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Basic Operation Procedures for the Asylum Research MFP

1. Force curves can be monitored with the headphones This is great for a variety of reasons You can have the headphone volume up to listen monitor to the force distance cycles while you work one something else across the room Large non specific multiple events curves can be heard through a sort of ripping sound when working with bio systems or you can hear if the tip comes off the surface by a constant static noise as opposed to static dampen static sound of a regular force distance cycle 21 Force Volume Mapping Chad Ray Duke Chemistry has written a macro to acquire force distance curves in an array Contact AR Support AsylumResearch com to acquire this procedure v Activate by going to programming load user functions UserChad0 It will load a window Figure 30 v choose the array size keep number of points and lines and equal value and what output you want the y axis to display i e Force deflection v leave all other parameters in their default settings v This procedure will also export curves to text files by clicking the button This creates a new folder called export in the folder where your ibw waves are in a comma delimited formatted This is great for exporting lots of curves in a batch type file One Note M makes only one parameter header in that specific folder full of ibw s This procedure is very good Chad has incorporated a feature called Update Start Dist W checkb
2. Height image Phase image 1 6 wm 1 4 1 2 Figure 42 Height Phase Overlay af 3 w 0 um o p2 4 XIII TROUBLE SHOOTING False Engagement in fluid decrease set point in AC mode decrease the set point increase drive amplitude until piezo extends fully 150 V Quickly finding tip in CCD camera with top view optics when you feel you have lost it mirror way out of whack Realigning the tip within the 10x top view optics can be quite an overwhelming trial and error undertaking if some user before you has completely lost the tip by cranking the mirror alignment knobs all the way and messing up the focus But it doesn t have to be that way With suitable amount of light the mirror can easily be realigned by looking into the mirror along the same vector as the top view optics path whether from below SA or above bio Turn mirror alignment knobs until you see tip chip and trapezoidal window align so it s in center Put MFP 3D back on it s scanner platform play with focus until you find some feature that should be in same plane as tip chip move mirror alignment Knobs slightly until you locate it Sum amp Deflection Meter Freeze This can occur if the cable connection isn t good With time and all that repeated cable twisting from putting the head on its back while attending to the sample the plug can wiggle its way out enough to cause the meter to freeze TO FIX IT power the controller off fo
3. Delete Sel Make Force Display Panel Figure 40 Master Force Panel load more curves those curves will included in any analysis you do i e a histogram Clicking the Edit button in the Force Review Graph brings up a table with the points if you want to copy it and dump it into another spreadsheet The Parm tab tells you the parameters of a file There are a series of pull down menus that lets you customize the panel to show only specific parameters The Cursor tab am not really sure what this does 35 The Modify tab allows you to zero the curve with respect to X and or Y individual curves or all the curves in the data set am not familiar with the actions of the Line Subtract button The WLC tab calculates worm like chain model for curves with multiple events The instructions for the analysis are located on AR s web page under the support MFP3D FAQ section This FAQ webpage has some good stuff on it The Analyze tab allows you to generate histograms and scatter plots of adhesion data select from pull down menu The histogram window has a feature in which you can fit Guassian Poisson or Lorentzian curve to it Sometimes you have to stretch the window out to see this feature on the right hand side of the window Clicking Edit brings up a table with the histogram points in it The number of bins can also be adjusted with a slider bar NOTE The procedure for this calcu
4. Integrate noise spectra to find biggest contributor From noise panel click Save In Test Results Noise rename folder to a unique name X01 not X FastOffSurf not Fast Make that the current data folder Duplicate O PSD Int Int Int omit this line if you are using true PSD from Mario s new panel old Noise panel gives amplitude spectral density as PSD and must be squared Integrate Int Display PSD AppendToGraph R Int ModifyGraph log bottom 1 ModifyGraph RGB Int 0 0 65535 ShowlInfo Now look for the biggest step s in the blue integrated curve Retrieved from http wiki AsylumResearch com Common_Commands 47 XV RF s QUICK REFERENCE Tip loading gently slide tip under tongue such that tip is in middle of clear trapezoidal window tighten screw finger tight with screwdriver see page 3 Set point determination in Contact mode after SLD laser alignment adjust PD to slightly negative value choose set point higher than this for gently engagement In contact mode higher set point voltage values translate to higher applied force see pages 7 8 Spring constant k determination 1 Force curve on a clean hard surface determine InvOLS cursors on slope of contact region gt Set Sens Deflection 2 Disengage tip perform Thermal tune Fit first resonance peak to get k see page 11 15 Set point determination in AC mode Pick a set point that is 10 to 20 of the free air amplitude value
5. ASYLUM ESEARCH Basic Operation Procedures for the Asylum Research MFP 3D Atomic Force Microscope Ryan Fuierer Ph D based on software version 060513 Section Table of Contents _ Disclaimer 2 Loading the cantilever 3 ll Laser Alignment i 4 lll Contact Mode Operation k 7 IV Image Channels oon Flattening 9 V Saving Images i a 10 VI Determination of Spring C 11 VII AC Mode Imaging 16 VIIB AC Mode i VIII D Force Spectrose en a 2005 2006 ia Ba i MFP 3D photo courtesy of Aleks Labuda The purpose of this Manualette is to offer new users some documentation on how to procedurally operate an Asylum Research MFP 3D AFM It s like a manual but more brief and to the point regarding procedures Its inception was to alleviate time spent training lab members and has evolved into a document to help new or sporadic users how to navigate the software and be more efficient at operating their AFM Essentially l d like your operational learning curve to be reduced so you can be more efficient in the lab It is assumed that the MFP 3D manual has been perused such that am not responsible for omitting such common sense precautions as don t stare into the SLD don t drop or abuse the equipment or don t leave the controller teetering on the edge of the bath tub It is an expensive high precision instrument and should be treated with great care This Manualette
6. Log Log Zoom Graph O zoom Center 0H JS Zoom Width 12 000kKHz SLS Max Samples 1000s JS OE Current Samples 346 Fesolution 7 default Do Thermal E1 Them al Graph puhtia ii Figure 13 Frequency vs amplitude plot from a thermal tune taken in air A full scale spectrum B zoomed Figure 12 Thermal Tab and fitted blue line v Click on the Show Fit M checkbox then the Fit Guess button this brings up a blue Gaussian shaped curve very near to the thermal resonant peak Click on the Try Fit button a blue curve will fit to this resonant peak Figure 13B The software automatically calculates the spring constant and updates the resonant frequency seen in Thermal and Main tab 15 NOTE when using a floppy cantilever IN FLUID I ve found that there is a dampening effect that causes difficulty computing this peak position see Figure 14B below There are ways around this 1 take the frequency spectrum before engaging taking force curve perform a try fit once you have the InvOLS value there will be some greater error in k determination with this approach 2 disengage the tip and manually retract of the tip a turn or two of the thumbwheel 20 to 40 um before taking the freq spectrum This seems to work much better with a low number of iterations kHz WOkHz TOOKHE IW Figure 14 Thermal tunes of contact mode cantilevers N FLUID A typical plot B one that has fundament
7. does not care if channel is displayed or not J Channel Save YDisp XDis Deflection f Ej P i anme OF oO Oo Phase LJ a Input an Inputa LJ Frequency OI Useringd O Wsernt CJ Mi i E z J J slololo Ly LL bo C0 i DSSS CH td Ld ou up to 5 channels can be plotted simultaneously t ejoj ojo o SIE SIES LJ serna OOl G C O Orc ae Lateral OO E ar E th E i j i DEES ae Current j i LI 0 0 0 0 0 0 UserCale 3 olololo miima Red Blue mode forward amp reverse pulls Red Mode forward pull tip starts cycle at surface and travels to free air then returns to surface Blue mode reverse pull tip starts cycle in free air and approaches surface then retracts Potential g g J J o By Ba Drive LVDT 5J OC 0 Ol LJ BeeB68 Force RawLVOT E Detlvalts O oje Ampalts L YDT Volts 3 lt Blue activation UserlndVolts Userini olts se ee OlO O O o CO OO O O O LJ 0 C1 ie es Wserln2 volts Oo Ue oo olo O G OO SIES Lateral valts SSES Dri
8. So now you have this force curve that looks similar to the one in Figure 10A It s time to determine the InvOLS inverse optical lever sensitivity of the cantilever from the slope of the contact repulsive region of the force distance curve This will complete the first step to determine the spring constant of the cantilever v With the force plot window active hit Ctrl brings up a panel on the bottom that has cursors A B K with there respective x y dx dy values v Move one of them by holding down with left mouse button on to a linear part of the contact region of the curve put the other cursor on another part of the linear region in the same fashion see cursors in Figure 10A v Make sure you have both cursors on the same trace use the keyboard arrow keys to assure they both move in the same direction If they move away or towards each other they are not on the same trace NOTE you can deactivate one of the cursors on the curve by going into the lower margin of that Igor window and clicking on the open circle square When it s deactivated it will turn black allowing you to finely position the other cursors on the curve When done reactivate and check that both cursors are on same trace You should have a plot that looks similar to the one in Figure 10A notebook Amp InvOLS is Defl InvOLS times Kappa In the Force tab click on Deflection in the Set Sens pull down menu Figure 11 T
