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NanoWizard User Manual - Section for Nanostructure Physics

1. Sensitivity Spring Constant N 0 4 0 4 If the sensitivity has just been meas C UNKNOWN 7 S 72 12 hmMvlluse tl ured the Calibration Manager should Use linearizing ee ES be open on the Sensitivity panel s 72 1 nm Accept Value Ss 00 on lt Check that the value is displayed in 5 the text box and use it M is selected a 262 02 5 F s Then click on the tab Spring Con Q ou 04 3 stant 0 6 0 6 Full Range Y Full Range X Select X Range eg ve nn 50 o 50 100 150 200 250 Height measured and smoothed nm Manual Sensitivity Selection Select Fit Range anager 0X m Sensitivity Spring Constant 10 10 Click Run or continuous in UNKNOWN SC Si finity symbol to start the measure Fit Values Settings r S ment If continuous is chosen each f a i ap aye i data packet is added together until it is na A na SI SZ clicked again to stop If Run or contin yaa uous are started again the data pack ertical K n a 3 geg S R i ets are added Reset erases all the a R stored cycles from the memory to start r a new measurement ae Note the cantilever must be retracted Full Range X Select X Range TT EE from the surface for this measurement dE If the Run buttons are grey and inac TERET Lait d ETT tive check that the piezo is retracted NanoWizard AFM User Manual Version 4 2 99 deift Instruments Sensitivity Spring C
2. There are various shapes of the glass block but the base part that fits in the AFM head is identical in all cases The alignment of the cantilever is also the same since the central optical path is always the same The glass block with straight cylindrical sides is designed for use with the top view optics setup It has a large polished region at the end that gives a large field of view for low magnification optics Straight sided glassblock for use with Top view optics Standard stage 30 NanoWizard AFM User Manual Version 4 2 Side cut glassblock for The glass block with the angled end faces is for using with an use with inverted optics inverted optical microscope In this case the field of view is much Life Science stage smaller and the end region has been cut down to give more space especially for the BioCell and easier handling in enclosed liquid cells and CoverslipHolder The extra long glass block with angled faces has been designed Longer glass block for the especially for the use with the JPK PetriDishHeater This glass PetriDishHeater block is of course compatible among all JPK sample holders This glass block houses a piezo ceramic incorporated into a PEEK composite So the cantilever can be driven directly using the JPK HyperDrive mode HyperDrive glass block for the HyperDrive software module Use the cantilever changing tool to mount the cantilever chip to the glass block This tool holds
3. pee Baseline 7 ends Pause pe eg Pause Z Closed Loop Duration el 05 4 s Duration v 0 0 a s Duration sl 0 5 a s Duration v oojals Z Start Option Distance from surface zl Length el L lalun Sample Rate Y Ol a lie Z Length el 1 0 4 un Sample Rate Y ol a lhe Height zl 1 0 4 jum ZSpeed 2 0 um s Sample Num 0 ZSpeed 2 0um s Sample Num 0 Velocity x 20 4 LM S Sample Rate sl 2048 sm Delay Mode Sample Rate sl 2048 a Hz Delay Mode Sample Num 1024 IGain el 150 0 a je Sample Num 1024 Setpoint 0 400 ajv PGain el 0 0048 a The Force Ramp Designer allows for the assembly of individual force spectroscopy experiments by sequencing differ ent force segments There are 6 different force segments available vg Two pause segments in constant force and constant height mode TTL Level Pulse segment to control TTL signals within a force distance curve please read section 0 Two extend and retract segments in absolute and relative mode Removes all segments from the force ramp TTL Removes the selected segment from the force ramp LP CECR el os ajs In F Extend and F Retract segments the piezo moves for the distance Z Length ZLength 1 0 a lun but stops if the Setpoint is reached If the Setpoint is not reached during Z Length ZSpeed 2 0 um s the piezo continues to extend retract until it is reached So Z Length is an expected Sample Rate v 2048 sl value that helps to set parameters like Z Speed or Sample Rate
4. wu 3381334 YBIaH ali Lb Horizontal axis xma 4 0 a um vun gt 0 0 a um Save Line Scan Vertical axis chi E Channel Display Y Max Y Min Leveling Color trace Color retrace Ch3 ch4 Height Both Linear red red dark _ Draw the last scan It is also possible to zoom into a smaller range to take a closer look at the curves The display range can be set using the text fields for the horizontal and vertical axis ranges LO Le Le JL La The standard oscilloscope toolbar can also be used to set the view ranges Note In Contact mode Error signal and Vertical deflection provide basically the same information but for Error Signal the Setpoint is always subtracted Also with a calibrated cantilever known sensitivity the Vertical deflection 72 NanoWizard AFM User Manual Version 4 2 signal is displayed in nm In Intermittent contact mode Error signal and Lock in amplitude provide the same information but for Error Signal the Setpoint is always subtracted and with a calibrated cantilever known sensitivity the Lock in amplitude signal is displayed in nm 5 5 Advanced imaging settings The Advanced Imaging Settings window can be opened directly from the Scan Control panel or under the Imaging menu in the main menu list This panel pro
5. ig AC Feedback Mode Wizard H Setup QControl for AC Feedback Mode Setup Q Control PreWous Finish Step 2 of 2 Manual cantilever tuning Click Select frequency range to zoom in around the reso nance peak Click on the left of the peak and drag the cursor to the right of the peak to define the range to display Typical resonance frequencies Stiff silicon cantilevers in air 100 400 kHz Soft silicon or silicon nitride cantilevers in liquid 1 50 kHz Follow the step Frequency and setpoint selection de scribed below Section 4 4 2 1 To finish the Cantilever tuning click on the Next button In the next step it is possible to setup Q control This is not necessary in the normal AC mode Therefore click Finish to save the Frequency and setpoint selection For Manual cantilever tuning the user sets their own values of the drive amplitude and gains The frequency range should be set as above the default values are the same as for Automatic tuning 42 NanoWizard AFM User Manual Version 4 2 jaa AC Feedback Mode Wizard Oo Setup AC Feedback Mode Enter a Drive Amplitude value In fluid a higher drive am aml 2 8 plitude is required because of the extra damping from the Lock in amplitude V water or other liquid Select the Gain for the expected range of amplitude e g select Gain 1 for lock in amplitudes up to 11 V Hap seyd ul 9307
6. LA o MJ fs Sample Num Setpoint 0 400 v 118 NanoWizard AFM User Manual Version 4 2 Zl 340 Retract Duration The Z segments extend retract the piezo for the distance Z length independent of Z Length the vertical deflection zZ Speed 2 0 pum s Sample Rate el 2048 sm Sample Num 1024 ZE Pause Duration w 0 0 a s Sample Rate fof a fe The F Pause constant force maintains the Setpoint of the previous segment for a Sample Num 0 defined Duration by adjusting the piezo height PGain 44 Pause TF a le The Z Pause constant height maintains the piezo end position of the previous of ae Sample Rate segment for a defined Duration Sample Num o Delay Mode Constant Height e The segments can be sequenced as required for the experiment The default sequence which appears when the force ramp designer is opened produces a standard force curve with the same settings as in Basic mode File Select default settings e Select default settings Several preset force ramps for standard applications like force clamp are availa ble in the File pull down menu Self designed force ramps can be saved using the save button absolute retract The experiment description can be saved along with the force ramp settings Change Force Settings Description x Rat Click Change Description to open Change Force Settings Descrip ge iert tion window Any comments can be ty
7. The manually saved optical images must be put in a known location The default name can be used or a special name set but make sure the folder that is set in the Data directory panel is known The second panel of the calibration procedure is the point at which the images must be manually acquired The calibration grid points are listed and the up and down arrow keys scroll through the list As each entry in the list is selected the cantilever auto matically moves to that position Images should be acquired at each point and saved with a se quential file name start with file names of the form 01 02 to en sure that the files are kept in the correct order when uploaded as the index counts up to 25 Any names can be used the only requirement is that they must have the correct alphabetical or alphanumeric sequence for the 25 AFM positions Most fluorescence camera or confocal software packages has the option for collecting a time series This can be a convenient way to save and manage all the images Set the time series running e g set 25 images at 2 3 seconds time interval As each image is acquired on the camera move to the next position in the list to put the cantilever in position for the next image NanoWizard AFM User Manual Version 4 2 m Optical Image Calibration Collect Images Once the files have been collected they have to be transferred to the Data directory chosen at the start of the calibration The SPM softw
8. YF E E a Basic Advanced Rel Setpoint 0 400 alv Baseline nA Adjust Baseline 1 zZ Length Extend Time Ext Delay Retr Delay Delay Mode Sample Rate Z Closed Loop _ In Basic mode the surface position is defined by the Relative Setpoint value of the feedback channel i e Vertical deflection in contact mode The piezo movement Z length is performed relative to this position This is particularly helpful when the lateral position changes between curves and the surface height is different from point to point Note The Relative Setpoint is independent from the normal feedback set point The setpoint in the Feedback Control panel does not influence the mo tion during a force spectroscopy experiment The top set of parameters control the distances moved by the z piezo and the features for limiting the repulsive forces between the tip and sample see Sec tion 6 2 1 Z Length defines the range of the force curve The middle part of the panel has the main parameters to control the timing of the force curves see Section 6 2 2 NanoWizard AFM User Manual Version 4 2 81 Ji 8 f Nanotechnology for Life Science Instruments The z piezo extends moving the cantilever towards the surface and retracting it again Tip movement 1 Approach of the tip from far distance 2 Tip snaps to the surface jump to contact 3 Increase of the repulsive force when the tip in very close contact with th
