Patent Abstract:
An atomic force microscope is described having a cantilever comprising a base and a probe tip on an end opposite the base; a cantilever drive device connected to the base; a magnetic material coupled to the probe tip, such that when an incrementally increasing magnetic field is applied to the magnetic material an incrementally increasing force will be applied to the probe tip; a moveable specimen base; and a controller constructed to obtain a profile height of a specimen at a point based upon a contact between the probe tip and a specimen, and measure an adhesion force between the probe tip and the specimen by, under control of a program, incrementally increasing an amount of a magnetic field until a release force, sufficient to break the contact, is applied. An imaging method for atomic force microscopy involving measuring a specimen profile height and adhesion force at multiple points within an area and concurrently displaying the profile and adhesion force for each of the points is also described. A microscope controller is also described and is constructed to, for a group of points, calculate a specimen height at a point based upon a cantilever deflection, a cantilever base position and a specimen piezo position; calculate an adhesion force between a probe tip and a specimen at the point by causing an incrementally increasing force to be applied to the probe tip until the probe tip separates from a specimen; and move the probe tip to a new point in the group.

Full Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
       [0001]     This application is a continuation-in-part of co-pending U.S. patent application Ser. No. 09/054,809 filed Apr. 3, 1998, which is incorporated herein by reference. 
     
    
       [[0002]]     This invention was made with Government support under contract number DE-AC02-98CH 10886, awarded by the U.S. Department of Energy. The Government has certain rights in the invention. 
     
    
     FIELD OF THE INVENTION  
       [0003]     The present invention generally relates to the field of atomic force microscopy and, more particularly, to control of an atomic force microscope.  
       BACKGROUND OF THE INVENTION  
       [0004]     The present invention relates generally to atomic force microscopy and more particularly relates to an atomic force microscope and controller which minimize contact forces between a probe tip and a specimen and is well suited for the study of biological specimens.  
         [0005]     In the study of biology, it is desirable to observe biological specimens under very high magnification in a native environment. Such observations allow scientists to monitor, in real time, biological processes at the molecular and sub-molecular level. Such processes include the interaction of proteins with DNA and with each other. Currently, these processes cannot be observed in real time with electron microscopes or x-ray crystallography techniques which are known in the art, as the specimens are not in their native environment when using these apparatuses. Accordingly, scientists have sought alternate methods to observe biological specimens. One such alternative is known as the atomic force microscope.  
         [0006]     Atomic force microscopes (AFM), which are generally known in the art, physically probe a specimen to create an image of the specimen&#39;s surface.  FIG. 1  illustrates a typical embodiment of an AFM known in the art. The AFM has two primary components, a scanner  10  and a flexible cantilever  12  having a probe tip  14  on a free end. The scanner  10  has a top surface  16  on which a specimen  18  to be imaged is placed. The scanner  10  typically employs three piezoelectric elements  20 ,  22 ,  24  to move the specimen  18  in three dimensions, X Y and Z, relative to the position of the probe tip  14 . The probe tip  14  is affixed to the free end of the flexible cantilever  12  and contacts the specimen  18 . The AFM includes a laser  26  directed onto the cantilever  12  and a photo detector  28  which is responsive to laser light to measure the deflection of the cantilever  12 . As the degree of cantilever deflection is proportional to the contacting force between the probe tip  14  and the specimen  18 , such force can accurately be calculated based on the angle of cantilever deflection.  
         [0007]     To create an image of a specimen, the scanner  10  directs the specimen  18  in a raster-scan fashion in the X-Y direction while continuously sampling the contour of the specimen  18  in the Z direction. The sampling is generally performed using one of two techniques known in the art, namely contact mode or tapping mode. In contact mode, the scanner  10  is controlled in the Z direction such that the contacting force between the probe tip  14  and the specimen  18  is substantially constant. As the contour of the specimen changes, the deflection of the cantilever  12  also changes and a servo system driving the scanner  10  adjusts the Z coordinate of the scanner  10  to restore the desired constant force. At each specimen point, the coordinate of the Z axis is indicative of the specimen contour. Because the probe is constantly contacting (i.e. it drags along) the surface of the specimen during the X-Y raster scan, significant lateral forces are applied to both the specimen  18  and the probe tip  14 . The probe tip  14 , which is typically 200-300 Angstroms in diameter is subject to rapid wear and breakage under these forces. Also, when used on soft specimens, such as biological specimens, the probe tip is likely to destroy the surface of the specimen, making accurate and repeatable measurements impossible.  
         [0008]     In tapping mode, the cantilever  12  is driven in an oscillatory fashion at the resonant frequency of the cantilever. This may be achieved by affixing the cantilever to a piezoelectric element  30  and driving the piezoelectric element  30  with a voltage signal at the resonant frequency of the cantilever. To determine the contour of the specimen in tapping mode, the scanner  10  moves the specimen in the Z direction until a predetermined reduction in oscillation amplitude is detected. The reduction in oscillation amplitude is the result of the probe tip  14  contacting the surface of the specimen  18  during each cycle of oscillation. Because the probe tip  14  repeatedly, but only momentarily contacts the specimen  18  at each point during the X-Y raster scan, the lateral force present during contact mode is substantially reduced. However, because the probe tip  14  is moving rapidly on arrival at the specimen surface, the contacting force, while short in duration, is large in magnitude. The force that results from tapping mode tends to be destructive to biological specimens. Thus, tapping mode is most useful in sampling hard surfaces, such as those found in integrated circuit manufacturing processes and the like. Also, tapping mode is difficult to use when measuring a fluid-based specimen. When the cantilever assembly is submerged into a fluid environment, the desired oscillation of the cantilever can be dampened and additional resonances are developed which can adversely affect operation and accuracy. Also, fluid flow induced by the tapping oscillation tends to erode the specimen. Because biological specimens tend to reside in a fluid environment, tapping mode is not well suited for measuring these specimens. Tapping mode is also incapable of separately measuring the contacting force and adhesion force at each pixel. Because tapping mode cannot separate the two forces, concurrent mapping of each force during scanning is presently not possible with AFM&#39;s employing tapping mode.  