9. The trigger point value is the amount of deflection the tip undergoes once the tip is in contact with the surface before the piezo switches direction for retraction This process can be monitored in real time by listening to the transducer headphones When the tip makes contact you can hear it go from a static noise to quiet dampened contact and back to static sounding upon retraction Master Panel OP p SetSens Vitual Def Virtual Defect Deflection Thermal Force Tune Amplitude Q Start Dist 1617 um S Virtual Defl 200 00 nm e O Scan Rate 0 66 Hz Ey Approach Vel 1 50 yms S Retract 997 34 nmd O Defl InvOLs 87 26 nm v E Kappa Factor 109 Amp InvOLs 85 12 nmv Spring Constant 120 99 p n e set Sens Deflection Virtual Deflection 3 64 mViun 6 Deflection Offset 227 31 mv JE Deflection Off Force Dist Yo oe amp DeflVolts Pos Slope Neg Slope Absolute Relative Trigger Point 0 200V 58D Channels Force Panel Trigger Channel O Defi olt amp Defoli Figure 10 A Force distance curve on a hard surface Cursors placed on linear region of contact repulsive regime Use Ctrl to get cursors B Set Sens parameter to deflection to update InvOLS value C resulting curve fit black line between cursors fit line Bebb bb amp amp Figure 9 Force Tab of Master Panel 13
10. can monitor the phase of the oscillating cantilever during imaging Mode hopping occurs when imaging switches between intermittent contact attractive and non contact modes repulsive This works well in air but doesnt hold as true in fluid Mode hopping occurs when the Phase flips between lt 90 and gt 90 resulting in kind of crappy images that s a technical term The sine of the phase at constant amplitude is the tip sample dissipation But sine is symmetric sin 80 sin 100 Thus there are always two phase angles consistent with a given dissipation So the second important part is whether the phase is above or below 90 Take home message Phase gt 90 attractive or low amplitude mode is very different than Phase lt 90 repulsive or high amplitude mode The colors represent what color the phase value regime will be in the S amp D meter text courtesy of Jason Cleveland When AC imaging in air you can following if the tip is net attractive or net repulsive forces with the surface by monitoring the phase value in the S amp D meter Typically you have to go into setup window use the pull down menu next to Phase and choose On from auto If the phase is lt 90 blue it s in the repulsive regime when its gt 90 red it s in the attractive regime This gets more complicated in fluid so am not suggesting you follow this trend in anything other than air The following ta
11. change data folders here From here a thumbnail Browse window comes up along with a List panel you can choose images from either The List panel not shown is convenient for saving real estate space on the monitor s screen just file names are shown plus some options like change directory etc The Browse window shows the image files in thumbnail form amp has more features Figure 32 You can choose the size data type trace or retrace etc lolx Vertical Number 4 show Data Type Height Whichimage Either v Page Start 0116 Horizontal Number 5 I show Force Data Deflection Next Page Previous Page Image Total 26 ya Figure 32 The display options of the Browse panel v Double clicking on file or image calls up a stored file Once you choose a file the image comes up with a bunch of analysis function buttons range value windows and pull down menus Figure 33 It s called a display window itt bigsphere0001 0 HeightTrace The tabs in the display window above the image represent the saved data channels Figure 33 shows height Trace amp retrace HtT HtR Amplitude Phase Z sensor The z range can be manually adjusted or auto scales by clicking the Auto button Range 500 00 ni M4 color Bar onset Erm JE similarly the z scale offset can be 15 adjusted The options under the Command pull E 10 down will be discussed below The
12. correction essentially levels the free air part of the force distance curve This can make curve analysis easier for example some rely on an analysis that averages some section of the free air to find the contact point of the retract This virtual deflection is a result of the mechanical path not being quite perfect resulting in a slight slope in the force curve Notice that this may only be a few nm s over several microns but if not corrected it can make a difference in measurement or analysis Procedure With low force distance value v Click button Force curve should like similar to one in Figure 27A a long free air in the approach curve is desired so you can fit a linear region of it v place the Igor cursors Crtl I on linear part of free air approach v go to Set Sens pull down menu in Cal Tab Figure 27B select Virtual Defl This calibrates the virtual deflection so you have level looking free air portions of your force curves Rename sae cat fl oFr tat A ae Set Sens Deflection Virtual Defieci Deflection Amplitude 105 j p Deflection Off J Virtual Defl 020 Figure 27 Calibrating virtual deflection upon the first force curve acquired place cursors on free air part of curve B go to Set Sens pull down and choose Virtual Defl C free air part of curve is fitted black line Force distance curves after this will have level free
13. down it contains the option to turn of the closed loop feature Making multiple copies of MFP software When updating MFP software but you want to keep your old version too make a copy of Igor See AR web site FAQs Copy the Igor pro folder in Wavemetrics rename it so you don t get confused Load the new software into this new folder When you want to use it go into this folder click on the Igor Pro icon to load Igor then go to File new MFP template This will load it properly have found that Igor can get mighty confused if you dont do it this way Contact Support for further insight Overlaying two image channels Requires knowledge of how to use ARgyle commands see section X What is displayed is one channel as a z scale while the other is displayed as a color scale See Figure 42 below 40 v Process your two channels to your liking i e flatten mask pick colors Create an ARgyle image for each image channel The example used below is a height and phase image of a bacterium One of the channels needs to be grayscale height while the other is some color phase Use aspect 1 checkbox to adjust z scale in this one vIn the command line type argl_bindmesh filename suffixHtT m0 filename suffixPhT m0 Example argl_bindmesh PAbacteria00O22HtTm0 PAbacteria0022PhT m0 The resulting ARgyle overlay will occur in one of the ARgyle windows Specular light was added shiny under Argyle light tab
14. in Sum amp Deflection meter In AC mode lower set point voltage values translate into higher forces see page 17 Auto Tune Drive frequency in AC mode Align laser on tip zero PD Do thermal to get approximate resonant peak enter frequency range that flanks this value Click Auto Tune Engage tip image see page 16 Picking Drive Frequency when imaging in fluid Align laser Do thermal approximate resonant frequency of cantilever amp enter in drive frequency value of Tune tab seslect Append Thermal M checkbox perform One Tune with a 15 KHz window use cross cursor to select point on black frequency plot that is near the resonant peak of the thermal red scatter plot select set drive frequency as Drive frequency updated in Tune amp Main tabs see pages 19 20 Alternatively perform thermal tune manually pick frequency by placing Igor cursor on left side of peak type this value into drive frequency Engage tip after engagement use Hamster wheel front of controller to fine tune drive frequency by monitoring the phase amp amplitude images and line traces see page 20 21 Gentle Engagement AC mode Pick a set point that is 5 of the free air amplitude Click Simple Engage to activate piezo Thumbwheel down until tip engages which is a false engagement Adjust set point with Hamster to hard engage tip you may have to use thumbwheel to adjust piezo further but it s already
15. red A dialogue comes up after you right click choose Set drive frequency as this updates the drive frequency i N l l 1H LORT i yha aiy Wy u yaf j W oy E Win F T E T s T m i i laf TOS 1 i Figure 18 Results from a One Tune frequency sweep in fluid with appended thermal tune red points The black vertical line indicates the user defined drive frequency v Click the Center Phase button once a drive frequency has been determined This will center the phase at 90 degrees which you can see in the S amp D meter This allows you to monitor whether the tip is in the attractive or repulsive regime however this doesn t hold as true in fluid as it does in air Section VII v Change the R filter Go to Programming make filter panel A new window pops up change R filter from 1 5 to 0 5 Hz 20 v Select imaging parameters and you ll be ready to Engage Choose the set point similar to the description in previous section v Look at the free air non engaged Drive amplitude in the Sum amp Deflection window See Figure 17 plateau part of curve v Choose a set point that is lower than that some percentage in volts If you want to engage gently make the set point 85 to 95 v Click Simple Engage on the sum and deflection meter top and manually thumb the approach wheel until the feedback servo is activated a
16. releasing their product Sa Tet Pane ole Single Measurement X Y z Fast A B input m O O M O m Res 4 OffsetiLever 000V E00 Eoo ql Fee ava 70 C Bandwidth Tooke C I owe ook Tee ee Courter 9 Sensitivity 2 53um V ETV Zaim 89 18nm V InvOLS Mine A Noise 2 6 nm 481 96pm 76 41pm 67 73pm 180 29mV 23 38mV haf Avg Noise 1 15nm 466 9 7pm 74 860m 64 160m 178 64mV 22 95m Save J Figure 43 Noise tab of the 3D Test Panel v determine the InvOLS of the cantilever on clean mica or graphite something atomically flat AR spec tests use a 100 um Si3N triangular cantilever The value will automatically be updated in the sensitivity value These noise numbers are sensitive to InvOLS which in turn is sensitive to the Sum and individual cantilevers Hence don t be sloppy with your InvOLS calibration when performing noise tests The Fast input is measured two ways engaged and disengaged free air Check the box below the fast column The free air will give you a sense of how noisy the system is The engaged measurement is going to give a real sense of how noisy the MFP s environment is i e Seismic acoustical mechanical electrical etc Free air this checks the noise of the system without the noise at the tip sample interface v Disengage the tip from the surface v Enter 70 V in offset lever v Hit the Start button A noise plot comes up which shows the sampled noise figure b
17. you can monitor the noise of these inputs at 10 70 amp 150 V low end middle and high end Only one can be tested at a time Values below 600 pm meet AR s specs for the X amp Y while values below 300 pm meet specs for the Z input have compiled the following table of AR acceptable specs Use 100 um triangular SiN tip InvOLS 30 nm V use mica or graphite surface for engaged test e k p e lt 600 pm lt 600 pm lt 300 pm Scanner calibration You can check the health of your scanner by clicking the Calibration tab of the 3D test panel Figure 45 Choose the scanner from the pull down menu make sure it is in the Drive Action and X Y or Z start 10 V and range 160 V Click Start button A graph similar to Figure 46 comes up You should have a nominal range of 14 V and a hysteresis value of 4 or less not sure on AR s specs on the percentage j hannel gjs 44 f Calibration _ Freq Analysis Images LYDT Piezo Calibration TE XBStart 10 0V 6 Range 160 0 6 a Action DKS Freq 1 0Hz i Start 10 0 A Ditaja J Phase 180 00 S Figure 45 Calibration tab of 3D Test Panel A properly functioning scanner will have a calibration plot that looks similar LVDT plot below typical voltages are of 14 V and hysteresis lt 4 ts X LVDT LVDT range 14 308V Hysteresis 3 7 Figure 46 typical LVDT X or Y scanner hystersis plot