9. select Gain 4 for lock in amplitudes up to 200 mV 200 Frequency kHz 250 Drive Ampl Drive Freq Start Freq End Freq Phase Shift Lockin Low e Click the infinity symbol to run continuously the instrument will repeatedly perform sweep curves while the values can be adjusted Manual Gain Next Cancel LQ JL Le I jE Lock in amplitude V 78 The display of the frequency curve is as above for automatic tuning Again click Full Range Y for a spectrum overview and click and drag using Select frequency range to zoom into the resonance peak Hap aseyd ul 307 Adjust the value of the Drive Amplitude get a good signal 82 83 Frequency kHz to noise ratio of 6 1 or better for the cantilever resonance peak Drive Ampl 6 023 a v Drive Freq 100 0 amp kHz Start Freq Y 77 948 amp kHz End Freq z o 7o amp kHz Phase Shift 4 deg Lockin Low 30kHz l z Manual Automatic Gain Gain 1 sl Max Ampl 11 08 V M DirectDrive gt Next Cancel 4 4 2 1 Frequency and setpoint selection The frequency and setpoint selection buttons enable the selection of the drive frequency and setpoint for imaging using the mouse Click inside the amplitude frequency plot area to get a cross hair of black dotted lines Holding the mouse but
10. Averaging time Current baseline 0 000 V Czer Rese Approach Parameters IGain PGain Target Height Timeout Approach Parameters SES Approach Type Approach with feedback on Approach with constant velocity Baseline Adjust No baseline adjust Baseline update at start Dynamic baseline update Averaging time 1 0 ms Current baseline 0 000 V zero A Reset Approach Parameters Velocity y 1 5 amp um s Target Height L um During the approach the z piezo extension and retraction can be observed with the Z Range display on the left The position of the red line should oscillate from right to left as it checks for the sample surface at each step If the red line does not cover the full range of the piezo then the approach will probably be unsuccessful The Approach Parameters window is displayed during the motion Choos ing Setup Approach Parameter from the main pull down menu allows you to open the Approach Parameter window as well There are two ap proach types Approach with feedback on and Approach with constant velocity Both approach types are using the same approach routine but there are different ways in adjusting the approach The approach gains and the approach velocity can be altered during the approach The gains must be adjusted in case that Approach with feedback on is selected as Approach type To make a slower approach
11. Delay Mode constant Force J The Delay Mode sets the feedback during the Extended Delay Pixel Rate Constant Height Constant Force For Constant Height the piezo height is held constant at the surface position zZ Closed Loop C For Constant Force the piezo height is adjusted to keep the cantilever deflec tion at the setpoint value Sample Rate 2048 A Hz The Sample Rate is the rate at which data values are stored through the whole force curve Combined with the times for each part of the movement this de fines the sample number for each segment of the force spectroscopy cycle A higher number produces better resolution but a larger data file If the rate is too low particularly for a long Z length value it may affect some control op tions e g Tip Saver or Relative Setpoint There must be enough data sam ples so the software can react to limit the deflection 6 2 3 Z closed loop If Z Closed Loop is enabled M the piezo movement during the experiment is defined by the linearized value of Height measured rather than the piezo voltage value Height so the nonlinearity and hysteresis of the piezo is cor rected during the movement Since the movement is relative to the surface in Basic mode the absolute position is not set in advance In open loop the piezo voltage is increased and decreased and the collected data is displayed against Height measured at the end so there are no errors in the position information in the
12. Display panel on the right hand side allows Vertical axis a the view to be modified the settings do not a SS change the experiment parameters Display All Y Max sl 0 5 aly A pena UONIaYap NUIA Y Min sl 0 7 a v Color extend red Color retract red dark Delta Y na Delta X na Set Line The standard oscilloscope toolbar can also be used to modify the display settings see Section 3 2 5 The normal Z Channel for displaying the force distance data is Height measured and smoothed This uses the linearized sensor channel Height measured rather than the simple piezo voltage Height so there is no hysteresis or piezo creep This channel is used for the Horizontal axis of the force distance display In contact mode the Vertical Deflection is directly related to the force and this is used for the Vertical axis of the force distance display Always make sure that the Vertical Deflection channel is enabled default setting If it is not available see Section 3 2 8 for details of the Channel Setup and Sav ing Settings controls For force spectroscopy in other feedback modes the feedback channel is usually used 80 NanoWizard AFM User Manual Version 4 2 6 1 3 Force Time Oscilloscope i t The Force Time Oscilloscope can be opened using this icon in the main toolbar when force spectros copy is active pt Force Time Oscilloscope at position
13. see the manufacturers manual for details If the connection fails the information about default settings can be helpful The information here only covers general cases as it is possible to change parameters such as the Baud Rate on the syringe pump itself The settings for the pump and SPM software have to be identical before the communication can be successful In general try combinations of serial port dev ttySO S1 S2 Address and Baud Rate until Connection successful is shown Additional pumps can also be added Once the configuration has been set the information will be saved for the next time the SPM software is used The configured pumps will now be shown in the Pump Control section All the pumps can be controlled with Start all and Stop all Individual pumps are controlled with their button bar lela e Version 4 2 REESEN Clicking on a pump in the list will CC select it and it is shown with a green Fast flow rate border The toggle button Show details and Hide details shows the full list of settings for the currently selected pump Pump mode continuous volume to pump O time to pump Status COMIN The Standard flow rate and Fast Selected syringe flow rate are the speeds used for the Add new syringe type dale ut dee Diameter 4 700 mm Total volume 1 000 ml sIngie an ouble arrows in the Dut Currently filled volume F 0 0 a fut Restore last ton bar Ehe ES The Sele
14. AFM User Manual Version 4 2 If you wish to know the spring constant more accurately for quantitative force experiments it should be measured in some way Within the JPK SPM software there is the option to measure the spring constant for softer cantilevers using the thermal noise method This allows an automatic measurement and calculation for cantilevers with resonant fre quencies up to 400 kHz which covers nearly all contact mode cantilevers used for force spectroscopy The method used is based on this paper Hutter JL and Bechhoefer J Calibration of atomic force microscope tips Rev Sci Instrum 64 1868 1873 1993 For more information about cantilever spring constant calculation see the NanoWizard AFM Handbook for example for information on using a reference cantilever to calculate the spring constant 7 3 2 Spring constant measurement The thermal noise method relies on measuring the thermal fluctuations in the deflection of the cantilever and using the equipartition theorem to relate this to the spring constant Essentially the thermal energy calculated from the absolute temperature should be equal to the energy measured from the oscillation of the cantilever spring See the Hutter and Bechhoefer paper for the assumptions and conditions of these calculations Since the deflection must be measured in units of length the Sensitivity must first be measured as described in Section 7 2
15. DN 18 Leveling FF Color trace red X t Color retrace red dark v 0 50 100 150 200 250 300 350 400 450 500 Frequency Hz 9 2 2 Real time oscilloscope Advanced iS Open Script JPK SPM Jython Console q Real Time Scan Logging Settings Advanced System Status Real Time Scan Oscilloscope Vertical axis Autosave Te chi che At Channel Y Max Y Min Error signal mV Color 2 9 3 0 aaa 4 0 4 5 5 0 5 5 6 0 6 5 7 0 Time s Ch3 ch Error signal z Full Range Y red NanoWizard AFM User Manual All the available channels depending on the imaging mode can be displayed as a function of temporal frequencies 1 time as normal or as spatial frequencies 1 distance if Spatial frequency is select ed The controls for the display in the Spec trum Analyzer are similar to the Oscillo scope window see Section 5 4 1 Multiple channels can be selected using the tabs in the lower part of the panel The time based oscilloscope is opened via Advanced Real time scan In contrast to the normal Oscilloscope window described in Section 5 4 1 this displays the channels continuously as a function of time rather than sep arately for each scan line The display is therefore more similar to the output from a conventional oscilloscope connected to the particular channel The Sample Freq is an important parameter as it defined the data collect