         [0009]     An alternative operating mode to both contact mode and tapping mode is described in U.S. Pat. No. 5,229,606 to Elings et al. Elings et al. refers to “jump scanning” where the probe is momentarily brought into contact with the surface to be measured. The probe is then lifted away from the surface as the specimen is moved in the X direction and the probe tip is then brought back down into contact to take the next specimen. By jumping over the surface of the specimen, Elings et al. teach a method of increasing scanning speed with reduced risk of probe damage. However, when the probe tip and specimen contact one another, an attractive force tends to hold the probe tip in contact with the specimen. To ensure that the probe tip is able to release, the cantilever  12  must be formed with a sufficient spring constant to overcome this attractive force. Unfortunately, increasing the spring constant of the cantilever  12  increases the magnitude of the contact force between the probe tip  14  and specimen  18  which is required to achieve a measurable cantilever  12  deflection. Such stiff cantilevers, i.e., in the range greater than 0.1 Newtons per meter (N/m), which are required for jump mode, are incompatible with the more sensitive biological specimens which are easily damaged under the application of such forces.  
         [0010]     The problem of overcoming the attractive forces between an AFM probe tip  14  and specimen surface was addressed in U.S. Pat. No. 5,515,719 to Lindsay. Lindsay operates in a mode where contact between the probe tip  14  and specimen surface is considered to be undesirable and recognized that when soft (low spring constant) cantilevers are used, the attractive interaction between the specimen  18  and probe tip  14  tends to draw the probe tip in and the probe tip  14  will contact and stick to the surface until enough force is applied to the cantilever base to release the probe tip  14 . To address this problem, Lindsay teaches the addition of a magnetic particle attached to the cantilever in combination with a magnetic solenoid located proximate to the cantilever. The solenoid generates a magnetic field which is variable and precisely regulated by a servo circuit. The servo circuit monitors the deflection of the cantilever and continuously adjusts the magnetic field such that the attractive force between the probe tip  14  and specimen  18  is substantially neutralized. In this way, the probe tip  14 , as taught by Lindsay, is kept at a distance from the specimen and never makes contact with the specimen. Therefore, it is impossible to measure an adhesion force using this method and difficult to measure the specimen profile at high lateral resolution because of the tip-specimen separation.  
         [0011]     The use of a magnetic particle affixed to a flexible cantilever and controlled by a magnetic coil as used by Lindsay was first disclosed in an article by Florin et al., entitled “Atomic Force Microscope with Magnetic Force Modulation”, published in the Review of Scientific Instrument, 65(3), March 1994. Florin et al. teach the use of a magnetic control system to drive the cantilever in an oscillating fashion such that the probe tip momentarily and adversely contacts a specimen, in a manner similar to tapping mode.  
         [0012]     Current AFM techniques tend to be destructive to biological specimens. Therefore, there remains a need for an improved atomic force microscope adapted for use in a fluid medium: for the observation of biological specimens in their native environment under conditions completely controlling and quantitatively measuring the interaction forces between tip and specimen.  
       SUMMARY OF THE INVENTION  
       [0013]     We have created an advance in the atomic force microscope field which improves operability and/or enables atomic force microscope usability in material science, genomics, proteomics, polymer science, and biomedicine by allowing observation of and data collection from biological specimens in their native environment.  
         [0014]     It is another object of the present invention to provide an atomic force microscope which provides a controlled, angstrom by angstrom approach of the probe tip to the specimen.  
         [0015]     It is yet another object of the present invention to provide an atomic force microscope which applies minimal vertical force to the specimen being measured.  
         [0016]     It is still another object of the present invention to provide an atomic force microscope featuring substantially zero lateral force applied to the specimen during a raster scan.  
         [0017]     It is a further object of the present invention to provide an atomic force microscope using a low spring force cantilever which overcomes the problem of probe tip retention resulting from adhesive forces between the probe tip and specimen.  
         [0018]     It is still a further object of the present invention to provide an atomic force microscope capable of using a probe tip with a diameter less than 100 Angstroms.  
         [0019]     It is still another object of the present invention to provide an atomic force microscope which is able to generate repeatable scan to scan measurement results on biological specimens.  
         [0020]     It is yet another object of the present invention to provide an atomic force microscope suitable for monitoring biological processes in real time.  
         [0021]     It is still another object of the present invention to provide an atomic force microscope which substantially continuously monitors both cantilever displacement relative to specimen and cantilever deflection.  
         [0022]     It is yet another object of the present invention to provide an atomic force microscope which is responsive to changes in cantilever deflection within five microseconds.  
         [0023]     It is yet a further object of the present invention to provide an atomic force microscope capable of recording and outputting complete force curves for all pixels in a specimen scan.  
         [0024]     For better understanding of the present invention, together with other and further objects and advantages, reference is made to the following description, taken in conjunction with the accompanying drawings, and its scope will be pointed out in the appended claims.  
