Difference between revisions of "Lab Manual: Atomic Force Microscopy (AFM)"

Contents 1 Objectives and learning goals 2 Roadmap scenarios and milestones 2.1 AFM mini-lab 2.2 Extended AFM work 3 The 20.309 AFM System 3.1 Hardware 3.2 Motion control system

AFM Apparatus

Objectives and learning goals

• Learn function of the 20.309 AFM and the relationships between its components.
• Understand how to extract quantitative information from this tool.
• Use the AFM to estimate the value of a fundamental physical constant.
• Possibly to take images, probe sample stiffness, and/or perform protein pulling.
• Analyze sources of uncertainty and noise in the system that limit the accuracy of measurements.

Roadmap scenarios and milestones

The AFM can be used both for A basic "mini-lab" homework problem, and also for more involved projects. This section will outline the mini-lab and provide one possible outline for extended, for instance as a final project. You are encouraged to devise much more creative uses for the AFM in final projects and beyond.

AFM mini-lab

1. Work with a TA or Instructor.
2. Learn the signal paths and connections of the system.
3. Practice aligning the AFM optics.
4. Learn how to calibrate the AFM to extract useful physical data.
5. Measure the vibrational noise floor in the AFM system.
6. Use the AFM to record the vibrational noise spectrum of a cantilever probe (Experiment ##)

Extended AFM work

1. Image several different samples with the AFM and measure physical dimensions of imaged features.
2. Use the AFM to measure the elastic modulus and surface adhesion force of several different samples.
3. Use the AFM and a gold-coated tip to pull and unravel proteins to determine their structures.

The 20.309 AFM System

This section describes the various components of the AFM you will use in the lab, and particularly how they differ in operation from a commercial AFM. In lecture we will discuss the operational principles of a commercial AFM. You may also ¯nd it helpful to review some of the References in Section 8 at the end of this module.

Hardware

A photo of our AFM setup is provided in Figure 1 for you to refer to as you learn about the instrument. Start by physically examining the setup and identifying all the parts described below.

Motion control system

To be useful for imaging, an AFM needs to scan its probe over the sample surface. Our microscopes are designed with a ¯xed probe and a movable sample (also true of some, but not all, commercial AFM systems). Whenever we talk about moving the tip relative to the sample, in 20.309 we will always only move the sample. The sample is actuated for scanning and force spectroscopy measurements by a simple piezo disk, shown in Figure 2. The piezo disk is controlled from the matlab scanning software, which is described in Section 3.3.

For vertical motion along the z-axis, there are three regimes of motion:

Manual (coarse): Turning the knob on the red picomotor with your hand (clockwise moves the stage up).

Picomotor (medium): Using the joystick to drive the picomotor (pushing the joystick forward moves the stage upward).

Piezo-disk (fine): Actuating the piezo disk over a few hundred nanometers using the matlab software. For x- and y-axis positioning (in the sample plane), coarse movements are accomplished with the stage micrometers, and fine (several nm) movements are also attained using the piezo disk.

3.1.2 Optical system

Our microscopes use a somewhat di®erent optical readout from a standard AFM to sense cantilever deflection. Rather than detecting the position of a laser beam that re°ects o® the back surface of the cantilever, we measure the intensity of a di®racted beam. To do this, a diode laser with wavelength ¸ = 635nm is focused onto the interdigitated (ID) \¯nger" structure, and we observe the brightness of one of the reflected spots (referred to as \modes") using a photodiode. This gives us information about the relative displacement of one set of ¯ngers relative to the other | this is useful if one set is attached to the cantilever, and the other to some reference surface.