16. Save Manipulation Pattern omer l Lee AUSGE CRU aea There are two standard scaling Look In ei manipulation 7 E E GI HE choices B circle svg tux svg Scale to fit scan region EE O Fixed metric scaling 1 Scale to fit scan region best for E grid10x10 svg Geier eise AAN 24e A x10 vector graphics created by external grid5x5 svg SVG file size 1000 0 m x 866 0 m Suggested scaling Biereg factor 4 446284200259807E 9 software packages jpk_new_line svg E square svg File Name itriangle svg Fixed metric scaling factor 1 0 to import the pattern at the same size it was saved best for paths exported from the SPM software Files of Type SVG vector graphics X Open Cancel Note manipulation paths exported from SPM are always defined over the full 100 x 100 micron scan area Re opening them using scale to fit scan region will generally reduce the size e g if the scan area is 10 microns the size will be x 10 smaller Additional Fixed metric scaling options allow factors of 10 10 and 10 to be quickly selected or any arbitrary num ber can be entered in the text box using the format 1 0e 6 The Suggested scaling factor in the information panel would be the scale factor used to fit the manipulation pattern to the scan area so this can be used as a guide for ap propriate scaling of the stored file The Arbitrary scaling factor default value is the va
17. The scanner in the AFM head has a maximum range of 100 x 100 um in xy This maximum scan range is indicated with a blue frame in the Data Viewer It is not possible to choose the new scan region shown here in red because it is partly outside the 100 um piezo range The region will be automatically reset inside the piezo range NanoWizard AFM User Manual Version 4 2 67 Ji F f Nanotechnology for Life Science Instruments Scan Control TESES Ks Line 0 511 gt amp jum H Line Rate Pixels X Offset Y Offset Scan Angle Advanced Imaging Settings It is important to remember the piezo range when selecting values directly in the Scan Control panel The scan area is defined by the x and y offsets and the scan size The origin of the coordinate system is the center of the 100 x 100 um region so x and y have the range 50 to 50 um e g Scan size 20 um and offset 40 um is possible Scan size 20 um and offset 50 um is not possible since the endpoint would be 60 um away from the zero position Rectangular non square scan regions can be set if the Square image option is un checked in the Advanced Imaging Settings For a rectangular image the pixel num ber is automatically updated in order to keep the pixels square 5 1 4 The image Scan List Current Scan Show Si Hold Current Scan Recent Scans Number of listed entries v a a Show 2 a
18. e AC Mode for oscillating the cantilever in water or other fluid Triangular silicon nitride cantilevers with a spring constant around 0 3 N m are suitable for this type of application For more information on intermittent contact mode AC mode and cantilever choice see the JPK AFM Handbook The resonance of the cantilever must be found and the drive amplitude set before an appropriate setpoint can be chosen for imaging Bai Make sure that the cantilever is retracted from the sample and click the Tune icon to open the AC Feed 7 T pack Mode Wizard window jg AC Feedback Mode Wizard o The default values for Start Frequency and End Fre quency are set differently for intermittent contact mode in air or fluid In air the default range is 0 400kHz max freq 2 MHz and in fluid it is O 50kHz The frequency range can be set by entering values in the text boxes at the top of the window Setup AC Feedback Mode LSK LQ JLB Laj IJE Lock in amplitude V Bap aseyd ul 9207 The user has the choice between Manual or Automatic tuning which is selected using the tabs in the middle of the window The buttons in the lower part of the window control the display of the frequency curves and the selec tion of the drive frequency and setpoint 30 Frequency KHz Drive Ampl Drive Freq Start Freq End Freq Phase Shift Lockin Low 30kHz Manual Automatic Target Ampl ly 1 0 EA
19. of the cantilever and the sam ple taken with the AFM NanoWizard in the standard version In this case the view of the sample and cantilever is from above oy tie The image shows the sample a polymer sphere and the approached cantilever from above During the scanning process the cantilever moves as indicated 4 1 3 Optical imaging hardware Inverted optical microscope In the NanoWizard Life Science version the AFM is placed on an inverted optical microscope The scan area can be selected by either looking directly through the eyepieces or using the CCD image displayed on the computer screen The inverted optical microscope shows the sample and the cantilever from below 4 1 3 1 Microscope alignment for Kohler illumination Modern optical microscopes are equipped for Kohler illumination This is important to get good contrast for advanced optical imaging techniques such as optical phase contrast or differential interference contrast DIC Note that if the NanoWizard AFM User Manual Version 4 2 29 Ji F f Nanotechnology for Life Science Instruments microscope is aligned for K hler illumination and then the NanoWizard is put in place afterwards the K hler illumina tion must be re aligned It is worth reading some background information about optical microscope adjustment and optimization since simple adjustments can make a large difference in the optical image quality and contrast There are many websites with h
20. 0 490 um 0 373 um 0 x Vertical deflection mV The main Spectroscopy oscilloscope shown in Section 6 1 2 shows the conventional force distance plot of the spectroscopy data aj aj aJ I Horizontal axis X Max v 1 0 a s X Min sl 0 0 a s Vertical axis chi Elle Channel Vertical deflection The Force Time Oscilloscope shows an alternative view of the same data Here the force data or other channel including the height is plotted against time Display All gt Y Max e 500 a mv Y Min x 400 a mv Color red The Force Time display is updated continuously during the movement while the main oscilloscope in Section 6 1 2 is only updated at the end of the cycle 0 x aj aje I Horizontal axis X Max el 2 0 a s The Force Time display also includes the data col lected during pause or delay segments while the oscilloscope in Section 6 1 2 only displays the ex tend and retract movements The example here shows the data curve for a movement with 1s ex tended delay see Section 6 2 2 for details X Min sl 0 0 a s Vertical axis chi chi2g chs chal Channel Vertical deflection Display All Y Max x 500 a mv Y Min x 400 a mv Color 6 2 Basic force spectroscopy mode Spectroscopy Control
21. 1 75 V k Lateral deflection 0 15 V E Sum 5 25 V WE Restart Force Mapping NanoWizard AFM User Manual In the Force Map Oscilloscope below the spectroscopy curve dis play region there is a tab button for pausing the force movement during a map The vertical deflection signal from soft gold coated cantilevers tends to change over time as the cantilevers bend in response to any envi ronmental changes This should generally be corrected for by using the Adjust baseline feature see Section 0 to adjust the Relative Setpoint during the map For long maps with many pixels the canti lever changes could be enough to move away from the center of the detector The Pause Force Scanning feature allows the map to be interrupted so that the detector signal can be returned to the center Always make sure that Adjust baseline is enabled first so that the new value of Vertical deflection will be accounted for in the Relative Setpoint The software will complete the current force curve movement and then wait in constant height mode Check that the piezo is not mov ing and then adjust the Vertical deflection using the normal position ing screws While the pause window is open nothing in the software can be changed everything is paused waiting for the laser adjustment When Resume Force Mapping is clicked the map will proceed as normal Version 4 2 93 Ji 8 f Nanotechnology for Life S
22. 165 nm NanoWizard AFM User Manual Version 4 2 135 Ji JI Nanotechnology for Life Science Instruments 8 6 4 Background patterns In some cases for instance the example above moving spheres it is useful to have a backgorund pattern for aligning the objects Arrays of lines or points or even geometrical figures can be useful for aligning the particles or other objects on the surface For this purpose patterns with the same format as the manipulation paths can be imported in a way that they remain visible in the software but are not used for the cantilever tip movement The tip path can be drawn over the top to move the particles to the right positions on the background pattern Import pattem for background The import dialog is very similar to the manipulation path import dialog see Section 8 6 2 for details of the options Look in CA manipulation sl ei A KR i circe swg Scale to fit scan region et me ag O Fixed metric scaling Below are two of the standard examples in the directory A square svg Arbitrary scaling factor ow x10 5 a TN triangle svg z S G file size 1000 0 mx 1000 0 m Suggested scaling Non svg factor 1 9999999999999997E 8 opt jpkspm data manipulation SVQ The background pattern is always shown in blue and can be turned off using the right mouse click menu in the Data Viewer The manipulation path can be drawn over the top the background pattern has no influ
23. 2 nm Z channel Height meas smth Vertical axis chi ch2 Channel Ch3 cha Vertical deflection Display All 10 a o 22 P InN Y Max sl Y Min sl Color extend mmm red oa NU penay UONayap ENUIA E ped dark WM Show Index sl 20 Index Selection Color retract 19 Show Current 95989 a The Quantitative Imaging Oscilloscope plots the force curve of each pixel index The Display panel allows the ad justment of the force curve display Section 6 1 2 When the Show Current op tion at the bottom of the Dis play panel is enabled the force curve of the current index is displayed Show Index gives the possibility to select a pixel directly within the active image or to type an arbitrary index position to display the corre sponding force curve The Analyze Panel displays the calculated values for Slope Adhesion and Relative Setpoint Values see Section 6 5 1 If the fit length for the Slope linear fit is changed during imaging the Slope image is updated from the next line 8 1 3 The Quantitative Imaging and Scan control panel Quantitative Imaging Control L sh al lt a Basic setpoint zZ Length Extend Time Retract Time Baseline 17 07 nN Grid Square image sow DEI 200 a un x Offset sel ool al um Y Offset sel oof al Um Pixels 512x Gr