18. ColorMap pull down gives you a lot 5 of color options including creating custom color maps 0 At the upper left of the image window you will see a series of small boxes with letters in them 3D D L A M They represent the following Modify Panel Figure 34 This panel is essentially contains the image flattening functions ColorMap Grays256 O G AZAR Amt PHT ZST ZER 0 a 20 200 Figure 33 Display Window un modified off line image recalled Under the Flatten tab you can exclude part of the image by clicking Exclude Points or Include Points which brings up a tool box right that has lots of shapes to use to make areas that won t be included in the area flattened When activating some of these shapes black squares on its periphery define it to move use arrows keys If you mouse click on it again it becomes fixed ES Modify Panel i Flatten Planefit Erase Mask Filter FFT History Prefs Ibigsphere0001 0 HeightTrace Figure 34 The tabs of the Modify Panel Misi ks nm 30 Alternatively by going to the Mask tab more complex shapes can by masked out think of the Mask function as something that with exclude all points pixels in the x y plane above a certain z threshold Using the image above as a very simple example the process is demonstrated vIn the Flatten panel click the Flatten button with
19. Deflection Scan Rate 05Hz Ae Amplitude F 5 ines Split velocity 600 00 nmd O Force j Misc Cal y Go There Save canal Defl InvoLs 56 43 nmi AmpValts Kappa Factor LVDT Volts Amp InvOLS 61 51 nmiv Figure 21 Trigger Channels Spring Constant 61 87 ninni Set Sens Deflection Virtual Deflection 56 72 u m Deflection Offset omy S Once k is determined the trigger channel can be switched to deflection or Force from the pull down menu and a distance or Force value can be entered if you are comfortable with this dimension Figure 21 Trigger Channel shows all channels you that can be selected for a trigger point Ek te te a Deflection Fos Slope Neg Slope Absolute Relative Force Curves are acquired individually by clicking Trigger Point 5 00 nm Or continuously by hitting the T 5 l button The amount of continuous curves can be limited by going to Setup Limit Contin To M checkbox and click Looks Good Enter amount of curves you want to collect continuously and do some force spectroscopy Figure 22 Force Tab showing Calibration Tab Be Go Bebe T Force Channel Panel This panel changes the data channels displayed and saved in Force Plots The Green Blue Red LEDS to the left of the channels describe the settings of those channels Red Channel is not on Blue Channel is ONLY displayed Green Channel is saved
20. ES choose a 17 character title it can t start with a number and type that into the Base Name slot in the Main Tab of the Master Panel see Figure 8 Click the Save Images M checkbox which brings up a dialogue to choose where to save it Use Browse button to change folders or type a new folder name The software is designed to do continuous capture which will increase the Base Suffix by one with every image The Note line allows an unlimited length of text to describe imaging experimental conditions this can be recalled in Show note option under the commands pull down of an display window offline analysis see Section X Save Partial is like force capture you ve changed the parameters yet you still want to save the image Browse lets you look at saved images also see Section X Save Prev saves the image scan previous it s temporarily stored in experiment memory Save Status tells you if its saving current scan or next Base Name Base Sufix 0018 I CS ooo save Images save Partial Browse Save Status Save Current Save Prev Figure 8 Capture part of Main Tab Psuedomonas Aeruginosa imaged in air 11 VI DETERMINATION of SPRING CONSTANT some of these procedures have been iterated in Section IlII The determination of spring constant k is a 2 step procedure 1 determine the slope of contact region from a force curve to d
21. O or 1 order flatten from pull down menu the following image occurs Figure 35A Notice the dark lines between the circular features v To remove these flattening artifacts go to the Mask tab choose Iterative from the Calc method pull down this automatically gives you the z range the operation chose after this calculation manual calc method automatically appears Figure 35B v Change threshold to some value that better represents surface threshold v Go back to Flatten tab click Flatten again to get result similar to Figure 35C v Click Reset Mask button to remove red lines EE Modify Panel 20 Flatten Planefit Erase Mask Filte bigsphere0001 0 HeightTrace O Global Mask 15 Calc Mask Batch Mask Calc Method Threshold L Range 4 Inverse ave Mask 0 0 0 5 10 15 20 0 5 10 15 20 Figure 35 flatten mask flatten process to remove flattening artifacts A 0 order flatten B Mask tab options C post mask Flatten with mask definition still present Iterative Bimodal Adaptive Fuzzy Entropy Fuzzy Mean Gray Tabs above the image with a 0 suffix indicate modification to that image Selecting Save Image in the Commands pull down will save this modification to the file the raw data will still be there The Modified image can be saved a number of ways 1 by going to the command pull down menu and selecting one of the many tiff expor
22. When done testing go to programming remove test procedures 45 XIV COMMON COMMANDS Here s a list of Some common commands that may be useful Bindmesh overlay images ArGL_BindMesh HeightIimageOHtT PhaselmageOPhT Render images in 3D anaglyphs using Argyle Argl_WriteValue anaglyph 1 turns ON Anaglyph in Argyl window Argl_ WriteValue anaglyph 0 turns OFF Anaglyph in Argyl window Monitor system parameters print td_ ReadValue Temperature Default Head reads temp at head print td_ReadValue Temperature Default scanner reads temp at scanner Turn ON OFF the blinking LED at the XYlvdt board and head print td_WriteValue LedBlinking Default XYLVDT 1 turns ON LED at XYLVDT board print td_WriteValue LedBlinking Default XYLVDT 0 turns OFF LED at XYLVDT board print td_WriteValue LedBlinking Default head 1 turns ON LED at head print td_WriteValue LedBlinking Default head 0 turns OFF LED at head Set read output voltage to XYZ piezo print td_writevalue Z Output Controller 70 sets output to Z piezo print td_readvalue Z input Controller reads z piezo voltage print td_wv y output 70 sets output to Y piezo print td_wv x output 70 sets output to X piezo Send voltage pulse output OutA using DAC eg 2V 2 print td_WriteValue A Output 2 print td_WriteValue A Output 0 print td_ WriteValue A Output 2 print td_WriteValue A Output 0 Print
23. act region called the inverse optical lever sensitivity or InvOLS a parameter necessary for the algorithm to determine the spring constant The white bar on the left side of the window represents the entire 15 um or 28 um range of the z piezo The red bar represents the Force distance a user defined approach retract cycle distance that the z piezo moves during the force distance curve acquisition As you change the force distance you can see the size of the red bar s length change This can also be activated with the Hamster wheel For a more detailed description of the Force Tab see Section IX Force Spectroscopy Initial Parameter adjustment v Choose a Force distance 0 5 to 1 um v Activate the Trigger from the Trigger channel pull down menu choose the DeflVolts channel v Enter a trigger point value of 0 2 to 0 4 V Most other parameters in Figure 9 are not important at this point v Choose a start distance with the red bar towards the beginning of its travel top Activate the relative trigger radio button v Click the Single Force button This makes the software choose a proper start distance on its own and you should have a force distance curve that looks similar to the one in Figure 10A The first force curve should have a very long approach red curve because the piezo was traveling toward the surface waiting to reach a force equal to the trigger point selected 12 NOTE
24. air approach retract data The Go There Tab This is where the user can designate points on an image for force curves This is very useful when you have imaged the surface and want to immediately acquire force curves at specific points features on your image The MFP 3D s Clear There closed loop makes the accuracy possible Procedure v Acquire image in AC or Contact mode v Use the Go There tab in the Force tab Figure 28 NO Marke v Under spot display pull down choose Numbered Markers Trigger Chan All Marke v Also check the Show Tip Location box a red dot will appear Pos Slop on the image 26 Pick Point spat Number 5 I Ll Show Tip Location Spot Display No Markers ee E OA AE iF A iL T aii k Numbered Markers Current Marker v Drag the symbol from lower left to the desired location Figure 28 Go There Force tab v Click on the That s Itl button which locks the point at that spot meanwhile the point number index is advanced one in anticipation of the next point being chosen v When finished you can enter what point you to return to for force curve acquisition hit Go There button The red dot should move to that cross hair see point 1 in Figure 29 NOTE Every time a new file name is chosen the numbered markers will refresh and you have to pick points over again Figure 29 Point and click Force curves IS9EGKBF
25. al peak dampened see note below Program has difficulty fitting to dampened fundamental peak At this point the software has determined the cantilevers spring constant by the equi partition method This is very useful when performing force distance curves or needing to Know how much force the set point is applying to the tip This value will be store in any saved data parameter file Determining spring constant without making contact with surface Keith Jones AR applications has a protocol to determine this Look for it in version X of the manualette 16 Vil AC MODE Imaging v From Main tab in the Master Panel select AC mode from the imaging mode pull down menu Figure 5 Adjust deflection PD to zero volts for AC mode no color in deflection meter Sometimes go a Sliver negative blue v Open the Tune tab of the Master Panel Figure 15 Make sure the Target Percentage is 5 0 The default Auto Tune high and low values are 50 kHz to 400 kHz respectively Most drive frequencies for AC mode cantilevers fall within this frequency range in air Click the Auto Tune button You can hear the shaker piezo sweep and an auto tune plot similar to the one in Figure 16 comes up The software automatically picks a drive frequency on the left side of the resonant peak at 5 of peak maximum at the minimum Drive amplitude needed to make a 1 0 V amplitude in the S amp D meter The target amplitude amp percentag