20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 As the cantilever de°ects, and the out-of-plane spacing between the ID ¯ngers changes, the re°ected di®ractive modes change their brightness, as shown in Figure 3. However, a complication of using this system is the non-linear output characteristic of the mode intensities. As the out-of- plane de°ection of the ¯ngers increases, each mode grows alternately brighter and dimmer. The intensity I of odd order modes vs. ¯nger de°ection ¢z has the form I / sin2 µ 2¼ ¸ ¢z ¶

and for odd modes, the sine is replaced by a cosine. The plot in Figure 4(a) shows graphically the intensity of two adjacent modes as a function of displacement. This nonlinearity makes the sensor's sensitivity depend critically on the operating point along this curve at which a measurement is done. To make useful measurements, the ID interferometer therefore needs to be biased to a spot on the sin2 curve where the function's slope is greatest - midway between the maximum and minimum, as sketched in Figure 4(b). Due to residual strain in the silicon nitride from which the cantilevers are fabricated, the relative planar alignment of the two ¯nger sets varies slightly over the area of the grating. This variation is typically a few hundred nanometers in the lateral direction. Therefore, the bias point of the detector's output can be adjusted along the sin2 curve by moving the incident laser spot side to side on the di®raction grating. (a) The non-linear intensities of the 0th and 1st or- der modes as a function of cantilever displacement (from Yaralioglu, et al., J. Appl. Phys., 1998). (b) The desired operating point for maximum de- °ection sensitivity is sketched here on the sin2 output characteristic of the ID ¯ngers. Figure 4: The characteristic output of the ID interferometric sensor. 3.1.3 Cantilever probes for imaging A few words about probe breakage: you will break at least a few probes { this is a normal part of learning to use the tool. We have a large, but not in¯nite supply of replacement probes. The cost of an individual AFM probe is not large, and the greater problem with breaking them is the time lost to replacing the probe and realigning the laser. 5 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 (a) (b) Figure 5: (a) Plan view and (b) SEM image of the imag- ing cantilever geometry. The central (imaging) beam dimen- sions are length L = 400¹m, and width b = 60¹m. The ¯n- ger gratings begin 117¹m and end 200¹m from the base. Exercise caution when moving the sample up and down, but don't let this prevent you from getting comfortable moving the sample around. Under most conditions, the cantilevers are surprisingly °ex- ible and robust. They are most often broken by running them into the sample (especially sideways) { avoid \crashing" the tip into the surface, or worse bumping the stage into the die or °uid cell. Make sure you're familiar with the motion control system (Sec. 3.1.1). The probes we use for imaging are shown in Figure 5 with relevant dimensions. The central beam has a tip at its end, which scans the surface. The shorter side beams to either side have no tips and remain out of contact. The side beams provide a reference against which the de°ection of the central beam is measured; the ID grating on either side may be used. When calibrating the detector output to relate voltage to tip de°ection, remember to include a correction factor to account for the ID ¯nger position far away from the tip. 3.1.4 Cantilevers for thermal noise measurements For noise measurement purposes, we'd like a clean vibrational noise spectrum, which is best achieved using a matched pair of identical cantilevers. The con¯guration with a central long beam and reference side-beams has extra resonance peaks in the spectrum that make it harder to interpret. With the geometry shown in Figure 6 the beams have identical spectra which overlap and reinforce each other. Using a pair of identical beams also helps to minimize any common drift e®ects from air movements or thermal gradients. There are two sizes of cantilever pairs available. For the long devices, L = 350¹m and the ¯nger grating starts 140¹m and ends 250¹m from the cantilever base. For the short devices, L = 275¹m, and the ¯nger gratings begin 93¹m and end 175¹m from the base. The width and thickness of all of the cantilevers is b = 50¹m and h = 0:8¹m, respectively. (a) (b) Figure 6: (a) Plan view and (b) SEM image of the geometry of a di®erential cantilever pair. Because the beams are fabricated so close together, we assume that their material properties and dimensions are identical. 6 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 3.2 Major operational steps 3.2.1 Power-on For our AFMs to run, you must turn on three things: (1) the detection laser, (2) the photodetector, and (3) piezo-driver power supply. The photodetector has a battery that provides reverse bias, and the others have dedicated power supplies (refer to Figure 1 for where these switches are located). When you ¯nish using the AFM, don't forget to turn o® the three switches you turned on at the beginning. 