24. 3 1 Cleaning and maintenance ccccseeseccccceeeeececeeeeeceeeceueeeeeeeeusececeseueeceeesaaeeesseaeeeeeeseaeeeeessaaeeeesenas 15 2 3 2 Cantilever Selection ANd handling ccccccccccseseeececeeeeeeeeeececeeeeeeeeeeeeeeeessaeeeeeeeeeeessaeaeeeeeeeessaaeeess 16 2 3 3 SEO e OU ME 16 Wee ER EE ee 17 3 1 SPM software introduction and indeX neesgeeee see ngeegeeueeeguE Seege A es 17 3 1 1 Elie ban eet EE 17 3 1 2 SOPTWALE OVELVIEW MA 17 3 1 3 WES TSU eege EE EE 18 3 1 4 ba SVCMCUE ICOM 100 e E 20 3 2 introduction to the main Controls iorsin aa i na aE a a aaia 21 3 2 1 Foen CEE ONNO E 21 3 2 2 imagmg settings Scan COM OM E 22 3 2 3 Scan PS te EE 22 3 2 4 WN Ate EE E 23 3 2 5 DS CIO eet 24 3 2 6 Increasing and decreasing values with the increment buttons 25 3 2 7 Personal display settings ccccccssececeseecceeseecceeseeceaeesseeeecseseeeseaueeessueeesageeecsueeessenseesseseeessaeees 25 NanoWizard AFM User Manual Version 4 2 i Ji 8 f Nanotechnology for Life Science Instruments 3 2 8 Managing and saving data 26 3 3 Software versions and updates ccccccccccsseseecceeeeeeceaeeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeesseeeeseeeeeeesseeeeeeeeeeesseaesaaeeeeeeesaas 27 3 3 1 Updating ASS OT E 27 SEL 28 4 Setting up and EEN 29 REE sesan e EN 29 4 1 1 The CCD camera JUNC cicer EE EEE EE R 29 4 1 2 Optical imaging hardware Top View ODpttcs 29 4 1 3 Optical imaging hardware Inverted
25. Instruments 3 2 5 Oscilloscopes DI Oscilloscope Height trace nm P Nanotechnology for Life Science Wu 3381334 YHIBH aj mj Le Li LE Horizontal axis X Max M 4 0 A um xmn gt 0 0 a um Save Line Scan Vertical axis chi FEbz Channel Display Y Max Y Min Leveling Color trace Color retrace Ch3 ch4 Height Linear red red dark Draw the last scan Non image data is usually displayed in some form of Oscilloscope window There are different kinds of Oscillo scopes for different measurement modes but some features are common to all The plot area is usually on the left with display settings on the righ The text fields can be used to set the for the horizontal and vertical axis ranges and displayed channels The standard oscilloscope toolbar can also be used to set the view ranges This is found in most oscilloscopes Zoom activates mouse tool to select a rectangular area in the plot which updates the axis settings Full Range XY sets X and Y axis to the full range of the current data Full Range X Full Range Y Autoscale XY toggle button the X and Y axes are reset to the full range of each new data set as it arrives Autoscale Y toggle There are some useful mouse shortcuts for changing the Oscilloscope display r
26. The software will now calculate the corresponding tip posi tion in the other 24 images The user must wait for all the values to be generated The progress can be monitored as the values appear in the Pixel X and Pixel Y columns Check the accuracy of the automatic tip location at the bottom of the panel The Standard deviation should be less than 1 pixel if the calibration is reasonable Scroll through the list using the up and down arrow keys When one of the images is selected in the list it appears in the top panel with the calculated tip location marked If the contrast is poor or if only a small part of the cantilever is visible in the selected image the calculation may be inac curate Images with an incorrect tip location can be dese lected with the Use tick box There are an excess of points used for the calibration procedure so the deselection of points will not disrupt the calibration Some images may have been automatically deselected When satisfied with the calibration points click Continue At this point the calibration is finished and the user should select an image to be imported into the background of the SPM program The Median image is calculated from all 25 calibration images When the size of the calibration grid is large enough this generates an image without the cantilever This median image is displayed on the final panel of the calibration procedure by default Alternatively the user can select one of th
27. adjustment in the same time Samples with very sharp changes in topography the feedback needs to react very quickly at the edges Low force scanning to give fast enough correction even for a small setpoint change 64 NanoWizard AFM User Manual Version 4 2 Decreased Z range lower z amplification In the opposite cases small slow or flat scans the gains do not need to be so high The feedback response depends on the setpoint since the gains control the amount of correction for a certain difference between the setpoint and the current feedback channel value Therefore for low force scanning high setpoint in AC mode low setpoint in contact mode the gains need to be set higher to react properly to very small changes in the setpoint value In some situations there is a large range of reasonable values that give very similar results for robust samples the feedback only has to react fast enough to follow the topography Scanning medium size regions in air for instance it is often possible to use the default values over a wide range of conditions particularly in contact mode If the sample is delicate then the feedback parameters really need to be optimized and it may be necessary to adjust all the parame ters many times during different scans In the SPM software the gains are applied relative to the current piezo range Therefore when the piezo range is re duced e g from the maximum 15 microns down to the 3 microns range the
28. curves from either the current measurement or a given index can be displayed Chose the index cell number and the spectroscopy experiment is displayed in the oscilloscope Click Full range X or Autoscale XY to get the force curve properly displayed Show current current spectroscopy data are displayed as the map progresses Show index spectroscopy data from other points are displayed With Index Selection the mouse can be used to click on a pixel in the data viewer to display the corresponding force curve The Index Comparison button in this panel opens a special oscilloscope window where the data from several pixels within a map can be compared Note that the force scan map file must be selected and focused in the Map List shown with a green frame to show the force distance curves from completed map images For further information about the tab Analyze see Section 6 5 1 6 7 4 Data types and file saving Force Map List OX Current Force Scan Map Show Force Mapping Grid Recent Force Scan Maps Number of listed entries Show E Old Force Scan Maps Show The Map List icon can be found on the main shortcut bar across the top of the SPM software The Map List is a tool for administering and saving Force Mapping files and is similar to the Scan List for normal images As with the Scan List see Section 0 for more details there are three sections for the Map List The Current Force Scan Map is hel
29. detergent can be added to the water When the glass block is put into the bath it should be set on its side otherwise the optical planes of the glass block can be seriously damaged Rinse the glass block with ultrapure water afterwards and dry it in a stream of nitrogen Try to avoid any contact with the optical planes 2 3 1 2 AFM housing and filters The NanoWizard AFM head does not require much maintenance From time to time clean the housing with a soft damp cloth The yellow reflective laser filter in the top of the AFM housing can be cleaned with a soft piece of clean lens paper soaked with a few drops of pure alcohol 2 3 2 Cantilever selection and handling The cantilever and tip are both susceptible to damage Cantilevers are expendable items and have to be replaced regularly The lifetime of AFM cantilevers strongly depends on the way they are handled If the tip is damaged the tip radius generally increases With worn or contaminated tips the image resolution will be reduced and the images may have serious artifacts Damage to the cantilever arm may also cause problems for imaging See also the more general discussion about cantilever choices for different imaging modes in the NanoWizard AFM Handbook Handle the cantilever chips carefully e Do not touch the cantilevers with fingers Use tweezers to handle them e Do not drop the cantilever chips The cantilevers are delicate and may break off from the chip e Only open the cantile
30. exactly the same area of the sample unless it can be located optically 68 NanoWizard AFM User Manual Version 4 2 Stepper Motor Position 99 099 um Coarse Step Size 500 00 um y Motor A 117 um Motor B 74 1 um Motor C 118 um v Y Zero counters View of the AFM head from the top Orientation of the cantilever A C in the AFM head E Gata Wawer Height Grace Slow scan direction y a Fast scan trace direction x Slow scan direc tion scanning bottom to top Slow scan direc tion scanning top to bottom Stepper Motor Position 99 099 um B Coarse Step Size Y Motor A 117 up Motor C 118 Open the Stepper Motor window to move the stepper motors independently The diagram shows the direction of the main scan axes to help translate the scan to the movement of the motors Note that if the tip is approached the down arrows of the stepper motor are inactive For a zero scan angle the fast scan direction x is front to back relative to the head and the slow scan axis y is right to left The orientation of the cantilever relative to the AFM head is shown here The movement of the fast and slow scan direc tions relative to the Data Viewer window and the cantilever is shown here Looking at the tilt of the scan lines in the oscil loscope is useful to see if it is necessary to re adjust the stepp
31. final force curve It is critical to use Z Closed Loop however where the speed of the motion is important In closed loop the Height measured position at each point of the force curve is used to correct the piezo voltage and the force curve has a constant speed Closed loop also becomes most important for larger Z lengths where the piezo nonlinearity is more significant Z Closed Loop can introduce a small amount of noise as there is an extra feedback This is not usually significant for low noise Z sensors such as the capacitive sensors in the NanoWizard 3 AFM heads 6 3 Advanced force settings The Advanced force Settings window can be opened under the Force Spectroscopy menu in the main menu list This panel provides extra parameters in addition to those found in the Spectroscopy Control panel NanoWizard AFM User Manual Version 4 2 83 Ji F f Nanotechnology for Life Science Instruments X Advanced Force Settings In the Advanced force Settings window the Z Extend Rate and Z Extend and retract rates E Retract Rate can be set to different values Use the same rate for extend and retract Z Speed Z Length and Z Time for extend or retract segments depend on each other Update time or speed helps to either maintain the duration of a segment Constant Duration if Z Speed or Z length is changed or to maintain the Z Speed Constant Speed if Duration or Z length is changed The updated values of Z Rate Z Time an