26. alitative Conceptual amplitude voltage vs distance for AC Mode imaging Lower set point values result in higher tip sample force higher set point values result in lower tip sample forces This is not to scale and not real data Gentle Engagement If you want to engage at the very lowest set point possible because of modified tip or an expensive super sharp tip the MFP 3D is perfect for it Here s how to do it v Perform an Auto Tune v Set the set point voltage for about 5 of the free air amplitude voltage i e for a 1 0 V free air use set point of 950 mV v Engage as you normally would However this engagement is a false engagement the tip is engaging on the inherent water layer on the sample Adjust the Z piezo voltage to 30 to 50 V halfway blue You can see the Z piezo voltage seems to kind of float as you move the thumbwheel v With the radio label for the set point activated use the Hamster wheel to increase the force decrease set point voltage value the Z piezo voltage will move to some larger voltage value 18 more red At some point you will see a hard engagement in the Z piezo continuing to decrease the set point value you ll notice the Z piezo will no longer move to some more positive value At the point of this hard engagement the tip has engaged at the lowest possible set point force therefore preserving the tip shape AC mode Phase Monitoring To prevent mode hopping while imaging you
27. at liquid flow falls over adhered tips usually on under side of chip reflective side Gently tap tweezers against desk to free adhered tips Blot dry with Chem Wipe Many times a biolever will pop back when in the fluid meniscus between tip holder and sample it may take several minutes for this to occur Icidentally AR application scientisthave discovered if the small biolevers have resonant frequencies below 7 or 8 kHz in fluid the changes of them imaging well are greatly reduced We suspect this is an Olyomus QC issue Reducing noise in your system have discovered that keeping anything with a cooling fan off the desk that your controller is on These items can translate their mechanical noise through the monster cable if all on the same desk Our lab have a system from 2004 when AR was using a more rigid monster cable which can transfer mechanical noise along it In efforts to reduce this noise have sandwiched it between to 50 Ib bags of sand Adhering samples to slides Asylum Research urges users not to use two sided tape because of the inherent creep and compliance that is associated with it Superglue or a 5 minute epoxy works very well However solvent volatility in these products can compromise a sample especially if you have a wet biological sample that has to stay wet In this case some Dow Corning vacuum grease works well put into a syringe barrel as a way to dispense it 38 XII Miscellaneous Software Proced
28. ble chip coupling to the tip holder i iHa al Wa ternal anv Figure 1 A loading the tip holder into the Cantilever holder stand B use tweezers to position tip in middle of polished quartz window C Loading tip holder into head Put tip holder into the MFP 3D head find it easiest to put the ball bearing on release lever side of the kinematic system first then ease the holder in the back Make sure the tip holder is parallel to the top of the head other wise it is not properly seated If you haven t already open the MFP 3D software the launch icon is on desktop TM Pe Program is loaded when Igor says its ready lower left hand side of window When Igor is busy it doesn t want to be interrupted while this is occurring typically the Abort button in the lower left of the MFP 3D software shows this Abort ll LASER ALIGNMENT There are two ways to initially rough align the laser on the tip 1 use IR card 2 use overhead CCD camera top down view optics ccd camera only The laser is actually a super luminescent diode but it s easier to call it a laser The top of the MFP 3D head is conveniently labeled Figure 2 The thumbwheel on the right moves the laser along the length of the cantilever while the one on the back of the head moves it across the cantilever The head has arrows indicating which way the laser will go as they are moved CCW v Turn on the laser diode by turning the key on th
29. ble was prepared to help you adjust your imaging or experimental parameters to eliminate mode hopping Lower amplitude Increase A ja Increase Ag Afa Decrease Asp Ava Decrease Asp Ar o Z where Arfa is the free air amplitude in volts and Asp is the set point in volts 19 VIIB AC mode IN FLUID The MFP 3D is designed to work in fluid very well The procedure for tuning the cantilever is almost is as easy as in air but differs slightly Determining Drive Frequency In Fluid There are two ways to determine a Drive Frequency for AC mode imaging the first is a bit more refined than the second but you can pretty much get to the same parameters following both 1 AR has worked a nice protocol into their software in which you can overlay the thermal power spectrum over the frequency sweep which allows you to determine a precise drive frequency see Figure 18 below which is associated with cantilevers natural resonant frequency v Determine a range to do the frequency sweep from performing a thermal tune v In the Tune Tab click the Append Thermal M checkbox v Click the One Tune button with the proper frequency range selected After the sweep you ll see a frequency plot black trace over top the thermal power spectrum red scatter points v Using the cross cursor right click at the most pronounced peak that corresponds to the left side to maximum of the resonant peak on the thermal tune scatter plot
30. e Button Capture What Inputi orev i dS Live Display Inputq ioga Gain in 0 F Oo cw Scope E Lateral Proportion Gain 000 Aa 3 Frequency Drive Amplitude 3416m J 2 Current Drive Frequency 386 297kHz G UserCalc inputGain OdB J6 Count Figure 5 Main tab of Master Panel On the Main tab Figure 5 adjust these parameters dependent on sample v Set point force minimum of whatever the deflection value on the S amp D meter is gentle low force to 1 0 V higher force the larger the value the larger the applied force if you need to know what kind of force you are applying you should determine the spring constant k using the procedure described in Section IV v Integral gain I 5 to 10 v Scan rate 1 Hz v Scan angle resolution scan points amp lines and image size is up to you At this point you are ready to engage for an imaging only application i e if you are not interested in knowing the spring constant of your cantilever If you want to determine spring constant see section Ill Tip Engagement Click Simple Engage in the Sum and Deflection window Figure 4 You will notice the z piezo voltage maximizing all the way red 150 V indicating the z piezo is fully extending in anticipation of engagement of feedback servo Slowly turn the thumbwheel counter clockwise towards the surface until the deflection value on the S amp D meter equals your user defined set point AND you here a chime sound co
31. e averaged by entering a value for Width i ie ae T v Straight or free hand lines can be chosen by selecting o Disposer from the Mode pull down menu Peron o Mode Line Line angles can be locked in by designating user defined angle Lange 87 3 M4 Full wicth iP 6 Up to 10 different sections can be made in an off line Eikcursors orive K cursors measure analysis v use section pull down to activate new one v designate line color with color pull down v repeat line drawing process multiple lines are displayed on same section plot 20 With the Full M checkbox checked the entire line will be shown in the section plot if unchecked only t5 distance between cursors on image will be shown Cursor Drive with this radio label activated you can adjust the position of the line after it has been 5 drawn Figure 37 click on cursor i e Kl and pull to new location Line and section plot are updated 0 0 5 10 15 20 0 5 10 15 20 Cursor Measure with this radio label activated i N you can measure distances on Sections Plot Move a the cursors on the section plot to the desired 0 location The cursors will move also on the image Figure 37 Cursor Drive to use the section tool on real time scans go to MFP IP Analyze Panel select the real time M checkbox Use procedure described above 32 Exporting Line sections In the line section grap
32. e bio modified tip Tune cantilever in AC mode like you are going to image with it start approaching with the thumbwheel Wheel down 10 um then do a Simple Engage if the tip doesn t engage hit Stop wheel down another 10 um and repeat Once engaged you can use the micron graduation on the thumbwheel to adjust to the middle of the piezo when the tip is engaged Once this is done you can disengage with Stop button and switch to contact mode to do force curves or whatever you want to do Alternatively you can choose an AC mode set point that is 5 of the free air amplitude hit simple engage wheel down until engagement which should be a false engagement move to some more retracted Z value 1 2 blue Then use the hamster with the set point radio button activated to dial in a lower set point smaller voltage value You ll see some movement in the z piezo movement but since it s under feedback and the AR has super fast electronics you can adjust the thumbwheel to your desired piezo value When it truly engages you ll see a sudden Snap in the Z piezo movement sum amp deflection meter and then it won t move anymore with decreasing set point This method works great and is efficient Both methods offer the tip to engage under a more controlled feedback Getting Biolevers to stay extended not folded under chip after wetting Grab chip at opposite end with negative tweezers Rinse with EtOH or water such th
33. e can also be defined by the user The drive frequency value will automatically be updated in the Tune and Main tab of the Master Panel and the Q of the cantilever is determined d The teal colored curve is the Phase which is set to 90 at the resonant frequency value FYI the Amp Freq and Q Main Thermal Force Tunes Values at the top of the panel are the values found for the Drive Frequency 72 0782 5 largest peak within the Sweep width in Figure 16 s case 5 KHZ Sweep Width 5 000 kHz Drive Amplitude 20 00 mV amp Cantil mmx O ET 5 Cantilever Tune Sweep Points SI Phase Offset 129 44 Input Gain 12 dB b Continuous Amp 1 036 V Freq 72 156 kHz 163 0 ne Tune Center Phase Auto Tune Low 50 000 KHz E Auto Tune High as Target Amplitude Target Percent b Append Thermal J Append SHO fit LJ Pure fit LJ fOKHz TikHz T2kHz T3kHz f4kHz Figure 16 Cantilever Tune results Teal line is the Phase S9G9999999999SS8HB8 Tune Panel setup Figure 15 Tune tab If you don t have any idea what the resonant frequency is of your cantilever but want to perform a thermal tune first to determine the resonant frequency of the cantilever as described in the 17 previous Section VI Examples can be seen in Figure 13 air or 14 fluid Bring up the cursors with Crtl and select the frequency of the fundamental peak As long as this number fits within the A