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0025]      FIG. 1  is a block diagram of an atomic force microscope formed in a manner known in the prior art;  
         [0026]      FIG. 2  is a block diagram of a sensing mode atomic force microscope formed in accordance with the present invention;  
         [0027]      FIG. 3  is perspective view of a cantilever and cantilever pulse release system, formed in accordance with the present invention;  
         [0028]     FIGS.  4 A-C are flow charts illustrating the operation of a sensing mode atomic force microscope formed in accordance with a method of the present invention;  
         [0029]      FIG. 5A  is a graph depicting the full range of cantilever base motion;  
         [0030]      FIG. 5B  is a graph depicting cantilever base motion truncated by the detection of probe tip contact;  
         [0031]      FIG. 5C  is a graph depicting cantilever deflection versus time and corresponds in time with the graph in  FIG. 5B ;  
         [0032]      FIG. 5D  is a graph depicting cantilever deflection versus time with the application of a reverse polarity pulse release signal, restoring the cantilever to a neutral position;  
         [0033]      FIG. 5E  is a graph depicting the full range of cantilever base motion incorporating a pause for incremental ramp up of the magnetic withdrawal force;  
         [0034]      FIG. 5F  is a graph depicting cantilever base motion truncated by the detection of probe tip contact and incorporating a pause after contact. The deflection occurring in  FIG. 5F  is stopped by contact with the specimen at a point intermediate to the full deflection illustrated in  FIG. 5E ;  
         [0035]      FIG. 5G  is a graph depicting cantilever deflection versus time and corresponds in time with the graph in  FIG. 5F ;  
         [0036]      FIG. 6  shows a sequence of responses of the cantilever to a given release force during engagement;  
         [0037]      FIG. 7A  is an example display of concurrent light and adhesion force information obtained from scanning a DNA sample available through use of the invention;  
         [0038]      FIG. 7B  is an enlarged view of image  772  of  FIG. 7A ;  
         [0039]      FIG. 8  is an enlarged view of the graphs of  FIG. 7A ;  
         [0040]      FIG. 9  is an image of several segments of DNA helix obtained using the techniques described herein; and  
         [0041]      FIG. 10  is a higher magnification image of DNA obtained using the invention. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0042]      FIG. 2  is a block diagram generally depicting an atomic force microscope (AFM) formed in accordance with the present invention. The AFM includes a scanner  10  formed in a conventional manner having an X-piezo element  22 , a Y-piezo element  20  and a Z-piezo element  24 . The scanner  10  has an upper specimen surface  16  on which a specimen  18  to be measured is placed. The X, Y and Z piezo elements each change dimension, in a conventional fashion, in three mutually perpendicular axes, in response to a received control voltage signal. An X-Y controller circuit  38  is included. The X-Y controller circuit  38  controls the X-piezo element  22  and the Y-piezo element  20 , generating a conventional raster scan of the specimen surface  16 . Several commercially available controllers can provide this function. For example, the NanoScope E controller, manufactured by Digital Instruments, Inc. of Santa Barbara, Calif. is suitable for this application.  
         [0043]     A sensing mode controller circuit  40  is also included and controls the Z-piezo element  24  in accordance with a method of the present invention. The sensing mode controller  40  can be implemented using a Pentium II Personal Computer and a DT3809 multifunction Input/Output Card, having a Texas Instruments C40 Digital Signal Processor (DSP) Integrated Circuit. It is contemplated by the inventors, and will be appreciated by those skilled in the art, that the functions of the X-Y controller circuit  38  and sensing mode controller circuit  40  can be performed by a single controller unit.  
         [0044]     The AFM further includes a flexible cantilever  12  which has a first end affixed to a vertically displaceable (Z-axis) cantilever base  30 . The cantilever base  30  preferably takes the form of a piezoelectric element which changes dimension in the Z-axis in a controlled fashion in response to a received control voltage signal provided by the sensing mode controller circuit  40 . The flexible cantilever  12 , shown in detail in  FIG. 3 , has a free end on which a probe tip  14  is affixed. The probe tip  14  is formed from a material such as silicon nitride or silicon, and is generally sharpened to a diameter less than 20 nanometers (nm). Suitable probe tips are currently manufactured by Digital Instruments, of Santa Barbara, Calif. Alternatively, single wall (1 nm diameter) or multiwall carbon nanotubes (5-20 nm diameter) or non-carbon nanotubes such as molybdenum sulfide (MoS 2, ) and tungsten sulfide (WS 2 ) (20-40 nm down to 1 nm diameter) can also be used. Advantageously, the nanotubes can be used in underivatized form or derivatized form. For example, defined functional groups can be added covalently at the end of the nanotube which has been oxidized, using known crosslinking reagents and linker molecules. A suitable coupling reagent could use carbodimide chemistry. Examples of functional groups include amines, methyls, benzyls and nucleosides. These defined functional groups will allow chemical-force probing on a molecular or pixel-by-pixel basis, i.e. at full resolution and in exact register with the height map. Advantageously, by using a single defined molecule on the probe tip, the binding energy between the molecule on the tip and the specimen can be measured.  
         [0045]     The 1 nm (10 Angstroms) single wall carbon nanotubes have great potential as probe tips. Mounted on the end of a cantilever, these tips can provide for force measurements and profile height measurements especially in fluids at a resolution previously unachievable with prior art AFM technology. In particular, interstitial molecular force measurement within lattice-type structures can be studied.  
         [0046]     As a general matter, Si tips have proved difficult to use in biological AFM. However, in applying the principles described herein, Si tips can be used to image DNA topography well even in buffer and with rescanning and a nominal force constant of the Si cantilever as low as 0.05 N/m.  
         [0047]     As noted above, probe tips made up of multiwall or single wall nanotubes can be used. Notably, when used as probe tips, nanotubes are susceptible to buckling rendering them less effective at detecting a surface contact. Advantageously, by applying the principles described herein a sufficiently low compressive force is needed to sense the surface of the specimen that even single-wall nanotubes can be used.  
         [0048]     During a specimen height measurement, the probe tip  14  is brought into contact with the specimen and a force results which deflects the flexible cantilever  12 . The magnitude of the force applied by the probe tip  14  onto the specimen  18  is proportional to the angle of cantilever  12  deflection. To measure the degree of cantilever deflection, the AFM further includes the laser source  26  which generates a light signal that is directed onto the surface of the flexible cantilever  12 . The light signal is reflected from the cantilever  12  and is directed by a mirror (not shown) onto a photo detector  28 . The photo detector  28  is preferably divided into four light responsive quadrants, a, b, c and d. Each light responsive quadrant generates an analog voltage signal which is proportional to the magnitude of reflected light incident upon it. A difference signal, which is equal to the signals (a+b)−(c+d), is calculated to determine the magnitude of deflection of the cantilever  12 .  
         [0049]     The scanner  10 , cantilever  12 , cantilever base  30 , laser  26  and photo detector  28  are conventional elements known in the art. These components are sold as an assembly by Digital Instruments of Santa Barbara, Calif. under the trademark, MultiMode SPM.  
         [0050]     As the probe tip  14  approaches a specimen, damping forces act upon the probe tip  14  owing to an effective viscosity increase of the fluid near the surface.  