3.2.2 Signal connections and data °ow Figure 7: Schematic of signal connections. The ¯rst key step to using the instrument is properly connecting all of the components to- gether. Figure 7 will help to guide you. The AFM itself requires two signal inputs (Xin and Yin) to drive the piezo actuator, which connect to the electronics board on the back of the headplate. They are provided by the computer's digital signal outputs (dac0out and dac1out). The computer also needs to read these signals in, together with the AFM signal output, so these become the three DAQ inputs. The output from the AFM's photodetector is a current signal proportional to the brightness of the laser spot, which needs to be converted to a voltage (a 100 k­ resistor to ground is su±- cient). It's good to be able to amplify and o®set this voltage at our convenience, so run this sig- nal through an AM502 ampli¯er before it enters the DAQ. Finally, during calibration, it's very useful to watch the detector signal as a function of stage movement in realtime, on the oscilloscope screen, so run those signals to the scope as well. 3.2.3 Laser alignment and di®ractive modes To get a cantilever position readout, the laser needs to be well focused on the interdigitated ¯ngers of the cantilever. Use the white light source and stereo-microscope to look at the cantilever in its holder. The laser spot should be visible as a red dot (there may be other re°ections or scattered laser light, but the spot itself is a small bright dot). Adjust its position using the knobs on the kinematic laser mount, until it hits the interdigitated ¯ngers (use the cantilever schematics in Figures 5 and 6 as a reference). When the laser is focused in approximately the right position, the white \screen" around the slit on the photodetector will allow you to see the di®raction pattern coming out of the beam splitter. Observe the spot pattern on this screen while adjusting the laser position until you see several evenly spaced \modes." Make sure you aren't misled by re°ections from other parts of the apparatus | some may look similar to the di®raction pattern, but aren't what you're looking for. When you see the proper di®raction pattern is on the detector, adjust the detector's position such that only one mode passes through the slit. Typically the 0th mode gives the largest di®erence between bright and dark. 7 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 3.2.4 Sample loading and positioning Correctly mounting a sample in the AFM is a key part of obtaining quality images. Our samples are always mounted on disks, which are magnetically held to the piezo actuator o®set post. The AFM can image only a small area near the center of the opening in the metal cantilever holder, so be sure that the area of interest for imaging ends up there. (a) A photo of the underside of the can- tilever mount, with the sample disk lowered for changing samples. (b) A close-up view of the opening into which the sample rises, showing the can- tilever die and sample disk. When changing or inserting a sample disk, the 3-axis stage must be lowered far enough for the disk to clear the bottom opening of the cantilever mount, as shown in the ¯gures above. This requires a large travel distance, so exercise caution when bringing the sample back up to the cantilever, and take care not to crash the tip. In addition, as you change samples, it is critical to reposition the o®set post as nearly centered as possible on the actuator disk, to ensure true horizontal motion in the x-y plane (Centering the sample disk at the top of the o®set post is not critical; rather, it's the position of the bottom end of the post on the piezo scanner disk. For instance, in ¯gure 8(b) above the sample disk is visibly o®-center, which is not a problem). 3.2.5 Engaging the tip The process of bringing the probe tip to the sample surface so we can scan images and measure forces is called \engaging." The aim is to get the tip in close proximity so it is just barely coming into contact, and bending only slightly. If the probe does not touch the surface, it is obviously useless, but if it's bent too much against the surface, it is equally useless. Before engaging, start the piezo z-modulation scan in the matlab software (see Sec. 3.3.3). Be sure the mode switch on the AFM electronics board is °ipped down to \force spec. mode," and make sure to turn on the piezo power supply. Carefully bring the tip near the surface, ¯rst turning the red motor knob by hand, then very slowly with the joystick. When you make contact, you will see the modes on the photodetector °uctuate in brightness. Because of the device geometry, only the central long cantilever with the tip will make contact with the sample surface. 