32. following sequence of colours can be observed at the rear panel 14 NanoWizard AFM User Manual Version 4 2 e bootloader on e program loaded e IP address assigned gt ready for operation It is alright to leave the PC switched on all the time When the SPM program is closed you can easily switch on off the controller At the log in screen enter the user name and password For new systems default account details are sent separately Once you have logged in with a valid user account the JPK icon can be found on the desktop to start the SPM software The PC can be left on all the time When the SPM program is closed you can switch the controller on off as required 2 3 Practical tips for using the NanoWizard 2 3 1 Cleaning and maintenance 2 3 1 1 Glass block cleaning and storage The flat top and bottom of the glass block are optically polished glass Only place these planes on soft tissue paper such as Kimberly Clark Kimwipes Lite 200 Do not touch the optical surfaces with tweezers or any other material that could scratch the surface and do not store the glass block lying on these surfaces When placed like this the polished optical surface When not in use always place the glass block on its of the glass block can be damaged side NanoWizard AFM User Manual Version 4 2 15 Ji F f Nanotechnology for Life Science Instruments The glass block can easily be cleaned with an ultrasonic bath If required some
33. for import Click on the Snapshot icon to acquire an image from the JUnicam software and import it into the SPM software The Snapshot icon is only active when the JUnicam software is open and an optical calibration is defined optical image is selected in the scan list The snapshot image is saved into the folder with the selected calibration optcal 2006 11 06 16 07 07 th te th th a zs D v S D a a o D median jpg optcal 00 jpg optcal 01 jpg L a a n 1 optcal 04 jpg optcal 05 jpg optcal 06 jpg optcal 07 jpg a a D e optcal 10 jpg optcal 11 jpg optcal 12 jpg optcal 13 jpg a A a 8 optcal 16 jpg optcal 17 jpg optcal 18 jpg optcal 19 jpg opti E R al a SZ date appr optcal 22 jpg optcal 23 jpg optcal 24 jpg optcal shifted snapshot 25029 jpg NanoWizard AFM User Manual 1 1 optcal 02 jpg optcal 08 jpg L optcal 14 jpg a optcal 20 jpg snapshot 25025 jpg Rre vewa The calibration folder that was set at the beginning of the procedure will now contain a number of files of differ optcal 03 jpg ent types Las The 25 calibration images are stored TT bere along with the calculated median eebe Both the original optical calibration file E optcal and the adjusted calibration file with the user determined offset optcal 21 jpg optcal shifted are found in this fold er snapshot 25027 jpg Lastly any snapshots taken while this calibration
34. gains need to be increased to give the same response The features will usually be smaller since the reduced range is chosen for very flat samples but for scanning at low force the gains can be increased a lot The Height image is obviously the first channel to use in setting the imaging parameters the quality of the scan may be obvious from the plain height image The Error Signal image Vertical Deflection in contact mode and Amplitude in AC mode is also vital for adjusting the feedback settings this shows the correction signal that the feedback is using to adjust the height If there are dark and bright patches of almost black and white around features then the feedback loop is not correcting fast enough and the error is large at these points If the image shows a more softly shaded shadowed image of the height then the feedback loop is adjusting the height within a reasonable time 5 2 2 Simple procedure to optimize IGain and PGain It is best to be able to see the current line scan in order to judge the effect of adjusting the gains or setpoint values on the imaging Therefore the Line Viewer in the Data Viewer window see Section 5 1 2 or Oscilloscope window see Section 5 4 1 should be opened so that the height profile for each line can be seen In this example the Line Viewer in the Data Viewer window is used to show the effect of adjusting the gains fice Jl zeck eh Sos Increase l Gain until the z feedback loop o 5
35. in force modulation mode in air The bright parts of the amplitude image have a higher cantilever deflection range and hence represent a harder Surface D 5 10 fast gam Height image Amplitude image The drive is below the resonant frequency so the measured lock in amplitude of the cantilever will never be more than the drive amplitude On a hard surface the extra deflection of the cantilever the amplitude is exactly the same as the drive oscillation On an ideally soft surface the tip would just sink into the sample and there would be no extra de flection at the tip so no lock in amplitude signal 5 7 2 Off resonance cantilever tuning The software for the Cantilever Tuning for Force Modulation mode is basically the same as for the normal proce dure for intermittent contact mode see Section 4 4 2 In this case however the aim is not to find a resonance of the cantilever or system gt Cantilever Tuning i The cantilever is modulated at a relatively low frequency well below its resonance frequency For instance a frequency for a typical contact mode cantilever in air would be around 5 kHz Find Resonance 3 Basic mm There are also special force modulation a Gain Gain 3 b i 3 medina Ee cantilevers available The resonant frequency _ Ey ey is around 75 kHz in air k 1 5 N m In Force modulation mode a typical drive frequency would therefore be 30 40 kHz
36. laser approached NanoWizard The blue approached LED should be illuminated on the front plate of the AFM head NanoWizard AFM User Manual Version 4 2 47 Ji F f Nanotechnology for Life Science Instruments Range The software panel Z Range should show the red bar in the centre as QO um Extended Retracted System Status sum Lateral Def 2 61 V Vertical Def 1 221 Y Status E 2 H Shown here or at the Target Height set in the Approach Parameter win dow if that is different The System Status panel at the bottom left hand corner of the software will show the Status as Idle This means it is waiting at the surface and ready to start a measurement sE Idle Sometimes the blue light is illuminated on the front plate of the AFM head but for some physical reason the cantilever is not in real contact with the surface If the Z Range shows the red bar at the ends of the range i e 15 microns fully retracted or 0 microns fully extended the approach has been unsuccessful Sometimes the routine may find the surface if Approach again is clicked but sometimes the value of the setpoint must be adjusted to reach the surface There are two common problems approaching which many occurs in Approach with feedback on 1 4 5 3 The feedback is too sensitive causing oscillations in the vertical deflection and possibly an audible ringing tone In this case follow the instructions to make a slo
37. location and content of data files saved 3 2 8 from the software Approach siarts the movement of the cantilever towards the surface 4 5 Run starts a scan in many modes this is only active if the cantilever is approached at 4 5 RUN the surface Retract moves the cantilever away from the sample using the Z piezo or the Z stepper 4 5 3 motors The Z Stepper Motor window controls the 3 motors for coarse approach or retraction 5 3 They can also be moved independently to change the tilt of the head Ep NanoWizard AFM User Manual Version 4 2 Contact mode Motorstage icon requires an optional software module which comes with the JPK Mo torized Precision Stages The stage must be connected at software start Start the CCD camera window to see the image from the optical microscope or top view optics Take a Snapshot and import a calibrated optical image using selected calibration re quires optional software module DirectOverlay The button is only active when an optical image is selected in the scan list Shows the status and switches the Laser on off this is a toggle switch and can not be switched off during scanning or while approached Open the Laser Alignment window to adjust the laser beam onto the cantilever and the photodiode The Cantilever Tuning icon is only shown when certain feedback modes are selected The tuning window is used to find the cantilever resonance The Feedback Mode is set with this
38. not been saved they are lost permanently vi Preview shows hides the scan in the scan viewer select the scan in the list and the name is highlighted with a green box cu Saving settings opens the window for setting the saving directory and channels Save all saves all the current files in the list Autosave File Filter Autosave No Filter Simple Filter O Advanced Filter 88 NanoWizard AFM User Manual The Autosave File Filter can automatically manage where and whether the files are saved Autosave must be switched on for the filter to be used If No Filter is active all force scans are saved using the default settings Version 4 2 Autosave File Filter Jl Autosave O No Filter lSimple Filte O Advanced Filter Adhesion Threshold Retract Current value n a Last min value in first retract Autosave File Filter Autosave O No Filter O Simple Filter Advanced Filte B Process file No process filter Accepted Not Accepted Unclassified Total COOC Autosave File Filter v Autosave O No Filter O Simple Filter Advanced Filter Process file filter process jpk proc force Accepted 65 Not Accepted Unclassified Total o LReset_ o Reset 6 The Simple Filter uses the value of Adhesion that is automatically calculated for all force curves see Section 6 5 1 If the Adhesion Threshold is not reached during retraction the curve is no