         [0051]     When the probe tip  14  contacts the surface of the specimen, adhesive forces frequently result which capture and retain the probe tip  14 . This problem is exacerbated when the cantilever  12  is formed with a very low spring constant, which is required for the measurement of biological specimens. Therefore, an important aspect of the present invention resides in the ability to disengage the probe tip  14  from the surface of the specimen. To accomplish this, a magnetic particle  32  is affixed proximate the free end of the cantilever  12 , as illustrated in  FIG. 3 . In a preferred embodiment, the magnetic particle is formed by bonding a particle of Samarium Cobalt to the cantilever  12  with a thermal epoxy. The particle can be previously magnetized by placing it in a magnetic field of approximately 3.5 Tesla.  
         [0052]     The AFM also includes an electromagnetic coil  34  which receives current drive from a pulse release circuit  36  and generates an inhomogeneous magnetic field in response thereto. The magnetic particle  32  and electromagnetic coil  34  are arranged such that the magnetic field generated by the coil  34  when energized drives the magnetic particle  32  away from the surface of the specimen  18 . Preferably, the coil  34  is formed and placed proximate the scanner  10  and the magnetic field is selected to repel the magnetic particle  32 . In other embodiments, the coil can be placed above the cantilever  12  and generate an attractive magnetic force with the magnetic particle  32 . In either placement, an opposite force used for restoring the cantilever  12  to a neutral position, can be generated by reversing the polarity of the current in the coil. The magnetic force is selected to be sufficient to overcome the adhesive force between the probe tip  14  and specimen  18 . Such a force is generally achievable when the coil is formed with 50 turns of 22 gauge wire about a 0.25 in. diameter core, and is energized with a current on the order of 200 milliamps. However, as the adhesive forces encountered are variable, it is preferred that the level of magnetic field also be variable to insure that the probe tip  14  can always be released from the specimen  18 .  
         [0053]     To achieve the desired objectives, the present invention employs a novel control method for an atomic force microscope, such as that illustrated in  FIG. 2 . The control method, which generally takes the form of a computer program, is illustrated in the flow charts of FIGS.  4 A-C. The control method of FIGS.  4 A-C results in a controlled approach of the probe tip  14  to the specimen  18 , a substantially constant range of motion of the probe tip  14  during successive measurement samples, very low contact force upon the probe tip  14  with no lateral force resulting between the probe tip  14  and specimen  18  and a sure release of probe tip  14  from adhesive forces presented by the specimen  18 . Accordingly, the method of the present invention is ideal for use when working with biological specimens.  
         [0054]     Through the above described continuous dual control of the cantilever base  30  and probe tip  14 , at each pixel, both contact force can be reliably measured and profile height of the specimen can be obtained as the specimen is scanned. Additionally, since release is obtained by incrementally increasing a magnetic field, the adhesion force is automatically available for determination and recording in memory at the increment preceding the release. As a result, adhesion force mapping can also be performed on a pixel by pixel basis during scanning. Thus, as the scan proceeds, complete data regarding the contact force, profile height and adhesion force of the specimen at every point in the raster scan is immediately and visually available.  
         [0055]     Measuring compressive (contact) and tensile (adhesion) force along with sample height at each pixel further allows for analysis of single-molecule reactions. To do so, the tensile (chemical) force inherently measured by the release pulse at each increment of the magnetic force ramp is calculated and stored. Thus, forces can be accurately measured at sub-nanometer intervals of separation between tip and specimen. Feedback may optionally also be introduced in order to stabilize the probe position after release, as the chemical force begins to decrease. In this way, the entire force/separation curve of two reacting molecules is measurable, with the integral of that curve being the reaction energy.  
         [0056]     Commercial DSP&#39;s provide a pixel scan rate of 400 Hz in a present embodiment and with obvious modifications can cover the range of scan rates between 100 pixels/sec or less and up to 1000 pixels/sec. Moreover DSP&#39;s becoming available are 30 times faster and will allow pixel scan rates well in excess of 1000 pixels/sec. The computer program implements both the force sensing and control aspects of the cantilever during the incremental approach and contact with the specimen, creates the magnetic force ramp during the first part of the withdraw cycle (while holding the specimen base fixed) and detects separation of the tip from the specimen while storing the adhesion force=magnetic force measured at that time.  
         [0057]     Referring now to FIGS.  4 A-C, operation of the AFM begins with the initialization of the X, Y and Z positions of the scanner  10  (step  42 ). The X and Y piezo elements are driven to a datum coordinate (0,0) and the Z-piezo element  24  is placed approximately the center of the range of motion. The approach value, which is provided by the sensing mode controller  40  to the cantilever base  30 , is also initialized to zero (step  44 ).  
         [0058]     After initialization, the sensing mode controller circuit  40  increments the approach value, thereby moving the cantilever base  30  in a direction towards the specimen surface  16  (step  46 ). After incrementing the approach value, the sensing mode controller  40  monitors the signals from the photo detector  28  and determines the cantilever  12  deflection angle (step  48 ). From the measured deflection angle, the sensing mode controller  40  calculates the force on the probe tip  14  (step  50 ). The sensing mode controller  40  then compares the measured force against a predetermined maximum force value to determine whether the probe tip has contacted the specimen (step  52 ). The maximum force value is preferably selected to be the minimum value which will reliably indicate a contacting force between the probe tip  14  and specimen. Suitable thresholds of between 35 and 150 piconewtons (pN) have been successfully practiced. Typically 70 pN provides an adequate threshold level. Advantageously, sensing mode permits selection of compressive force at detection of contact (i.e. detection threshold) typically in the range 35 to 150 piconewtons, although a lower threshold can be used. As a result, a lower compressive force on approach results in a lower adhesive force on pull-off, aiding in preservation of biological specimen structure. Threshold values lower than 35 pN are considered achievable as significantly softer cantilevers become commercially available.  