3.2.6 Calibration and biasing At this point, it's worth pausing to review the de¯nition of calibration, as well as the distinction between sensitivity and resolution { terms which will often used frequently in this context, but 8 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 whose precise meaning isn't always made explicit. Be sure you're clear on the di®erences between them. Calibration - ¯nding the relationship between the output of your instrument to the physical quantity you are measuring like distance or force; in our case, relating the mode brightness measured by the photodiode to cantilever tip de°ections Sensitivity - a numerical expression for the calibration, the \slope" of a transducer output e.g. mV/N, W/ºA, or in our case nm/V Resolution - the minimal measurement or change in signal that an instrument can detect; depends completely on the noise and the frequency and bandwidth of the measurement It's a good idea to run a calibration before performing any measurement, because they varies from AFM to AFM, and may be thrown o® by drifts or disturbances. We calibrate our AFM in force spectroscopy (or z-modulation) mode, in which the sample is only moved straight up and down (see Section 3.3.3 for details). Watching the AFM signal on the oscilloscope in x-y mode, (with detector output on the y-axis, and the stage actuation signal on the x-axis) you should see something like the plots shown in Figure 9: a °at line that breaks into a sin2 function at a certain x-value (whether it starts upward or downward depends on the mode you choose). The °at line is the cantilever out of contact, and the oscillating section is the cantilever bending, after making contact with the sample. If necessary, use the o®set on the voltage ampli¯er to position the sin2 so that it is centered around zero. Then, set the out-of-contact bias point by moving the position of the laser focus on the ¯ngers until the °at section of your force spec. curve is approximately at zero volts, halfway between the maximum and minimum, as in Figure 9(c). (a) Bias too high. (b) Bias too low. (c) Bias set to optimal sensitivity for out-of-contact. Figure 9: Proper setting of the bias point. The goal of this calibration process is to relate the detector's voltage signal to physical tip de°ection { i.e. how many nm is the cantilever tip bending for every volt of signal. We can take advantage of the fact that a mode's brightness goes from fully bright to fully dim (peak to trough on the sin2) as the ¯ngers de°ect through a distance of ¸=4 (¼ 160 nm). This should allow you to calculate the relationship between de°ection and y-axis voltage in nm/V. Finally, you will also need to multiply the calibration by a correction factor Acorr to account for the location of the di®raction ¯ngers with respect to the tip of the cantilever. Acorr can be estimated most simply by assuming a quadratic shape for the bent cantilever Acorr = 1=m2 ID, where mID = LID=LT is the ratio of the distance of the ID ¯ngers from the cantilever base to the total cantilever length. A more precise expression is Acorr = 2=(3m2 I D¡m3 ID) (derived from the equation for the shape of a simple rectangular beam, with an applied end-load). 9 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 3.3 Software The software that interfaces with the AFM is an application that runs in matlab. Its graphcial user interface (GUI) is launched by typing `scannergui' in the matlab command window. Its main function is to systematically scan the probe tip back-and-forth across the sample, recording the cantilever de°ection information at each point, line by line, and assembling that data into an image. Figure 10 shows a screen capture of the scanner control window, and an overview of its operation is provided below. Figure 10: The Scanner GUI window. The AFM is scanning a 12 £ 12¹m area, at a rate of one line per second, and is currently near the bottom of the image. 10 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 3.3.1 Controls overview Many of these are self-explanatory, such as the start imaging and stop buttons, as well as the image area in the lower right, which displays the image currently being scanned. Some notes are given below on features that are not immediately obvious. To begin with, it's easiest to simply use the default settings on all these controls, and to experiment with changing them as you become more familiar with the tool. Scan Parameters - The Scan size sets the length and width of the image in nanometers (always a square shape), but its accuracy depends on having the correct value for Scan sensitivity (which should already be set for you, but may require calibration). The Scan frequency (lines per second) sets the speed of the tip across the surface, and together with the Number of lines a®ects the amount of detail you will see in the image. Setting the Y-scan direction tells the scanner whether to start at the top or the bottom of an image, and the trace/retrace selector determines whether each line is recorded as the tip scans to the left or to the right. Scope View - As the tip scans back and forth, this plots the tip de°ection data for each line. Useful for quantitative feature height measurements. Scanner Waveforms - Shows the voltage waveforms driving the piezo scanner, for each scan line that is taken. Helpful for knowing where in the image the current scan line is located, and the output level of the waveforms driving the scanner. Z-mod Controls - These are only active during a z-mod scan, and have no e®ect when taking an image. For more on this mode, see Section 3.3.3. 