39. not been moved e Scanner Retracted means that the retract button has been clicked more than once and the motors have been moved Advanced System Status SE In addition to the System Status the Advanced System Status win Scanner Retracted dow displays the Sum Vertical and Lateral Deflection of the photodiode SE The Advanced System Status window can be opened under the Ad Vertical Def 0 843 V vanced pull down menu Lateral Def 0 63V User mueller Saving on Filename home mueller jpkdata DO force Force scan Oofl 0 of 20s 4 5 4 Advanced approach using Baseline adjust For users who wish to have a more sensitive approach in some feedback modes there is a special feature available in the software for approaching with a variable setpoint Feedback Control In the standard approach routine the Setpoint value in the main Feed e 130 0 amp HS back Control is used directly as the desired value for the adjustment of the feedback system The setpoint is an absolute value that remains constant for the whole approach The setpoint is often chosen far from the surface however and as the tip approaches the forces between the tip and sample change the response of the cantilever The standard routine does not take account of these changes and only looks for the point where the abso lute value of the feedback channel reaches the Setpoint value Example for contact mode If the Vertical deflection on the photodiode at
40. of the screen A window including a menu running and scripting Temperature Controllers controls as well as the scripting console is opened Voltage Output Settings q gt ExperimentPlanner 126 NanoWizard AFM User Manual Version 4 2 ExperimentPlanner File Edit Run Control structures Alignment Temperature Controller Spectroscopy FluidicsModule Optical image JPK Script checkVersion SPM 4 0 99 lt MT Help Script output while condition A while loop describes a loop that will be executed as long as the condition given after the while All commands that will be executed in the loop have to be indented Our suggestion is to use 4 space characters to indent the commands In the file menu existing scripts can be opened and new scripts can be generated New Ctri N and saved Open Ctrl o Examples Save Ctrl S El Save as Clase Under the edit menu tools for editing the script can be found Undo Ctrl Z o Redo Ctri d Cut Ctrl x F Copy Ctrl C Paste ctrlv The scripts are started with the Run button and can be interrupted with the Stop but ton NanoWizard AFM User Manual Version 4 2 127 Ji F f Nanotechnology for Life Science Instruments Control structures Alignment Scanner Temperature Controller Spectroscopy FluidicsModule Optical image The ExperimentPlanner is designed to select the commands needed to perform a complex experiment from l
41. optical mcroscope 29 4 2 Set up the cantilever and optical detection system 30 4 2 1 Mount th cantilever E 30 4 2 2 Setting up the laser detection system 33 4 2 3 Adjust the laser beam onto the end of the cantlever 34 4 2 4 Adjusting the mirror for large Changes in deflection cccccccecsseeeeeeeseeeeeeseeeeeeeeeeaeeeeessaeeeeeeaas 34 4 2 5 Adjust the spot onto the centre of the detector nsnssnnsennnnsreosrtnrtrrnsrtnrtrrrrsrtnrttrresrnnttnnennn nnn 35 4 2 6 SS UM UD Mi le e WEE 36 4 2 7 Troubleshooting alignment porobleme 37 4 3 Mounting the ue UE 39 4 3 1 Lie Science Stage saissetiae E EEA AEREE 39 4 3 2 El Ee e 40 AA SEIECHONMO TECODACK INO E 40 4 4 1 COn NOUE eee eee eee ee eee te A E E E 40 442 AG ee 40 4 4 3 Using intermittent contact mode in liquid c cceeccceeceeeeeeeceeeeeeeeceeeseeeeesseaeeeesaeeeeeeessaeeeeessaneeees 44 4 4 4 Force Modulation INO CG E 45 ES leit Ten UL EE 45 4 5 1 Ge ee e c 61 EE 45 4 5 2 PAO TING KO ACM E 46 4 5 3 Retracting the tip from the sample 48 4 5 4 Advanced approach using Baseline acdust 49 4 6 Environmental Control for experiments cceccccseeeecceeeeeeceeeeeeneeeeeceeueeeseeeeeeaeeeeeeeeeeeessaeeesseueeesseeeessaeeesseeeneas 50 4 6 1 Temperature Control ANd data SAVING cccccccccseeeceeceeeeeeeeeeeeeeceeeeeeeeeeeeseaeeeeeeeaeeeeeesaeeeeeesaaeeeeeeeas 50 4 6 2 Pump control for syringe Dumpe ss snsssnsesennesernesrrrrerrer
42. part of the help tab exceptions that can be thrown due to incorrect use of the command are given NanoWizard AFM User Manual Version 4 2 129 Ji f f Nanotechnology for Life Science Instruments Unsaved Script ExperimentPlanner Eile Edit Run Control structures Alignment Temperature Controller Spectroscopy FluidicsModule Optical image checkVersion SPM 4 0 99 Scanner approach lt M Help Script output Scanner approacht Approach the scanner to the surface moving the piezo and the stepper motors The maximum moving distance is the currently set approach target height Throws ModeConflictException SPMscript ApproachFailedException if the surface is not reached in the scanner range HWException InterruptedException Invalid ParameterException In the other tab of the lower part of the Exper Help script output imentPlanner window the Script output is Script started placed Start and end of the script are docu SCript finished mented here as well as any error appearing during the execution of the script Errors in the syntax of the script are listed including the line of the error Due to the command selection characteristics of the ExperimentPlanner little to no knowledge on Jython program ming the scripting language of the Planner is necessary Nonetheless there is a very good online tutorial on www python org Within very short time this language can b
43. screws Optical path schematic AFM head adjustment Laser Alignment Window Detector Prism Laser FE Laser Alignment Adjustment Vertical Def J 0 00 V Lateral Def 0 01 V x 7 BI Sum Mirror S d 1 19 V Detector Adjustment Detector Cantilever Screws NanoWizard AFM User Manual Version 4 2 35 Ji F f Nanotechnology for Life Science Instruments The Laser Alignment window in the SPM software shows graphically the signal from the different quadrants of the detector Adjust the detector adjustment screws so that the red spot is in the centre of the detector in the Laser Alignment window This means that equal intensity is reaching all four quadrants Note that the spot in the blue re gion shows the centre position of the laser beam relative to the four quadrants not the representative size Laser Alignment This is how the Laser Alignment should look like after successful Vertical Def adjustment 0 00 V Lateral Def HE 1 Sum is at the maximum value for that cantilever Sum 1 19 V 2 The red spot is in the middle of the crosshair 3 Vertical Def and Lateral Def are close to zero 4 2 6 Setting up in liquid The NanoWizard AFM is optimized for performing experiments on samples in a liquid environment The instrument is designed to protect sensitive parts of the microscope but particular care is still needed when working in liquid When the bottom face of the glass bl
44. slider Scale or turn the image with the slider An gle Using the right click menu in the Data Viewer the view can also be shifted and zoomed using the mouse Note Neither function affects the motion or scanning angle of the cantilever only the data display Focus Rescales the selected image to full size The selected image is usually the current scan but other images can be se lected using the Scan List This quickly brings a selected image into view as in the before and after screenclips below NanoWizard AFM User Manual Version 4 2 59 Ji F f Nanotechnology for Life Science Instruments Line Viewer Scale Autoscale WC E a LA m K T E a ac Save Line Scan POSS Sf99900r om MAM Oo hm Bo oo The Line Viewer displays the current line scan as it is acquired The channel and leveling settlings are taken from the data viewer The buttons on the right hand side can be used to adjust the scaling or to save a single line scan Scan Control By default the first image is started from the bottom of the Data Viewer window as ad H A Led marked below This is the case when the right arrow is active in the scan control box E Ks lineO 511 as marked with the asterisk here on the left If the left arrow is active the image is Pan al gi scanned from top to bottom If the software is set to perform repeated scans then the Slow v m Se gr
45. small tool must be installed with administrator rights Log in as jpkroot open a terminal window and type the following command apt get install timer applet The timer can then be added to the toolbar of each user individually Click on the desktop with the right mouse button and choose the option Add to panel Select the tool Timer This now adds a small icon to the taskbar at the bottom of the Ubuntu desktop This opens a timer with simple count down settings 9 2 Advanced SPM software options 9 2 1 Spectrum Analyzer Imaging The spectrum analyzer is opened using Imaging Spectrum Analyzer i Open Saved Image With the help of the spectrum analyzer acoustic or electronic noise on the canti lever can be detected The calculation is made using an FFT of each scan line Advanced Imaging Settings and combining the data from several consecutive scan lines to give an average Hover Mode Settings noise spectrum Scan List k Oscilloscope oo The maximum frequency is limited by the so called Nyquist Frequency which CES Spectrum Analyzer depends on the scan rate duty cycle image size and pixel number p Calibration Manager 144 NanoWizard AFM User Manual Version 4 2 zA Spectrum Analyzer X Max z 512 a Hz SCH Full Range X Select X Range Spatial Frequency 15 Vertical axis chi tha chs cha 16 Channel Height Display Trace ze gt 17 S Y Min k 22 a m i Full Range Y
46. some point was 0 52 V and the Setpoint in the Feedback Control panel was 1 0 V then the effective setpoint the difference between the initial deflection and the final setpoint would actually be 1 52 V NanoWizard AFM User Manual Version 4 2 49 Ji F Nanotechnology for Life Science Instruments In the advanced approach using the Baseline Adjust it is possible to perform the approach with a relative setpoint instead of a fixed one Approach Parameters Approach Type 2 Approach with feedback on Approach with constant velocity paseline Adjust 2 No baseline adjust Baseline update at start Dynamic baseline update acaqging time Current baseline 0 000 V Zero Reset Approach Parameters IGain PGain Target Height Timeout The Baseline Adjust within the Approach Parameters window allows choos ing different advanced approaches The options are no baseline adjustment baseline update at start and dynamic baseline update When Baseline update at start or Dynamic baseline update is active in the Approach Parameters window then the number in the Setpoint text field is used as the difference between the offset value of the feedback channel cur rent baseline and the actual setpoint used for the approach The Approach Baseline update at start is adjusted after each motor step to the current val ue of the feedback channel The value s also