         [0059]     If the probe tip  14  has contacted the specimen, the approach value is stored as the detection coordinate to calculate the profile height of the specimen (step  54 ). The specimen profile height is then determined by combining the current Z coordinate position of the scanner  10  with the detection coordinate (step  56 ). At contact, a lower Z-value for scanner  10 , and a lower detection coordinate on the ramp, each signifies a higher profile height. So these two variables must be added (after multiplying each by a constant to convert digital value to height) and finally the sign changed. The specimen profile height value represents the height of the specimen at the current X, Y position and can be displayed graphically as the intensity and/or color of a pixel at that two dimensional coordinate, in a manner well known in the art of atomic force microscopy.  
         [0060]     Returning to step  52 , if the probe tip  14  has not contacted the specimen, the controller then determines whether the approach value has exceeded an allowable limit (step  58 ). The range of approach values is determined by the required force resolution and the range of motion achievable from the cantilever base  30 . Preferably, a digital to analog converter within the sensing mode controller  40  provides at least 200 incremental approach values as illustrated in  FIG. 5A  (approach maximum=200). This allows an angstrom by angstrom approach of the cantilever base  30  toward the specimen at every pixel. In general, the timing is such that each 5 μsec the DSP issues a command to the piezo that drives the cantilever base, and also to the coil that provides the magnetic coil drive. Accordingly, there are typically about 200 pairs of commands during the advance part of the cycle alone. Also each 5 μsec, the DSP learns the cantilever deflection angle by analog to digital conversion of the reflected laser beam&#39;s detector output, and the cantilever base position by similarly reading the cantilever base piezo voltage.  
         [0061]     If the approach value is within the acceptable range, generally between 10-50 nm (typically 15 nm), the program returns to step  46  where the approach value is incremented.  
         [0062]     Steps  46 - 52  and step  58  are performed in such a way as to generate a controlled approach of the cantilever base  30  towards the specimen  18 , as illustrated in the graphs of  FIGS. 5A and 5B . The approach begins with a gradually descending portion  84 . The curved initial approach, which can be generated by a sinusoidal function or other suitable function, minimizes the initial acceleration of the cantilever base  30 . This minimizes the excitation of oscillations in the free end of the cantilever  12  and enhances measurement accuracy. The approach then is characterized by a linear segment  86 . The linear approach of the present invention together with a minimum practical approach distance minimizes the velocity of the cantilever at contact and allows the probe tip  14  to contact the specimen with minimal contact force, essential for low damage to biological specimens and the soft high polymers of materials science.  
         [0063]     If the approach value exceeds the maximum value (step  58  is true) the sensing mode controller  40  withdraws the cantilever base and applies a signal to the Z-piezo element  24  of the scanner  10  to reduce the separation between the cantilever base  30  and specimen surface  16 . A high voltage amplifier  42  is preferably interposed between the sensing mode controller  40  and scanner  10  to generate the ±200 volt signal required to drive the piezoelectric element. A suitable high voltage amplifier, model number PA87, is manufactured by Apex Microtechnology of Tucson, Ariz. After adjusting the scanner height (step  60 ), program control is directed to step  44  where the approach value is again initialized to zero.  
         [0064]     After the probe tip  14  contacts the specimen  18 , adhesive forces act to hold the probe tip in place, even when the cantilever base  30  and specimen  18  are moved away from each other. This problem is especially acute when soft cantilevers (k&lt;0.1 N/m) are used. The method of the present invention overcomes this problem by coordinating the withdrawal of the cantilever base  30  with the application of a force on the probe tip  14 . Moreover, the magnetic force can be applied in precise increments enabling the measurement of adhesion force versus separation.  
         [0065]     After the specimen profile height is determined (step  56 ), the magnitude of the approach signal is reduced, thereby moving the cantilever base  30  away from the specimen (step  62 ). Preferably, the approach signal is initially reduced ( FIG. 5A ) or paused and then reduced ( FIG. 5E ) gradually to effect a gradual change in slope, thus avoiding instability of the cantilever  12 . In order to release the probe tip  14  from the specimen  18  either during the pause ( FIGS. 5F and 5G ) or when the cantilever base  30  starts to withdraw ( FIG. 5C ), the sensing mode controller  40  applies a pulse release signal to the pulse release circuit  36 .  
         [0066]     In response to the pulse release signal, the pulse release circuit  36  generates a suitable current pulse which energizes the electromagnetic coil  34 . The electromagnetic coil  34  generates a magnetic field which repels the magnetic particle  32 , and thus the probe tip  14 , away from the specimen  18 . The sensing mode controller circuit  40  monitors the deflection angle of the cantilever to ensure that the probe tip  14  in fact is released (step  66 ). If not, the sensing mode controller circuit  40  either requests intervention by the operator to increase the pulse size for this experiment or increments the value of the release pulse release signal (step  68 ) and reapplies the pulse (step  64 ). When the sensing mode controller circuit  40  is adjusting the pulse release signal, steps  64 - 68  are repeated, typically for a maximum of 64 increments, until the magnetic force overcomes the adhesive force between the specimen  18  and the probe tip  14  or a predetermined maximum pulse release signal is reached. The speed of commercially available DSPs allows the use of 64 levels of current in the coil which provides a measurement precision of approximately 2%. If higher precision is desired, faster DSPs are becoming available and can be employed.  
         [0067]     The coil current is increased from zero in a defined stepwise manner at defined time intervals under program control. The force scale is calibrated by measuring the cantilever deflection voltage at the maximum (i.e. kth level current where k=the maximum number of levels) and separately producing the same deflection voltage by advancing the specimen piezo—which is calibrated absolutely in nm. In this way the nm deflection of the cantilever at maximum coil current is determined. Multiplying by the force constant of the cantilever gives the magnetic force at maximum coil current, with the steps being in increments of 1/k of the total if all levels are used. Although the magnetic force required to achieve release is a function of tip  14  to specimen adhesion, typically a few hundred pN or less will suffice. Moreover, for non-low adhesion probe tips, a magnetic release force of approximately 10 times the contacting force has been found to be a useful estimate of the actual release force.  