3.3.2 Image mode operation This is the primary operating regime of the AFM, and provides a continuous display of the surface being scanned, as the probe is rastered up and down the image area. To use this mode, the switch on the back of the AFM must be °ipped upward. Remember that the maximum scan area is only abut 15 ¹m square, and adjusting the position of the sample under the tip requires only the smallest movements of the stage micrometers. Finally, keep in mind that there is always a delay after pressing start imaging before the scan begins, as the actuator drive signals are bu®ered to the I/O hardware. 3.3.3 Z-mod (force spectroscopy) operation In this mode, the piezo moves the sample only along the z-axis { i.e. straight up and down (hence z-mod, short for z-modulation). To use this mode, the switch on the back of the AFM must be °ipped downward. The default frequency and amplitude of 2 Hz and 8 V provide a nice force curve. Besides being critical for calibrating and biasing the readout, this mode is used to perform force spectroscopy experiments, in which tip-sample forces can be measured as the tip comes into and out of contact with the sample. (Note that the red stop button is also used to stop a z-mod scan). 3.3.4 Saving AFM data The software allows you to save the raw data of both images as well as force curves. Unsurpris- ingly the \Save Image" and \Save Force Curve" buttons do this. In both cases an instantaneous \snapshot" of the current image or force curve is written to the ¯le location speci¯ed in the entry box at the bottom of the window. 11 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 An image is written to a ¯les as a square matrix (interpolated to have the same number of rows and columns), with the value at each point representing height data. Force curve data is saved as two columns: x-axis (stage de°ection) data in column one, y-axis (mode intensity) in column two. If you intend to save an image, it is best to set the ¯lename before starting the scan { the ¯lename box behaves . . . elusively while the scanner is running due to some peculiarities of the software. As a ¯nal note, a \quick and dirty" way to save an image is by simply doing a screen capture while the AFM is scanning (press \Print Screen" on the keyboard). The captured image can then be pasted into MS Paint (Programs ! Accessories ! Paint) and cropped to leave only the scanned AFM image. These images can be imported into matlab for analysis/processing (we'll do this in later parts of the course). 3.4 Additional instrumentation 3.4.1 Di®erential ampli¯er Figure 11: An AM502 di®erential ampli¯er. The gain is determined by the central red knob, to- gether with the ¥100 button in the center above it. The Tektronix AM502 is a di®erential amp, so it ampli¯es the voltage di®erence between the two in- put signals. Both inputs have DC, AC, and GND input coupling, like the scopes. The amp can be used single-ended if the ({) input is left unconnected and ground-coupled. The gain (ampli¯cation fac- tor) ranges from 1 to 10,000, and is set by the red knob together with the \¥100" button above it. The instrument also has high- and low-pass ¯l- ters. These are somewhat confusingly labeled \HF- 3dB" and \LF-3dB" (see Figure 11). This doesn't mean \high-pass" and \low-pass," but refers to the \high [cuto®] frequency" of a LPF and the \low [cuto®] frequency" of a HPF. Therefore, HF-3dB is the low-pass ¯lter, and LF-3dB is the high-pass ¯lter. Both ¯lters are considered \o®" when the low- pass is turned all the way up, and the high-pass all the way down. Finally, the lower (high-pass) ¯lter knob also has two settings for controlling output signal DC level: dc and dc offset. When set to dc, the amp outputs the actual DC level of the input signal, multiplied by the gain. Set to dc offset, you can manually adjust the DC level using the knob at the upper right of the AM502. In both cases, the AC component is simply added on top. 12 20.309: Biological Instrumentation and Measurement Laboratory Fall 2006 3.4.2 LabVIEW VIs for signal capture The spectrum analyzer Figure 12: The LabVIEW Spectrum Analyzer VI runs continuously until the stop button is pressed. Each PSD is averaged with all the data that precedes it, so the spectrum gets cleaner the longer you acquire. Note that the \PSD controls" (N®t, downsampling) can be changed on the °y, but to change the sampling rate you must stop and re-start the VI. Pressing \Stop and Save Data" pops up a window in which you can choose a ¯le name to store the spectrum currently displayed. Time-domain signal capture Figure 13: The LabVIEWWaveform Acquisition VI does a \one-shot" waveform capture in the time domain. Pre-set your desired parameters, including sampling rate, length of captured waveform, and ¯lename to save to, then press the Run (arrow) button to do the data capture. What you've captured appears in the waveform