34. e front of the controller 90 degrees clockwise Deflection PD adjustment Camera focus X y camera translation for top view optics Engagement thumbwheel aser adjustments Figure 2 MFP 3D head and its thumbwheel locations top down view model shown 1 Laser Alignment via IR card for use with heads without camera or for the MFP purists This is done on a stand that is J kinematically machined for the legs or a table top just A somewhere your sample isnt v Place the card under the tip holder Figure 3 right attempts to depict this procession If the laser spot isn t being blocked by anything you can see it on the IR card If you can t see it move the laser along the length of the cantilever until you do Then follow this procedure v Move the beam back onto the support chip no spot visible 1 Figure 3 laser alignment pattern 5 v Move the beam forward a bit until you see it 2 At this point the laser is just past the edge of the chip v Next move the laser perpendicular to the length of the cantilever 3 When you encounter the cantilever you will see the Sum voltage signal increase VAt this point use both laser alignment thumbwheels to maximize the Sum voltage 4 Place the head onto stage with sample Make sure you ll have enough room such that you don t crash your tip OR 2 Laser Alignment via top view optics CCD Turn CCD camera on look for ccd icon on low
35. e pull down menu Figure 5 Image channel selection Before scanning you must choose what kind of data you want shown during imaging i e image channels v Click on a channel tab in the Master Channel Panel Figure 6 VAt the Input pull down menu to choose what data you want for contact mode imaging i e height deflection lateral etc If you want lateral data but don t change these tabs from the default AC mode imaging windows i e Ht AM amp Ph seen below you are just going to have some noisy looking image that isn t representative of your surface For more on the Master Channel Tab see Section IV Master Panel OK Main Thermal Force Tune Scan Sie 2500um_ AO Potential Slow Scan Disabled LJ Imaging Mode BaseName mfp_logo _ COO sses JS ao Save Images Mf Gave Status Eae Giman Adjust deflection value to slightly negative for contact mode main Panei Semp deflection meter is a little blue like O to 0 20 V This is done to compensate for a ca zero deflection once tip is engaged Figure 6 Master Channel panel with Channel input options 2 I Scan Rate 1 28Hz S LJ Auto Input Height EES 11206ums __ isg Data Scale x ofset 731um o TO AEI ee Deflecion Scanangle 0 00 ColorMap 7Sensor ScanPoints 512 Live Flatten Userin0 scan Lines 512 dS UserIni Width Height ai Radio Save PlaneFit Userln EI Delay Updat
36. ed click on export button look for the 0 processing thing in the lower left of the MFP3D window This feature automatically creates an export folder in the same folder where the Force curve data is ibw files filled with converted tab delimited files This way is much easier only flaw of the program is that it doesn t put name of individual curve at top like you can in single export protocol have Single Note checked M this gives only one file with parameter info Exporting Waves in Igor You can export any wave in Igor 90 of the battle is finding the wave For any wave displayed on a graph 1 Double click on the wave You get a modify trace dialog 2 Hold Down the left mouse button on the name of the wave that looks like the correct wave The box below then shows the data folder that is holding that wave 3 Go to the Data Data Browser menu Locate the data folder that was shown by the dialog Alt left click to the left of that data folder There should now be a red arrow pointing at that folder and letting you know that you are sitting in that folder 4 Go to the Data Save Waves Save Delimited Text menu Select the wave from the list of waves in that data folder 5 Click Do it Then select where you want to save that dat file Deactivating Closed Loop Scanner Go to in Main tab of master panel Towards the bottom click on the Show M check box next to Scan Mode pull
37. elow The fast input free air value should be less than 30 pm to meet AR s specs Engaged this measurement monitors the noise at the tip sample interface v Engage tip in contact mode v Adjust Integral gain to 0 01 deactivates feedback loop This step is important 43 v Click Start This will be an inherently noisier system because the tip is in contact with the substrate The Fast input the 5 MHz ADC engage value should be less than 60 pm to meet AR s specs Figure 44A shows a typical noise spectrum of a system that meets AR spec Figure 44B is an example of one of an MFP 3D s Fast input Engaged noise that is very noisy You can see there is some period noise in the time plot red That works out to be around 32 Hz noise which can be seen as a broad peak in the frequency plot black lines in red box this suggests some mechanical external noise In our case suspect it s caused by some air handling equipment outside the old lab s window The spikes and peaks in the frequency plot show various types of external noise 200 0 I gt 00 To 0 i E One liad 400 rire 1 0 12 4 ai rT n uf r HUD my iri H r 4 3 4 5 amp 7 8A 2 3 4 5 7 8 9 1 Hz 10 Hz 100 Hz 1kHz 10kHz 100 Hz 1000 Figure 44 A Typical fast engaged noise test meets spec B Fast Engaged spectrum showing mechanical noise in lower frequencies and all over X Y Z inputs With the tip disengaged
38. er left of MFP 3D software focus camera on tip using focus adjustment wheel on back of AFM head The two knobs in the back of the head are for x and y position adjustment of the camera the large wheel in the ccd icon housing is for focus CCW focuses down CW focus up in the plane of the tip See Figure 2 for location of adjustment knobs on MFP3D head usually follow a pattern remarkably similar to Figure 3 except you follow the laser spot in the camera v Move the laser back onto the support chip to locate it in the camera view and then move it towards to the end of the desired cantilever v This results in a Sum signal on S amp D meter v Move the laser in x amp y to maximize the sum Adjust Photodector PD Use the thumbwheel on the left side of the head Figure 2 to adjust the PD whose value is seen in the Sum amp Deflection meter Figure 4 This meter window is set up rather conveniently If you have red in the deflection meter you need to go negative to adjust to ca zero The top of the head is labeled to tell you what way to go positive or negative When aligning the laser just pay attention to the Sum and Deflection meter voltage values v For contact mode imaging the deflection should be slightly negative slightly blue for AC mode it should be zero no color v At any point clicking the Withdraw button will disengage the tip retracting the piezo to a reading of 0 0V mostly blue on the m
39. eservoir around sample This can be done a variety of ways have found that a silicone based adhesive works pretty well to fabricate a make shift fluid barrier on a glass slide put a small amount of the adhesive into a 5 mL syringe so can control the amount coming out much like what is used when piping frosting onto a cake also draw a template onto a piece of paper to get consistent sizes during application Figure 41A When choosing a silicone check the ingredients to insure the solvents won t interfere with your sample Alternatively a slice of glass tube stock can be adhered to a glass slide shown in right figure below PAP pens can also be used to create a hydrophobic barrier around your sample but they are more expensive There are Teflon coated standard microscope slides with circular bare glass regions that could also be used Figure 41C A variety of these slides can be purchased from the EMS website below Plastic under sample offers more protection against fluid reaching bottom side of scanner vww emsdiasum com microscopy products histology slides aspx 634 14 Figure 41 Cost effective alternatives to keeping the X Y scanner dry when working in fluid A silicone barriers B glass tube stock C Teflon coated slides from Electron Microscopy Sciences 37 Gentle engagement learned this from Chad Ray Achremitchev group Duke Chemistry This is a good approach for a very gentle engagement i e you hav
40. eter Figure 4 shows the piezo fully extended anticipating set point to be achieved Sum and Deflection Meter OK Stop Meter Sum 6 05 Se Deflection 0 10 Amplitude 0 98 E a Z Voltage 150 0 Fully In Middle Fully Retracted Extended TOY 10 150 V Figure 4 Sum amp Deflection Meter all values in volts 6 At this point you are ready to engage the tip depending on what imaging mode or application you are doing If you are working in fluid you may have to wait out an equilibration period You will see significant drift in the deflection value without equilibration seen in deflection value of the S amp D meter CAUTION When adjusting any of the alignment thumbwheels if you feel resistance turning them don t force it Its probably at the end of its travel If A you over torque it it becomes very difficult to reverse its direction Regarding the software the help menus can be helpful for explaining functions in the instrument Anywhere a occurs to the right of a panel dialogue there is a link to the help menu describing its function Quite often the help menu is descriptive yet doesn t necessarily describe functions in a procedural sense p a r f PERF m EF 4 at i Wi itt HHE f J AVA SEBS polymer imaged in air lil CONTACT MODE Operation From Main tab in Master Panel select Contact mode from the imaging mod
41. etermine the sensitivity of the lever in nm V 2 perform a thermal tune to determine resonant frequency of your cantilever An algorithm computes the spring constant using the equi partition theorem Engage in CONTACT MODE on a clean hard surface like freshly cleaved mica v Select Contact from the Imaging Mode pull down menu on the Main Tab of the Master panel v Choose a set point force low force is slightly larger than the value of the Deflection value volts in the S amp D meter I usually choose 0 2 to 0 5 V v Integral gain 5 to 10 integral gain is usually sufficient v Click the Simple Engage button on the S amp D meter Engage tip by turning the thumbwheel until you here a bell sound the z voltage goes from railed in the red 150 V to some other lower value v Slowly adjust this down to around 70 V no color in z voltage meter NOTE if you need to engage as gently as possible it s best to do a false engagement 2A in AC mode first then increase the set point force until the tip hard engages at the minimum possible force then switch over to Contact mode once engaged See Section VII for this gentle engagement procedure v Click on the Force Tab of the Master Panel Figure 9 This panel is set up so the user can define the specific distance the piezo travels during the force distance cycle called Force distance in Force tab The objective in this step is to measure the slope of the cont