47. the Spectroscopy Grid Manager will be greyed out The last grid can be restored using Set Grid NanoWizard AFM User Manual Version 4 2 85 deift Instruments 6 5 Other Features Nanotechnology for Life Science 6 5 1 Analyze Adhesion and slope Display i Analyze Slope Linear fit Fit Length le Slope extend 12 81 Wum Slope retract Show extend fit Show retract fit 12 93 Vum Adhesion extend 1 631 V Adhesion retract 1 489 V Relative Setpoint Values Height extend 3 481 um Height retract 3 495 um Height measured ext 6 427 um Height measured retr 6 419 um le gt Fit Length Tt ff E Adhesion trace t Adhesion retrace In the right hand panel of the Spectroscopy Oscilloscope display there are also options to perform simple online analysis using the Analyze panel All the analysis operations give values for both the extend and retract parts of the force curve The set of values is also used during Force Mapping to generate the images in the Data Viewer The Relative Setpoint Values show the Height or Height meas ured value when the relative setpoint was reached These values can be used offline with other software to calculate sample volume for instance The Slope value is fitted with a linear fit over a fixed range starting from the end of the force curve at the sample The single fit parame ter is the Fit length which must be sho
48. the glass block fixed and avoids damaging the glass block when changing the cantilever Additionally the changing tool corrects for the tilt of the chip holding part of the glass block to facilitate cantilever mounting Put the cantilever holder into the cantilever changing tool The screw side of the cantilever holder is opposed to the downward tilted part of the changing tool The notches in the glass block must be lined up with the metal tabs on the changing tool Turn the glass block by 90 degrees into position and turn the two finger grips on the rim of the steel disk in order to lock the glass block This is basically the same mechanism as used to mount the glass block into the AFM head NanoWizard AFM User Manual Version 4 2 31 J f f Nanotechnology for Life Science Instruments Mount the cantilever chip and clamp it to the glass block as described below 8 Cantilever St om groove Cantilever spring Alignment marks cw amp G glassblock C cantilever S spring Note that the polished top and bottom surfaces of the glass block must not be scratched Always use the cantilever mounting tool or put the polished glass surface on a soft lens tissue free of dirt or dust Place the substrate chip of the cantilever onto the inclined part of the glass block This has an angle of 10 degrees to ensure that the tip at the end of the cantilever reaches the surface first The cantilever chip s
49. the piezo diagram The current piezo position is marked as a red line and the recent range cov ered within the scan line is marked in blue Z Range zZ Range 0 Em 8 111 um 13 Hm 0 um 5 000 um 15 um es 7 ee Extended Retracted Extended Retracted 0 15 um maximum range 0 5 um useful range for small samples MER GU SECHER t is also possible to view the Z 15 um Retracted Piezo display vertically which is more intuitive This extra panel is opened from the View menu Autosave scan Repetitions ER Laser Alignment Range 15 000 jum zeg gt Approach Parameters Li saving Settings e Channel Setup 0 um Extended Sp AC Feedback Mode Wizard Exit Ctrl G 5 3 2 Independent movement of the stepper motors Most samples are not mounted exactly parallel to the sample stage and sometimes the interesting region of a sample is at an angle to the AFM head In this case the feedback has to correct for this general sample tilt as well as the local changes in height due to the roughness of the sample If the sample is parallel to the AFM head over the region that is being scanned the feedback only has to correct for the roughness of the sample and this can lead to better imaging particularly if it allows the Z Range to be reduced see Section 5 3 1 Note that generally the exact location on the sample is changed when the stepper motors are moved independently of each other Therefore it may not be possible to find
50. to calibrate the sensitivity and spring constant YF See Chapter 7 for more information Shortcut to open the Spectroscopy Grid Manager to create grids of spectroscopy points See Sec tion 6 4 2 for more information de Shortcut to open the Force Scan Repetitions window to set repeats of force curves See Section Keng 6 4 1 ZX O 0 Number of Scans _ Go through X Position list Repeat mode Repeat each point Either Single scans an arbitrary Number of Scans or Infinite scans can be recorded when the Run button is clicked Go through XY Position list goes through all the positions in the list If Number of Scans is active and greater than 1 there are two settings for the measurement sequence Repeat each point makes the set number of scans at each point before moving Repeat whole list makes one force measurement at each point before moving and goes through the point list the set number of times 6 1 2 The Spectroscopy Oscilloscope _ Spectroscopy at position 0 490 um 0 373 um Vertical deflection extend V 250 Height measured amp smoothed nm KSE Horizontal axis xma 7 lz Z channel Height meas smth ertical axis chl fi i Channel Vertical deflection e The Spectroscopy oscilloscope opens au tomatically The force distance curves will be EES ce plotted in the main area of the window The X Min el 50 a eg Z channel Height meas smth
51. with the smallest available calibration standard Over small height ranges the nonlinearity of the piezo material is not very noticeable nonlinearity is more of a problem in the Height channel when the range is a significant fraction of the full piezo range The Height measured channel is better over larger z ranges because it is linear but over very small ranges the noise of the capacitive sensor is notice able and may be seen in very flat images So overall the choice of which channel to use depends on the height range of the sample topography e For flat samples with height features in the range of a few nm it is recommended to choose the Height chan nel with a manually calibrated z piezo which will have a lower noise e For rougher surfaces with height features over 100 nm or in the micron range it is better to use the Height measured channel to get linear operation over the greater height range For non imaging applications where the piezo makes large movements it is strongly recommended to use the z linearization since the piezo movements may be in the micron range Make sure that the linearization is enabled in e Spectroscopy mode e Force Mapping mode e Sensitivity calibration NanoWizard AFM User Manual Version 4 2 95 Ji F f Nanotechnology for Life Science Instruments 7 2 Sensitivity calibration The movement of the cantilever is measured by the deflection of the reflected spot position on the photodiode T
52. 0 100 150 200 250 300 350 400 450 500 starts to oscillate Offset nm height nm en Wl 220 0 ae Reduce IGain slightly until the oscillations are eliminated 5 Scale 50 100 150 200 250 300 350 400 450 500 Offset nm E we ebe LS w f m NanoWizard AFM User Manual Version 4 2 65 Ji 8 f Nanotechnology for Life Science Instruments j Poan Il 0 007 Fain F 0 007 zi 5 Scale S EA Increase PGain until z feedback oscilla 10 height nm 0 50 100 150 200 250 300 350 400 450 500 tions are observed again Offset nm 5 a 7 ze JW ei 5 Scale iG Reduce PGain until the oscillation van 0 50 100 150 200 250 300 350 400 450 500 i ishes Offset nm 5 2 3 Scan speed and feedback adjustment Generally you can achieve better results with lower scan rates in these cases e The sample is rough or strongly corrugated a large z piezo range is used in scanning e The scan size is large a larger x y range is used for scanning e The scan is made in intermittent contact mode in liquid e The sample is very soft and easy to damage A combination of several of these features means that the scan should definitely be in the lower end of the range For example when scanning living cells in liquid there is a very soft sample that has a large height range and is often scanned with x y scan sizes up to 100 um In this case the speed should be around 0 3 0 5 Hz For smaller sc
53. 3 2 2 9 2 6 9 2 5 9 2 2 9 2 3 4 5 3 5 1 5 5 5 4 1 9 2 1 7 6 6 1 2 6 1 3 6 3 7 6 7 6 7 3 6 1 3 7 19 Ji F f Nanotechnology for Life Science Instruments Window Tile Vertically Tile Horizontally Settings to allow tidy alignment of open windows Cascade X Close All Frames v Auto save Window Layout lf Auto save is enabled then sizes and positions of internal 3 2 7 windows are remembered wv Avoid Opening Frames Forget Saved Window Layout The personal settings can also be deleted with Forget ne EET ARAHAN Select from the list of other currently open windows Help About Information about the software version and related projects Credits 3 3 2 3 1 4 The shortcut icon toolbar Below the menu bar you can find icons to launch the most commonly used features A short explanation for each icon is shown in the table below Some icons or options only appear if software extension modules have been purchased am 8 an e Re ob S HIH The icons on the left hand side of the toolbar are always shown regardless of the feedback or measurement modes that are selected Contact mode Lab Imaging Details in Shortcut icon Brief explanation Section LA Autosave this toggle button both shows the status and activates the Autosave func 3 2 8 CR CS tion so that all scans are automatically saved Saving Settings this is used to set the name
54. 5 00 3 optcal 03 jpg 17 50 35 00 4 optcal 04 jpg 35 00 35 00 5 optcal 05 jpg 35 00 17 50 6 optcal 06 jpg 17 50 17 50 7 optcal 07 jpg 0 00 17 50 8 optcal 08 jpg 17 50 17 50 optcal 09 jpg 35 00 17 50 RE 35 00 O00 17 50 0 00 2 0 00 0 00 3 17 50 0 00 35 00 0 00 5 35 00 17 50 3 17 50 17 50 7 0 00 17 50 3 17 50 17 50 35 00 17 50 20 35 00 35 00 21 17 50 35 00 22 0 00 35 00 23 17 50 35 00 24 35 00 35 00 Cancel Optical Image Calibration Locate Tip Positions Reference EI men 2 E 35 bel 3 E 35 wl A E 35 00 35 00 wl 5 E 35 00 17 50 v 6 E 17 50 17 50 EI 7 E 0 00 17 50 wl 8 E T750 17 50 v 9 E 35 00 17 50 wl 10 E 35 00 0 00 EI 11 G 17 50 0 00 Jl 12 IT 0 00 0 00 wl 13 E 17 50 0 00 wl 14 35 00 0 00 wl 15 G 35 00 17 50 wl 16 E 17 50 17 50 Je 17 IT 0 00 17 50 EI 18 Il 17 50 17 50 wl 19 E 35 00 17 50 Je 20 E 35 00 35 00 wl 21 E 17 50 35 00 wl 22 E 0 00 35 00 EI 23 P 17 50 35 00 wl 24 E 35 00 35 00 v Click Calibrate to calculate tip positions C D Cancel This panel needs no input from the user when automatic calibration is being used In this case the tip moves auto matically to each of the 25 grid points and an image is transferred automatically from the camera The size of this grid corresponds to the size of the calibration area selected in the first step of the procedure Since each image is saved it is displayed in the ca