         [0068]     As will be described in greater detail below, a maximum of 64 steps may be chosen to encompass  100  pN and up to 1000 pN or more. At the lower setting, sensitivity (1.5 pN) and precision of approximately 2% provides very satisfactory knowledge of the adhesion force in the range exerted for weak chemical forces such as the hydrogen bond. The upper ranges are appropriate for covalent force interactions. These force ranges are appropriate for detecting non-covalent forces between molecules and characterizing covalent bond formation during chemical reactions.  
         [0069]      FIG. 5C  illustrates exemplary cantilever deflection versus time for the approach depicted in  FIG. 5B . During the linear portion of the approach 86 the cantilever  12  experiences little deflection from a neutral position  92 . Depending on the material being sampled and the construction of the probe tip  14 , attractive forces may be present which draw the probe tip into the specimen. This results in a negative deflection  94  of the cantilever  12 . Upon contact of the probe tip  14 , the cantilever  12  begins positive deflection  96 . This is the condition detected by the sensing mode controller  40  at step  52 . At that point the cantilever base  30  is withdrawn and the pulse release signal is applied, deflecting the cantilever  12  in a positive direction  98 . Because the cantilever  12  is operating in a fluid environment, the cantilever deflection  98  is damped and is characterized by an exponential function. Upon termination of the release pulse  100 , the cantilever returns to the neutral position in an exponentially decaying fashion  102 . In order to accelerate the return to the neutral position, a reverse polarity pulse signal can be applied to the coil  34 . This creates an attractive force sufficient to overcome the spring constant of the cantilever  12  as well as the fluid damping. An example of this is illustrated in  FIG. 5D .  
         [0070]     After the probe tip  14  is released and the approach signal is decremented to zero ( 90 ,  FIG. 5B ), the program determines the distance traveled by the cantilever base  30  during the last sample. Since it is preferable for limiting the contact force on the specimen during measurement to use a minimum practical approach distance, the specimen height change that can be stored in the variable representing the detect position is limited. Therefore, the sensing mode controller circuit  40  generates a delta value by subtracting a predetermined “set value” from the actual value traveled by the cantilever base  30  during the last approach (step  70 ). The set value is normally chosen near the midpoint of the linear ramp of  FIG. 5A . The sensing mode controller  40  then adjusts the height of the Z-piezo element  24  of the scanner  10  to reduce the delta value to zero on the next approach (step  72 ). In this way the Z-piezo element  24  functions as the accumulator or integrator of deltas generated by the detect position which therefore equals the Z-value or specimen height at this particular x-y position or pixel. Z is stored for each of the X-Y pixels in memory contained in Sensing Mode Controller  40 , where it is available for analysis and display. The range of heights that can be generated by the Z-piezo element  24  is several thousand nanometers, which is entirely adequate for the range of specimen heights encountered in biological experiments. A useful by-product of this method of operation is the achievement of substantially constant timing and substantially constant distance of travel in the approach of the cantilever base  30  to the specimen  18  during successive samples.  
         [0071]     While the method of FIGS.  4 A-C has been described with the cantilever base  30  being controlled to effect the approach and withdrawal of the probe tip  14  and the scanner  10  being controlled to minimize the delta value, those skilled in the art will readily appreciate that these functions can be exchanged without deviating from the improved methods of the present invention. To effect such a change, the range of motion of the cantilever base  30  is preferably increased from several hundred nanometers which is commonly used, to several thousand nanometers so that a broad range of delta values can be corrected.  
         [0072]     Upon completion of step  72 , the X-Y controller  38  then operates to effect a conventional raster scan of the specimen relative to the probe tip  14 . The X-Y controller  38  increments the X coordinate value (step  74 ) of the scanner  10  and determines whether the end of the row has been reached (step  76 ). Typically, each row contains either 256 or 512 coordinate values.  
         [0073]     If the end of the row has not been reached, the program control returns to step  44 . If the end of the row has been reached, the X coordinate value is reset to zero and the Y coordinate value is incremented (step  78 ).  
         [0074]     Alternatively, the program can increment the Y coordinate value and decrement the X coordinate value for successive measurements until X=0. In this way, a serpentine scan is achieved. The X-Y controller  38  then determines if the final Y coordinate value has been reached (step  80 ). Generally, the Y axis will include the same number of coordinate values as the X-axis, thereby generating a square image matrix. If the final Y coordinate value has not been reached, control returns to step  44 . If the final Y coordinate value has been reached, the image scan is complete and the program ends (step  82 ).  
         [0075]     The process described in FIGS.  4 A-C is referred to by the inventors as “sensing mode.” This is because the sensing mode controller  40  senses when initial contact is made between the specimen and probe tip  14  and immediately (typically within 5 microseconds) begins retraction of the cantilever base  30  and probe tip  14 . The method and apparatus of the present invention significantly reduces the velocity of the probe tip  14  at contact with the specimen  18  and therefore reduces the resulting contact force between the probe tip  14  and the specimen  18 . By reducing the contact force, specimen destruction is minimized. The inclusion of the magnetic particle  32  and pulse release coil  34 , operated in accordance with the sensing mode method of the present invention, allows the use of a cantilever  12  with a spring constant significantly less than 0.1 Newtons per meter (N/m) without the risk of the probe tip  14  being retained by the adhesive forces exhibited by the specimen. With sensing mode, the lower limit on acceptable cantilever spring force is bounded only by cantilever manufacturing constraints. The use of such a soft cantilever further reduces the compressive force between the probe tip  14  and the specimen  18  during contact, making it possible to reduce specimen damage even further.  
         [0076]     As an additional benefit of the significantly reduced contact force, significantly smaller probe tips  14  can be used in sensing mode with reduced risk of tip breakage. Probe tips significantly less than 10 nm, for example, 6 nm (60 Angstroms) in diameter have been successfully used in fluids with the sensing mode scanning method of the present invention.  