42. ging v Click the Stop button to withdraw the tip S amp D meter piezo reads 0 0 V v Retract thumbwheel some clockwise turn and turn off SLD turn key counterclockwise v Turn off ccd camera window and camera fiber optic light v Remove sample from stage and tip from tip holder v If operating in Buffer rinse tip holder off with water to remove salts Dry in stream of No v Leave Controller and PC running unless expecting power outage 22 IX Force Spectroscopy FORCE CURVE AQUISITION Force distance curves can be acquired by a variety of ways The first step to acquiring an accurate force distance curve is to determine the spring constant of a cantilever on a clean hard surface Section IV To reiterate v Select Contact mode Main Tab v In Force Tab Figure 9 or 22 Set Trigger to DeflVolts Figure 21 v Activate Relative Radio button v Choose a piezo velocity and Force distance like 1 um s and 600 nm to 1 um v Hit the button this gives a Force curve and Force Channel Panel see Figure 23 and description below v Determine InvOLS of contact region place cursors select Deflection under Set Sens pull down v Withdraw tip v Perform a Thermal tune v Fit curve spring constant determined Wl Master Panel ie ool x P Main Thermal Force Tune I Trigger Channel DeflVolts PG Start Dist 1 34um AE Pos Slope None Force Dist 600 00 nm Je O Absolute amp
43. h panel that comes up click the button which brings up a new table with one column of data in it Kill it v go to Windows New Table v Select the four waves in the Columns to edit that end with the following suffixes Filename suffix IHtT QHtT XHtT amp _HtT click FYI the T stands for the Trace Data Retrace data would have an R suffix A new table comes up that has two columns that you can export to another spreadsheet or use in Igor Clicking this D button brings up the Display manager this helps organize display windows when many are open There is a great description in the help file L Clicking the L button brings up the List panel the same that is presented after selecting a file to browse Commands Y The Command Menu There are many features in here Modify Analyze Display and list do the same thing as what was just Display Panel described above Modify Panel Commands Extract Insert Delete Layer sales joes Show Note this is where all the imaging parameter information is ae xtract Layer Incidentally the following binaries mean Scan down 1 downscan slow scan goes from top of image window to bottom Scan down 0 up scan slow scan goes from bottom of image window to top Imaging Modes Contact 0 AC 1 Change User Names Save image after modifying an image clicking this will save that modified Insert Laye
44. he Defl InvOLS deflection ee atom inverse optical lever sensitivity value will automatically be KappaFactor 1 09 6 updated in the force panel and connects the two cursors Amp InvOLs 79 94 nmv 6 X in the deflection vs LVDT plot with a thick black line Spring Constant 120 99 prnje Figure 10 C Its a good idea to record this InvOLS in your Set Sens Vitual Def saan Amplitude should be selected if doing force distance curve in AC mode Figure 11 selecting sensitivity to update InvOLS value v Disengage tip by clicking the Stop button on the bottom of the Main tab of the master panel or by clicking the Stop button to the right of the deflection meter on the Sum and Deflection Meter window or by clicking the Withdraw button in the Force tab All do the same thing the S amp D meter should show a piezo voltage of 0 0 V in its withdrawn position STEP 2 Click on the Thermal tab on the Master Panel Figure 12 v Adjust the deflection on the AFM head so it reads zero deflection in the Sum amp Deflection meter v Click the Do Thermal button at bottom of panel This detects the natural resonant frequency of the cantilever by doing an iterative series of frequency sweeps and averaging them A real time amplitude vs frequency power spectrum plot comes up like the one seen in Figure 13 see Figure 16 for thermal power spectrum in fluid Let it go through many cycles the more yo
45. he parameters using the Hamster wheel on the front of the controller Any parameter with a radio button next to it Figure 5 can be changed during a scan when it is activated looks like black dot in circle with the Hamster The Hamster gives digital control with analog feel The toggle switch to the left of the Hamster allows you to toggle between 9 radio buttons in the panel This is a GREAT feature for tuning on the fly and you don t need to use the mouse The Hamster is also functional in the Force panel described later in the force spectroscopy section Image quality can be monitored by image resolution and amount of noise in the line traces located below images Rotating the scan angle results in counter clockwise rotation with positive rotation angle values NOTE Scan angle has to be at 90 for lateral force microscopy LFM This may be obvious but the author mentions it because some other commercial scopes scan perpendicular to the lever at 0 When finished scanning see shut down procedure below Section V IV IMAGE CHANNELS SCALING FLATTENING The z scales of the images can be auto scaled by creating a square with the mouse on the image right or left mouse clicking amp selecting the fit scale option That will auto scale using the pixel values within the square it will auto scale in that image only You can also select Fix all scales and have the auto scale update in al
46. if working in fluid clean tip holder dry see page 10
47. is broken down into sections that are in a logical progression of obtaining AFM images or simple force spectroscopy experiments and the subsequent data analysis only discusses operation of the MFP 3D itself and does not include any optical microscopy techniques if your MFP happens to be sitting on an IO is in continual development so more insightful updated versions occur every few months The next version will have descriptive procedural electrical characterization and lithography techniques in it as well as a section describing what most of the features in every software panel do in a procedural sense Regarding the latter for now just look in the help menus they are there for a reason If you are not familiar with Igor you should read through the getting started manual that comes with the software also Suggest when updating new software from Asylum read the what s new read me files they are often very descriptive The MFP3D Help Files under the Igor Help menu are also very useful especially for image analysis and display some features of this manualette EE ST et T OS Ae ae Orange text represent topic sections AC mode image of Fibrin on methyl termin Any where there is a gray bold text in single quotes means its SAM nai an execution button in the software example Simple Engage looks like this button Simple Engage Bold text represents new software features or tabs denotes a new c
48. l open image channels Alternatively you can manually select z values in the Master Master Cha OK Channel window Figure 5 or 7 with Data Scale or hit the Fix button to auto scale The Data Offset can be adjusted the same RAA PRA ANN ona LJ Auto Input Data Scale 10 00 nm S Data Offset Onm 6 ColorMap Grays256 Live Flatten Live Flatten options can also be selected from pull down menu Figure 7 Similar pull downs exist for Save Planefit Trace or Retrace on the image display is chosen in this window Capturing the trace and retrace data usually increase Save PlaneFit your chances when looking for that publication quality image Capture What The Auto M check box if checked auto scales the trace retrace lines below the image with every slow scan line added If this box is unchecked it scales to what ever the Data Scale value is For example if the Data scale value is too low then trace retrace will be clipped this also depends on the Data Offset Live Display Show Scope Mw Ei Auto bf Channel 1 Setup 3 t G3 t3 b t3 Bee Figure 7 Flattening options above 1 um than does the height which is based on the piezo values You should this NOTE Due it its linearity the Z sensor LVDT gives much more accurate heights for features channel as often as possible in addition to the height channel 10 V SAVING IMAGES TO SAVE CAPTURE IMAG
49. lation is that is averages the last five or 10 points of the retraction curve and finds the difference between that and the minimum of the retraction curve In other words the histogram represents only the largest single event in each curve If you have curves with multiple events that you are concerned about this histogram will not be representative of your data set The Spot tab correlates individual force curves to surface features on an image that you picked points from It places the force curve suffix respective to their position on the image My description of the Master Force panel are brief More complicated questions can be directed to Support where someone where more seasoned force spectroscopists who wrote the software code can answer your questions 36 XI Empirical Tricks Stuff I ve learned along the way for better imaging Imaging in fluids PRECAUTIONS When imaging in fluids it is important not to allow buffer to get to scanner or you have a 9000 problem minimum What can happen is that fluid can fall down onto the top of the stage get sucked into the bottom of the scanner through the back plate via capillary action then into the flexor channel via capillary action Fluid can ruin the LVDT sensors and compromise corrode the flexor channels from moving properly IF YOU DON T HAVE A CLOSED FLUID CELL To protect the scanner place a piece of plastic under the sample see Figure 41B below Also make a r
50. lyze W Spot Review button brings up the Master Force Panel Figure 40 Load up some Force distance curves Clicking Load Curves in the Display tab of the Master Force Panel will also get you there This will put all the curves in that folder which could be a data set The Display tab lets you choose what channel you want your data displayed i e Force deflection how many axis extension retraction part of the curve The Show Notes button brings up a table with all the experimental parameters associated with that file The y axis can be chosen at the top deflection force etc Individual curves can be advanced by clicking the lt or gt buttons lt 10 and gt 10 advance 10 slides ahead or back and Rand randomly pick curves the adds the next curve in the file and overlays it on the force plot removes one The filenames are displayed in the window at the bottom Clicking on one brings that force curve up holding the shift key down lets you choose multiples When ready to load new data set delete loaded curves by clicking Delete All if you don t and o x Number of Axes A i 2 i LJ x Axis LOT She Stir T a ha Fet o Suffix 0000 JE naex D000 J a RS Force Plate 1000 O Contiguous EXPT_EOO01 EXPT_EOOO2 EXPT_EOOOS EXPT_EOO04 EXPT _EOOOs EXPT _EQOOG Delete all
51. manageable value for my stuff like 0 03 Alternatively you can click the Aspect 1 M1 checkbox which scales the Z range relative to the X and Y dimensions quite often this makes it look flat but this is good for image overlay see Section XII page 40 Figure 39 shows the progression described above 48 im Figure 39 A 2D image B initial ARgyle image C after Zoom Z scale adjustment new color map and adding specular lighting v Adjust other values such as the Data scale values light rotation zoom and offset Color map found in Display tab background color found under Prefs tab Specular lighting occurs in the Lights tab click the shiny M checkbox Hotkeys Crtl the mouse does zoom Shift mouse does offset To export images from ARgyle make the 3D image window the forward most image active window Copy to clipboard and dump into another program Paint Photoshop PowerPoint etc 34 To plot a real time image in 3D go to MFP IP 3D surface plots this brings up the ARgyle Master Panel Figure 38 From the pull down menu under the New tab select RealTime and click the Do Ift button You can also make movies with your ARgyle image Contact Support AsylumResearch for this instruction file Bruce Wallace Force Curve Analysis In the Force tab of the Master Panel clicking the im Master Force Panel Display Pref y Parm Cursor Modify y WLC Ana
