this function, the chaperonin GroEL double rings were imaged head-on adsorbed onto the mica support at four different well-defined imaging forces as shown in FIGS. 7a and 7b.

(55) GroEL is a barrel-shaped chaperonin of about 14 nm in diameter and about 16 nm in height, consisting of two stacked rings (14 nm in diameter and 8 nm in height). Under many conditions the chaperonin adsorbs in end-up orientation on the mica surface. In GroEL, the two rings are only weakly bound and can be dissociated as function of the applied force and hence makes it possible to evaluate the sensitivity of the force controller.

(56) Sample Preparation:

(57) GroEL from the company Abcam, France was diluted in a buffer solution (20 mM Tris, pH 7.3, 30 mM KCl, 10 mM MgCl.sub.2) to a final concentration of 100 nM. 1 ul of GroEL was put onto freshly cleaved mica for one hour at room temperature in a humid chamber. Then non-adsorbed GroEL was rinsed off with the same buffer.

(58) Imaging:

(59) The imaging parameters were 200200pixels, 400400 nm, 2 s/frame, and A.sub.free=1 nm.

(60) During the first 50 lines (part A), the setpoint value of imaging amplitude A.sub.set=97% of A.sub.free, the second 50 lines (part B) were imaged with A.sub.set=95% of A.sub.free and the third 50 lines (part C) with A.sub.set=93% of A.sub.free and the last 50 lines (part D) with A.sub.set=91% of A.sub.free.

(61) In accordance with the present invention, A.sub.free was set to 1 nm and kept constant at this value. A.sub.set was modulated to the above-mentioned fractions of A.sub.free every 50 lines. The estimated average applied loading forces are 33 pN, 55 pN, 76 pN and 97 pN for each section, as estimated following equation eq.1.

(62) Data Analysis:

(63) HS-AFM images were analyzed by using the particles analysis plugin in the software Image J.

(64) The results as illustrated in FIGS. 8 and 9 indicate that in the low-force regime (<76 pN) initial dissociation rate is force-independent and reports about the protein-stability, while the following dissociation rates seem to rather report protein dissection due to X-scanning process related lateral forces that is constant at low forces.

(65) This suggests that amplitude modulation AFM should be operated at A.sub.set0.93A.sub.free, (here A.sub.free=1 nm and with optimized feed-back parameters) because under such low amplitude damping the vertically applied force does not cross-talk into lateral forces leading to destruction of the biological sample.

(66) The e capacity of imaging at various forces in one image allows to test for ideal aging conditions within a single frame and hence avoid long force adjustment experiments that risk to contaminate the tip. Moreover, it makes it possible to analyze the effect of force on the biological sample and derive biophysical parameters from it.

(67) The present invention is not limited to the embodiments and examples illustrated.

(68) For example, oscillation of the cantilever may be achieved with an AC magnetic field (with magnetic cantilevers), piezoelectric cantilevers, or periodic heating with a modulated laser beam.