         [0077]     As a further advantage resulting from the continuous sensing process of sensing mode, a high-speed algorithm can compute a running linear least squares fit for as many as 20 or more successive readouts of the cantilever deflection signal (angle), with update of the least squares fit each 5 microseconds as a new digital value for deflection value is presented to the program. Thus, continuous sensing makes possible a low-noise running fitted average to both tip position and tip velocity (slope of the least squares fit). Because the averaging time of the fit can extend over several cycles of the damped vibrational period of the cantilever, thermal noise in the averaged cantilever signal is strongly reduced and noise in the detected position of the surface is lowered correspondingly. The running least squares fit can be used in a variety of sensitive algorithms for detecting contact with the surface at low force. One such is to define contact with the surface as that point during the approach where the velocity of the tip, which is the same as the slope of the least squares fit, changes from negative (down) to positive (up). Tests with this definition have shown an ability to detect the surface with substantially smaller force exerted on the specimen.  
         [0078]     Still another advantage of the present invention is in the mode of adjusting the specimen piezo Z to reduce the deviation in contact position from the set position. In contrast to contact mode and tapping mode, sensing mode as described herein can be configured to carry out adjustments to the Z specimen piezo without a differential equation. Once each x-y location, the electronics determines how far the surface detection point deviates from a pre-assigned vertical or Z set point (typically at the midpoint of the total approach distance) and immediately corrects the deviation by adjusting the specimen piezo. One measured constant, the ratio of sensitivities in nm/volt of the cantilever base piezo and specimen piezo suffices to determine quantitatively the correction.  
         [0079]     Depending upon the particular implementation, it may be desirable to make corrections over a multiple of pixels to avoid the introduction of noise associated with making the entire correction at once. Thus, correction can be introduced over a few pixels, e.g., 80% the first pixel, arriving at 96% correction the second pixel, etc. A simple, but exact, theory enables one to know the final correct height at the first pixel even though the lower piezo has not yet reached the final height. Therefore the corrected height can be recorded each pixel along with the achieved height. The formula for the correction is S n =Z n +D n (γ 0 −γ); where S n  is the corrected surface height, Z n  is the height actually reached after one application of the partial correction (80% in the example), and D n  is the deviation of the surface detection point from the set point, all at pixel “n”. By convention, S n  and Z n  are in volts applied to the specimen piezo or in counts applied to the D/A converter that produces that voltage. Similarly, D n  is volts applied to the piezo attached to the cantilever base or counts applied to its converter. γ 0  is the factor converting cantilever piezo counts to sample piezo counts, measured experimentally and found to be within a factor of 2 either way of unity. If the sample piezo is corrected by γ o D n  for a deviation D n , then the whole height deviation would be corrected at once by the specimen piezo motion. γ is the factor, smaller than γ o , that produces the correction actually used, 80% of γ 0  in the example. As a result, the true height, S n , is the actual height Z n  plus the extra correction not yet applied in the first correction because γ was chosen too small. In practice, Z n  contains the low frequency variation of the specimen surface and D n  the high frequency. D n  and S n  can be displayed in neighboring windows for visual analysis.  
         [0080]     By employing the techniques described herein, relating to adhesion force, through iterative application of an increasing coil current to effect release of the probe tip at each pixel, we necessarily identify the time at which the release force is overcome. Advantageously, measuring the force at the time of pull-off quantifies the adhesion force. Moreover, since the adhesion force is measured during the same approach/withdraw cycle that measures specimen height, the adhesion force can be mapped in exact register with the topography and at the same resolution, whatever resolution is used. As a further advantage resulting from the continuous sensing process of sensing mode, since a quantitatively ramped magnetic force is applied just after surface detection, determination of the point at which the adhesion force is overcome also isolates and quantitatively measures the adhesion force. Moreover, because both the approach and release are done incrementally, storing the forces for each increment at each pixel enables mapping of force as a function of distance from the surface of a specimen. This feature thus provides concurrent mapping of topography with adhesion force at high resolution.  
         [0081]     An atomic force microscope formed and operated in accordance with the present invention can also be used to reduce the time for the cantilever to approach an equilibrium angle at certain phases of the approach/withdraw cycle. By applying a reverse current pulse in magnetic drive coil at the termination of the cantilever pulse release signal, the return of the cantilever to its equilibrium angle is accelerated so the next approach can begin immediately. A brief current pulse of the same sign as the main release pulse but at the beginning of the main approach ramp speeds up arrival at the new equilibrium angle associated with linear approach, simplifying subsequent detection of the surface.  
         [0082]     Another valuable feature is an optional pre-scanning or zoom mode. Since the detailed behavior of the cantilever is controlled by the DSP program for each pixel, one can make a change to the x or y position corresponding to each pixel simply by changing the appropriate variable at the end of tip withdrawal and before the next advance. In this manner, areas of interest can be quickly located. To do so, the program that controls the tip advance and withdrawal maintains a running count of the 1 through 256 pixel number, for example, and the 1 through 256 scan line number and creates a 256×256 table in memory. This allows looking up in the table the voltages that should be applied at any given pixel to the piezo scanner of the microscope. These voltages are output to the microscope, first x and then y, by a single D to A converter and are held separately in two sample-and-hold circuits for application to the microscope. The lookup table itself is prepared by having the microscope scan a calibration grid. Separate lookup tables can be stored and used for each scan size and for any other scan variables found necessary, since the storage required is minimal.  
         [0083]     After a molecule or interaction region of interest is located in a field, reduction in the scan size to encompass just the event of interest can be done. This makes the machine more responsive in studying biological function. In one embodiment, a scan of 400 pixels per second is equivalent to a field of 40×40 pixels (200 Angstrom×200 Angstrom at 5 Angstrom pixel separation) each 4 seconds, for example, to allow following the evolution of structure in biochemical reactions. Since biological rates can often be manipulated by changes in temperature, pH or concentration, this invention provides means of observing changes in physiologically relevant biological structures.  
         [0084]     A further advantageous byproduct of the sensing mode configuration and method is that it also reduces the possibility of damaging a probe tip on initial engagement. We have been able to make use of a highly characteristic change in cantilever response to the magnetic release pulse (which occurs each pixel) related to the increase in effective viscosity near the surface. This change allows reliable prediction of when contact with the surface of the specimen will occur. Initial tip engagement occurs only once each measurement session. Activation of the stepping motor for engagement requires repeating a sequence of four pulses phased properly in time. However, according to prior art systems, the operator has no way of knowing when the surface will be encountered, which can induce a painfully slow approach to avoid tip damage. With sensing mode operation, this problem is overcome. A repeated approach/withdraw cycle, typically 15 nm in amplitude of the format shown in  FIG. 5E , is initiated by the program before engagement starts. The cycle includes the magnetic release pulse as part of each withdrawal.  FIG. 6  shows the cantilever deflection signal for approach/withdraw cycles at several stages of engagement.  