52. mputer speaker volume must be on You will also notice the z piezo voltage meter goes from railed in the red 150 V to some lesser value If you have the volume up on the transducer front of controller headphones plugged in you can hear a frequency change immediately before engagement occurs Carefully adjust the front thumbwheel down to around zero 70 V no color in z voltage meter This indicates the piezo is in the middle of its z range 7 5 um standard head 14 um extended head At this point the tip is engaged and just sitting on the surface it does not begin rastering until you tell it You can now being imaging see next step or determine spring constant Section IV NOTE If the z piezo voltage is railed all the way blue 10 V upon clicking simple engagement the piezo is fully retracted because it thinks it has crashed This indicates a false engagement and adjustment must be made Do this by clicking Stop and adjusting the set point voltage or drive amplitude if in AC mode Section VII to allow extension of the piezo This often occurs when imaging in fluids high humidity To begin scanning click the Do Scan button on the Main Tab After a brief moment imaging will begin Tune the parameters with set point force integral gain 1 and scan rate You can use the arrow clickers to adjust parameters opposed to typing values in Alternatively you can fine tune t
53. ocedure if you have difficulty getting a decent image from the above protocol From the Thermal power spectrum plot place an 10 lgor cursor on the left side of the first or i second peak about 5 to 10 of the maximum amplitude The areas indicated by the red arrow s are where you can manually pick the drive frequencies by entering the X cursor value in the drive frequency window of the Tune tab Notice the second peak isa Eigen frequency 100 Hz ikHz 1OKHz 1OOkHz Figure 19 Thermal tune Freq vs amplitude plot N FLUID Peaks become broader due to lower Q value and resonant peaks to lower frequency v Engage as you normally would v Once imaging you can also adjust the Drive Frequency with the Hamster wheel until you find the sweet spot ThAHz 21 v Figure 20 shows the results of changing the drive frequency on the fly to find the sweet spot During parameter tuning monitor the image quality in the height and phase images as well as the scan traces below the height image The Phase image can be monitored for noise as seen in the left image A slight adjustment reduces this noise for better image quality The image is of a protein pattern lighter color and poly ethylene glycol terminated thiolate dark region WA W l Ue 11 026 kHz 10 578 kHz Figure 20 Monitoring the Phase image while changing the drive Frequency on the fly VIII Shut Down Scanning Cessation When you are done ima
54. oncept or comment v checkmarks denote a procedure typically these are in a series of instructions and are included to help guide the eye through specific operations Blue text represents a Note or empirical trick something have picked up along the way and include so it may not take you as long to figure it out as it did me it is also accompanied by a little note icon in the margin I tried to incorporate as many software and hardware images to help describe from a more visual point of view There are no equations in this document I LOADING THE CANTILEVER Load tip holder into loading apparatus Figure 1A which offers stability when loading tips The tip holder has a kinematically machined ball bearing port that snaps into place using the lever on the apparatus and head port The tip holder apparatus works great for rinsing the tip holder after imaging Slide cantilever chip under tongue of tip clamp Position cantilevers so that they are more or less centered in the clear trapezoidal shaped Kel F window Figure 1B The Kel F tip holder was designed to be resilient to scratching from tweezers so don t be paranoid For best results DO NOT push tip chip substrate all the way back in the pocket it can cause the chip to lift off the floor of pocket which then compromises the deflection signal i e no signal Tighten the screw in the center of the stainless steel tongue assembly finger tight This generally allows for suita
55. ore clicking capture it won t make an image You must first capture an image un magnified before capturing ccd images that are magnified Also the New Image is inverted vertically Exporting curves to txt format create a place to dump the curves for them someplace that will show up in the path box in the save delimited text dialogue box v Go to Misc gt New Path v make Filename of your choice v click path choose where you want to dump it v click Do it 1 Exporting single waves Note go to data folders click review force curve v checkbox Go to data load waves gt Igor binary brings up browsing dialog select the curve you want then a new dialogue asking if you want to copy and you say OK Data gt SaveWaves gt Save delimited text find wave name in Wave s menu amp choose path in Path box click Do it 39 Check to see if it s there and tab delimited by going to the place where you dumped it Open it w notepad to make sure its tab delimited 2 Exporting as Batch file The easiest way know is to use Chad Ray s Force Volume Mapping macro you can do it a lot quicker with his program The name of the program is ChadoO Contact support asylumresearch com for the ipf file Go to MFP controls gt Force Mapping this brings up a small dialogue with a Duke logo click on Export choose place where you want curves to get dump
56. out what 50 pN is in Volts for your given Spring constant and Invols Useful for setting a trigger during force curves Variable MyForce 50e 12 I want 50 pN Print MyForce GV SpringConstant GV Invols Display captured video image First set the data folder to root or create one like root Video or root Capture Then click capture button Newlmage capture Better way of displaying captured video image Display AppendImage capture ModifyGraph height Plan 1 left bottom Rename captured video image so it doesn t get overwritten example Rename capture pollenA Generate a piecewise continuous wave Make N 65535 wave0 wave1 wave2 wave3 46 wave0 p lt 32 68 0 1 wave p lt 16384 0 p lt 32 768 5 p lt 49152 5 0 piecewise linear wave2 p lt 32 768 5 p 10 32768 15 p 10 32768 Output a wave to Out0 and collect from In0 in sync write crosspoint to connect OutO to OutA and In0 to InA print td_ Stop usually not necessary but safest print td_ xSetOutWave 1 2 2 A Output Controller wave2 2 print td_xSetinWave 1 2 2 A lnput Controller wave3 print 42 2 print td_WriteString 2 Event Controller Once wait for it to print 42 Display wave3 Note be sure to read the help for td_xSetiInWave and td_xSetOutWave on banks decimation and interpolation In particular you want the decimation and interpolation to be the same if you want to correlate input with output
57. ox that automatically adjusts the start distance as the tip traverses the sample collecting the array This way you don t have to be concerned about the tip not making contact because the surface is not level Number of Points WE Mf Update Start Dist Number of Lines CA Do Force Volume Stop Force Volume A Combine Output If One Note Y Data Force Y Data None Y Data None Data Raw Force Volume by Chad Ray at Duke University Figure 30 Chad Ray s Force Volume Mapping acquisition ipf For force curve analysis see Section X 28 X OFFLINE DATA ANALYSIS NOTE This analysis has a lot of options so describe what have primarily used in efforts to possibly aid a new user suggest exploring this software to get a sense of its flexibility and power Image Analysis v Click on Browse Images in the Main tab A Path Dialog Figure 31A will come up asking what directory to load images from By clicking That s It is like clicking return b c the path is acceptable If you want to change folders click the Browse button A new dialog comes up Figure 31B by clicking the Browse button in this dialog you can change folders Final selection made by clicking OK MM ARPathDialog ji Je lolx Path To Load Data Path to folder C Asylum example data a Ce Cae Coos OK Figure 31 Left Browse dialog box for image recall Right Path to Load Dialog
58. play with the trigger point here because a very low one can put you down in the noise level of your system cantilever Figure 25 below is an example of two curves obtained with different absolute trigger points The upper curve red has a lower trigger point than the lower black Low relative trigger values appear noisy because they are auto scaled in the y axis NOTE Whatever Trigger you are using if your tip is modified it a good idea to use low trigger values to prevent unnecessary grinding of the tip into the surface during contact Relative triggers 95 3 nm trigger vV VY T S 100 nm 105 110 15 nm trigger 650 7 00 750 800 850 900 950 nm Figure 25 Relative Triggers in deflection channel 3 nm red 15 nm black The Misc Tab Figure 26 this is where the tip piezo dwell setting can be varied In blue mode this lets the tip dwell at trigger point if one for a defined amount of time before continuing through the retract motion Dwell time and filters settings can be chosen here No Dwell In Red mode the dwell occurs away from surface ad Toward Surface Away From Surface Figure 26 Misc Tab of Force Channel Panel 25 The Calibration Tab In this tab the Defl InvOLS AmplnvOLs or virtual Deflection should be calibrated upon an initial force distance curve The former two have already been discussed in Section IV Virtual Deflection Calibration The virtual deflection
59. r Delete Layer Show Note Change User Names Save Image Save Then Kill Image work under a new 0 tab Kill Image Save Then Kill Image same as above but closes it too TIFF Export 1x Kill Image closes image TIFF Export 2x TIFF Exports 1x 2x 4x refers to image size TIFF export 4x Crop Image to crop an image used the mouse to Expand on an area then a click on Crop Image this will make image that size of expanded area i e Crtl A doesn t take you back Before using this feature like to make a copy of that file just so have the original raw data Setup Help Help 33 ARgyle three dimensional display brief introduction Clicking the 3D button in the display window brings up the ARgyle software feature This lets you plot saved images or real time scans in a quasi 3D rendering The menu is vast so suggest exploring these parameters on your own However just to get you started have an example of how use it to look decent am by no means an ARgyle expert v Start with appropriately flattening an image v Click on the 3D button the ARglye menu Figure 38 and the 3D image will come up Notice the z scale is huge Make Aral Mer Panel Figure 38 Master ARgyle Panel Notice that holding the left mouse button down while in the 3D image rotates the image v Go to the View tab and select Zoom from the pull down menu Use the right hand bar to lower the Z Scale to a
60. r 10 to 20 seconds then turn back on While you re waiting wiggle the plug back in a bit more have noticed any time one of the levels in the S amp D meter aren t responding properly it s a good idea to do some variation of the above procedure Rescan Bus Sometimes if you have a freeze or the system is hanging because Igor has been confused click the Rescan button lower left This pings all the hardware systems to make sure they are all communicating properly with each other as far as know Reducing noise in your system have discovered that keeping anything with a cooling fan off the desk that your controller is on These items can translate their mechanical noise through the monster cable if all on the same desk Our lab have a system from 2004 when AR was using a more rigid monster cable which can transfer mechanical noise along it In efforts to reduce this noise have sandwiched it between to 50 Ib bags of sand 42 NOISE MEASUREMENTS This scope has the ability to test how much noise the system is experience both internal and external AR has a series of noise tests regarding the x y amp z inputs at different input voltages and the 5 MHz A D converter known as the Fast input v Go to programming load test procedures This creates a new pull down menu item called testing test panel The window below comes up AR has specific testing parameters values that meet their specs before
61. s described above Typically in fluids the drive amplitude will have to be somewhat high to avoid a false engagement Z piezo goes all the way blue 10 V because it thinks it has crashed NOTE When imaging in fluid there are a few things you have to look out for depending on your cantilever s floppiness j False engagement will occur if the set point isn t low enough and or the drive amplitude isn t high My experience for imaging some proteins has been drive amplitudes of 1800 mV and set points of 200 mV Picking the second quasi harmonic can result in larger drive frequencies to circumvent this false engagement issue Change filter Go to Programming make filter panel A new window pops up change R filter from 1 5 to 0 5 Hz You can also change the Q gain in the Tune tab have had limited experience success with this feature As you thumbwheel down you will notice that the amplitude value is increasing This is believed to because of the liquid being compressed between the tip and sample as it approaches Use the Hamster wheel to decrease the amplitude to pre approach value to maintain the proper set point of the free air amplitude As the tip gets very close to the surface you will notice the amplitude decrease and will engage at the set point value you defined v Move piezo into middle of Z range v tune parameters as described above 2 Alternatively you can follow this pr
62. t options or save image 2 By going to File Save Graphics 2A NOTE you must have a path chosen ahead of time to tell the software where to save modified image This is described more below in Tricks section The Planefit tab does a plane fit on the image chose x plane with X button y with Y button or x y with XY button The Erase tab is where aberrant noisy slow scan lines can be eliminated from the image v Click on the Draw Lines Button a little hand g comes up use tip of index finger to place where you want to erase scan line sometimes you have to increase the line width value 31 v Click Erase Lines to execute v Clear Lines gets rid of the red line RE is still not familiar with the FFT and Filter tabs A Analyze Panel This is where the RMS data line section and image histogram stuff can be found v Click on the A button at the top of the image The Roughness tab This tab will give RMS and associated stats You can also you the masking feature to calculate areas or RMS of the masked area or RMS of with the masked area excluded The Section Tab Figure 36 v to draw a line for section analysis click on the Draw button go to the image and draw the line in the desired location on image von the image hold the mouse button down over the el Igor cursor and draw the line through the desired areas for section analysis sah a Lines can b
63. u collect the more it filters out noise 14 How fast the Thermal tune proceeds is dependent on the resolution value 3 is very good but Slow 7 is pretty efficient but produces a noisier plot v Hit Stop Thermal to cease sampling when plot looks like it has definition enough to fit a curve to it On the power spectrum plot Figure 13A expand the first large peak red dash line Do this by holding down the mouse button and creating rectangle around peak then right or left clicking the mouse button select Expand to give a plot similar to Figure 13B Hit Ctrl to get the Igor cursors x at bottom place one on the peak of the first resonant peak Read the value from the cursor X value which is in Hz and type this value in KHz into Zoom Center parameter on the thermal tab Figure 12 If working in fluid the peaks are much broader see Figure 14A and their resonant frequencies shift to about 1 3 their in air values The red vertical lines in Figure 13B correlate to the Zoom Width value in the Thermal tab In igor Ctrl A will get you back to full spectrum in any expanded Igor window image For example this would get you from Figure 13B to 13A Mi Master Panel Mima ea lain Thermal Force 4 Tune Thermal DC 8 36e 13 JS Themaajas Jg Frequency e 18 White Noise 3 86e 13 JS twon poo 1 mors Eam 5 Spring Constant 141 48 pNinm JS Show fit M4 Graph Log
64. under feedback so you re pretty safe see page 17 Monitoring Phase during AC mode imaging While imaging in air you can monitor the Phase levels Sum amp Deflection meter to determine whether the tip is in the attractive intermittent contact or repulsive non contact regime with respect to the sample ATTRACTIVE gt 90 gt REPULSIVE see page 18 Saving images File Save Graphics Filename image size and file type of your choice click path gt choose where you want to dump it click Do it see page 38 Saving images from ccd camera top down view model Create path to dump file Misc gt New Path choose path Do it Select Capture on ccd image Data data browser right click on capture select new image Save image see page 38 Creating a Path in Igor to save images Misc gt New Path name your path click Path click Do it see page 38 Exporting Line sections n the line section graph panel that comes up click on Edit which brings up a new table with one column of data in it Kill it Then go to Windows gt New Table Select the four waves in Columns to edit that end with the following suffices Filename suffix IHtT QHtT XHtT amp _HtT click Do It see page 32 Real time Section analysis During imaging go to MFP IP Analyze Panel Select Real Time M checkbox follow directions on page 31 Shutdown Withdraw tip turn off laser
65. ures Saving Graphics First you must Create a path Misc New Path create name and then choose a path the folder you are going to want stuff dumped when you save it Then bring what you want to save an image of as the forward most window File Save Graphics dialogue comes up in which you can choose what kind of file you want to save image as actual dimensions name Also this is where you choose the path that you just created in the path pull down menu Otherwise it gets dumped deep within the bowels of the Wavemetrics folder in the program files Capturing images from ccd camera top down camera _ o x model only Current Data Folder v Click capture button on camera window gt root v Go to Data gt data browser right click on capture select T new image This produces a new window with an image in a root Z it M variables v save this image by going to file gt save graphics save in z a Bea perea what ever format amp size amp resolution you want remember Plat ethan Poreet ures that you need to first designate a place to put your data by eE l af Images going Misc gt new path select path and click do it oe E fd Scannerlnfo H S_tileName It s best to rename each image you capture before you Browse Expt capture another Help H S_path i fy S _waveNames NOTE Its been my experience that if you magnify A your ccd image bef
66. uto Tune high and low values click the Auto Tune and the software will determine the Drive Frequency If not adjust the Auto Tune high and low values accordingly Tip Engagement Once a Drive Frequency has been determined adjust the following parameters and youll be ready to engage the tip v Because the Auto Tune automatically adjusts the Free Air amplitude to 1 00 V adjust the Set Point Voltage about 20 less than that i e 800 mV the default value Jo get a better understanding of this relation see Figure 17 v Adjust gain to 5 to 10 v Click the Simple Engage Button on the S amp D meter and wheel the thumbwheel on the front of the MFP 3D head counterclockwise As the tip nears the surface you will notice the amplitude value on the S amp D meter decrease until it reaches the same voltage as your user defined set point At this point there is a chime sound and the Z piezo voltage will go from 150 V to some lesser value less red on the S amp D meter v Begin imaging make sure proper image channels are set in Master Channel Panel see Section IV page 8 While monitoring the images and scan re traces tune imaging parameters set point drive amplitude scan rate gain to obtain best image quality Free Air Amoilitude Oscilatian voltage Set Point Voltage l I l Lower force Paint of I interaction with i tip and surface Higher force l Lo 0 Distance Figure 17 Qu
67. ve volts a wai LI re yii 3 f Time lt Red activation Reverse Axis is a Figure 23 Force Channel Panel Split Sync piezo velocity modes This button allows the Approach Vel 1 50 yms S user to select different approach and retract piezo velocities u 397 34 nmd G figure 24 Retract Figure 24 Sync mode allows different piezo approach and retract velocities Trigger points The Trigger point is a user defined value detected on the PSD that tells the piezo to switch directions even if the force deflection curve hasn t achieved the completed Force distance deflection value v Activate it by choosing what channel you want it to trigger on Figure 21 for example like to use the deflection channel which will be in nm alternatively your can choose DeflVolts which will be in Volts or Force in Newtons 24 v Type in your desired trigger value just don t pick femptometers v Select what kind of trigger you want the difference between the two are Absolute trigger is was the default trigger My understanding it takes whatever trigger value you choose and adds it to what ever the free air deflection happens to be What you get is Force curves with larger deflections or other channel plotted then you might expect Relative trigger is more sensitive and should be used for more accurate force distance curve acquisition You have to

Basic Operation Procedures for the Asylum Research MFP

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