         [0085]     When the cantilever tip is nowhere near the specimen, the cantilever response as indicated by the deflection signal, has a certain shape,  662 , that includes one or a few cycles of ‘ringing’ after the release pulse is over. However, as the engagement motor continues to run and the tip approaches the sample, the ringing decreases  664  and nearly disappears,  666 , just before contact,  668 , is made. This is due to the increased viscosity seen by the cantilever in the immediate vicinity of the sample owing to restriction of flow lines. While the initial engagement portion of the program is running, the program looks once each approach/withdraw cycle for the amount of cantilever ringing and reduces the engage motor speed appropriately. In addition, because the approach/withdraw cycle is in continuous operation, feedback from the position (fraction of the 15 nm range) at which the tip encounters the surface to the position of the specimen piezo, can also be in operation so that when the surface is encountered, the specimen piezo retracts smoothly to keep the surface encounter position at the set point. The net effect is that it is practically impossible to crash the tip into the specimen or damage it by excessive force.  
         [0086]      FIG. 7A  is an example display of information available through use of the invention to scan a DNA sample. As shown, the upper graph is the force data at pixel  3  of scan line  93  (where pixel zero is to the left and scan line zero is at the top) out of a 256×256 pixel image. The lower graph contains a representation of the specimen piezo position and the deviation from a set position.  
         [0087]      FIG. 7B  is an enlarged version of the Height image  772  from  FIG. 7A . The image area,  776 , is contained within a field of 256 by 256 points (i.e. pixels). To the right of the image area,  776 , is a scale,  778 , correlating a shade (color) with a count value, representing, in counts, the height of the specimen at each of the points.  
         [0088]     Above the graphs in  FIG. 7A  are pull-down menus through which the various program features may be accessed. Along side the graphs are three images which are obtained concurrently during scanning. The upper image,  770 , labeled “Detect” plots the surface detection position at each pixel expressed as deviation from the set position. The middle image,  772 , labeled “Height” plots the specimen height at each pixel using the corrected specimen piezo position (Sn referred to above). The lower image,  774 , labelled “Adhesion” plots the measured adhesion force at each pixel using the pull off step, converted to piconewtons. In each, the corresponding scan line  93  is indicated.  
         [0089]      FIG. 8  is an excerpt of the graphs of  FIG. 7 . In the upper graph, the “Force Curve” is the heavy black line,  882 , plot of cantilever deflection vs. time. Each unit of the abscissa represents 5 μsec, which is the interval between computer interactions with the microscope. The units of the ordinate represent picoNewtons. The horizontal dashed line,  884 , (red), is the detect threshold. As shown, for a 15 nm approach distance, each interval represents ¾ of an Angstrom. From 0 to 98 units the cantilever is approaching the specimen under control of the cantilever piezo. At 98 units, cantilever deflection crosses the detect threshold (here 70 pN), and the program brings the cantilever base piezo smoothly to a halt. The magnetic force ramp then begins incrementally increasing. At approximately 138 units, i.e. after approximately 40 steps in the ramp, the tip pulls loose,  886 , and the cantilever in this case goes to 2 Volts. This 10-fold increase in deflection is detected by the program. The increment or step at which the deflection crosses a second threshold (here set at twice the height of the detect threshold line) is defined as the pull-off step, and the magnetic force at this point is the measured adhesion force for this pixel.  
         [0090]     The lower graph in  FIG. 8  describes what happens over the entire scan line  93 . Two lines are superimposed over each other. The fine line (blue),  188 , records the deviation of the detect position ( 98  units for pixel  3  above) from a set position, typically 120 units. The heavy black line,  890 , is the specimen piezo position, which is obtained by feedback from the detect position as described above. Line  93  passes through three segments of DNA in the Height image,  772 , and these crossings appear as three pronounced dips in the lower graph.  
         [0091]      FIG. 9  is an image of several DNA strands imaged in Hepes-Ni 2+  buffer on Aptes-mica with Si3N4 tips obtained using the techniques described herein. As shown, the image  904 , on the left is a height profile of a portion of the DNA. The bar  906  represents 24 nanometers for the x or y directions. Original field size was 220 nm. The scale,  908 , below the height profile image  904  represents the heights in nanometers (nm) converted in a color or grey-scale representation. Image  902  is an enlarged portion of the adhesion force image  910 , showing structure in a segment of DNA occurring at the highly significant spacing of the 10 base pair repeat distance of DNA. An adhesion force image  910  is in the same register and x-y scale as the height profile image,  904 . The scale  911  below the adhesion force profile image  910  represents the adhesion force in nanonewtons (nN) converted in a color or grey-scale representation. The adhesion force map of  FIG. 9  (right panel) was acquired simultaneously with the topography map of  FIG. 9  (left panel) in real time at the same resolution as the topography. Such maps provided by this invention, to our knowledge, represent the first real time high resolution adhesion maps available as a consistently present adjunct to topography for interpretation of the image.  
         [0092]      FIG. 10  is a higher magnification image of one DNA strand imaged in  FIG. 9 , which was obtained by a new scan done at a smaller field size. The five ridges marked “A” in the height image of  FIG. 10  correspond to the five repeats of the double helix marked “B” in the X-ray image of the DNA. This rescan demonstrates that the initial scan using the dually controlled cantilever of this invention leaves the DNA sufficiently intact to provide higher resolution on the succeeding scan. The original field was 50 nm. The bar represents 5 nm.  
         [0093]     While there have been described what are presently believed to be the preferred embodiments of the invention, those skilled in the art will realize that various changes and modifications may be to the invention without the departing from the spirit of the invention, and it is intended to claim all such changes and modifications as fall within the scope of the invention.

Technology Classification (CPC): 8