55. 5 4 1 should be used to display the height profile NanoWizard AFM User Manual Version 4 2 57 Ji I Nanotechnology for Life Science Instruments d The Outline function makes the cantilever repeatedly perform a square or rec tangle movement around the edge of the scan region that is currently set during the outlining process the scanner is in piezo retracted mode In conjunction with an optical microscope or top view optics this feature helps to set a suitable area prior to scanning and the user can decide if the selected scan region is the right size or in the right location before approaching and imaging 5 1 2 TheData Viewer window Data Viewer Height trace The Data Viewer displays real time the image that is currently being scanned and also old scans from saved images using the Scan List see Section eee 3 2 8 Managing and saving v Show Manipulation Pattern E Se Multiple data viewer windows can be opened simul taneously to show data from different channels or ee with different settings Select Cross Section More options for moving the scan region and dis playing information can be found in the menu that appears when you right click with the mouse within the data viewer window see also Section 5 1 3 Channel Settings View Focus Line Viewer The controls on the bar at the bottom of the Data Viewer are used to set the selected channel the scale and orientation of the image view and t
56. 90 deg and Run Result good inflection point hits drive frequency Dap sseyd ul y307 In this example the drive frequency is 295 8 kHz and the target amplitude is 1 0 volt To image the phase contrast from the sample sensitively the inflection point of the phase curve must be at the drive frequency The phase curve can be shifted manually by adding a shift offset to the phase Enter a value between 180 und 180 deg in the textbox Phase shift By toggling Run or continuous the infinity symbol the new resonance Curve can be seen alal 100 1 0 80 0 9 60 0 8 40 5 H o eE 20 a e 3 a CH E 0 6 0 Fd D x 0 5 20 a ei 2e a 0 4 40 0 3 60 0 2 80 0 1 100 295 4 295 6 295 8 296 0 296 2 296 4 Frequency kHz Enter 90 deg and Run Result Inverted phase curve The color scale in the result ing phase image would be inverted Change the phase shift by 180 deg to 130 deg instead When it is well adjusted the phase decreases with in creasing frequency and crosses 0 degrees at the drive frequency Note that each experiment will require a different adjust ment the actual phase shift numbers here do not apply to each case Adjust the phase shift until the correct curve shape has been obtained NanoWizard AFM User Manual Version 4 2 75 Ji F f Nanotechnology for Life Science Instruments 5 6 3 Variation of the drive amplitude In intermittent contact mode the drive a
57. Advanced Imaging Settings Hover Mode Settings ki Oscilloscope ki Spectrum Analyzer p Calibration Manager Force Spectroscopy Force Scan Series List ye Force Spectroscopy Oscilloscope D Force Time Oscilloscope a Advanced Force Settings y Calibration Manager Force Mapping scan List yc Force Mapping Oscilloscope vt Force Time Oscilloscope a Advanced Force Settings ze Calibration Manager NanoWizard AFM User Manual Imaging Open Saved Image load AFM images into the scan list Scan list manage holding and saving of scan data Advanced Imaging Settings for scan speed and overscan Open the Oscilloscope window Open the frequency Spectrum Analyzer window Calibration Manager set calibration of height vertical deflection and spring constant Force Spectroscopy Force Scan Series List manage holding and saving of Force Spectroscopy data Open the Force Spectroscopy Oscilloscope Window Open the Force Time Oscilloscope Window Advanced Force Settings for spectroscopy rates and delays Calibration Manager set calibration of height vertical deflection and spring constant Force Mapping Scan List manage holding and saving of Force Maps data Open the Force Mapping Oscilloscope Window Open the Force Time Oscilloscope Window Advanced Force Settings for spectroscopy rates and delays Calibtion Manager set calibration of height vertical deflect tion and spring constant Version 4 2
58. Auto save window layout is enabled default is on then the positions and sizes of the internal windows are remembered when they are closed so they appear the same when they are opened again If it is off the layout can be saved directly with Save window layout now The default has Avoid opening frames turned on This means that the positions of windows are remembered when they are opened by hand but the windows are not automatically reopened when the software is started or the mode is changed The personal settings can also be deleted with Forget saved window lay out This resets all the saved settings but does not change any windows that are currently open The list of windows at the bottom gives the choice to bring any of the current ly open windows to the top level E coniguraton File Browser File Edit View Go Bookmarks Help al SI E lt ES rachel jpkdata configuration Places x d H rachel T Desktop ipgui user properties ipgui user frame EJ File System properties ES Network Servers E Trash Documents Music Pictures Videos 4 items Free space 338 4 GB Back Up Reload Home Computer Search The personal display settings are saved for each login account under username jpkdata configuration e a A 100 View as icons The files here store display and channel settings sav bi reien ing settings selections etc for both SPM and IP To reset everything to default thes
59. C mode 87 6 6 Managing and saving spectroscopy CUmves 87 6 6 1 FS MO ME 87 6 6 2 ONCE SCAN oae EE 88 OF FOCE M PDN E 90 6 7 1 IMtECGUCTION to Force Mapping secaanicuriociinrnie esine aaee a aT e 90 6 7 2 The Force Mapping Control poanel 91 6 7 3 The Force Map Oeclloscope 92 6 7 4 Data types and file SAVING BET 92 6 7 5 Pausing force mapping E 93 NanoWizard AFM User Manual Version 4 2 II Ji F f Nanotechnology for Life Science Instruments E SE e NEE 94 EROM CUD O WEE 94 7 1 1 Calibration Drocecdure 94 7 1 2 Hardware z linearization Height Measured 95 7 2 Sensitivity calibration ccc ceeeeeccccceeceaeeeeeceeeeeeaaeeeeeeeeeeecaaeeeeeeeeeseseeeeceeeeeesaeseeceeeeeseeeaseeeceeesseeeaeeeeeeessaaeeeeeeees 96 7 3 Spring constant calibration thermal NOISE measurement 98 7 3 1 General Wal OMAN le ecse o EE seeta neeossed sate S 98 7 3 2 Spring constant MEAS UTE CIM E 99 7 3 3 Using thermal noise to calibrate soft cantilevers in Tlud nn 101 8 Available Software Extensions cccccccsececeececeeeeceeeeceeceseeeesaeeesaueeeaeetseesaneeeas 104 61 Quanitative Imaging NEE 104 8 1 1 DAEN ENEE 105 8 1 2 The Quantitative Imaging Oscilloscope nnaannnnnnnnnennnnnnnnnennnnnnnnnennnrnrnnsnnnrrrrnnsrnnrnrrensennrrrrennne 106 8 1 3 The Quantitative Imaging and Scan control panel cece cccceeceeeeeeeceeeeeseeeeeseeeeeeeessaeeeeeeeessaaaaeess 106 81 4 e eer Ico die GE 107 8 1 5 Alia types a
60. EE h a Old Scans Show C Saving settings 62 There are often a large number of interrupted scans that would fill up the memory over time The Scan List is manages the saving and removal of images The image files are held in three levels Current Scan The scan that is still in progress is held in Current Scan This is the only scan with a yellow outline in the Data Viewer Recent Scans When an image scan has finished the file is automatically moved to the Recent Scans section of the Scan List Only a limited number of scans are held here older scans are removed automatically as new scans are finished The default number of scans is four but this can be increased using Number of listed entries If the scans have been saved either manually or with Autosave they are still stored on disk even if they are removed from the Scan List If they have not been saved they are lost permanently if they reach the bottom of Recent Scans Old Scans To store good scans for reference during the session they should be moved to the Old Scans section by clicking Hold Here they are kept until the software is closed or they are removed manually they will not fall out of the list Unsaved scans will still be lost if the software is closed An image will be sent straight to the Old Scans list if Hold is clicked while it is still scanning If the Direct Overlay feature is installed then imported optical i
61. FM User Manual Version 4 2 SA Setting up and approaching 4 1 Optical images 4 1 1 The CCD camera JUnicam Most NanoWizard AFM systems are equipped with a Firewire camera The same camera can be used iS with either an inverted optical microscope Life Science system or the top view optics standard system If using an inverted optical microscope with the optional DirectOverlay feature the optical image can be imported into SPM see Section 8 2 Simply connect the Firewire cable or USB cable from the AFM computer to the camera Consider video labelled Firewire USB ports The connection will automatically be recognized by the software Start the camera viewer with the CCD button from within the JPK SPM software icon bar The software for the optical camera is called JUnicam Further detailed information about JUnicam and software support for different camera families are given in JPK Software Integration for Cameras as separate manual 4 1 2 Optical imaging hardware Top View Optics The top view optical device for the NanoWizard standard version allows you to adjust the laser spot on top of the cantilever as well as helping you to navigate around your sample Start the camera viewer with the CCD button or Linux command as above The magnification by turning the wheel just below the steel ring that holds the camera e E en G CZ ER e Dis e i SN P bk a e Sr This is a CCD camera image low magnification
62. Help and Support Shut Down About GNOME JPK EI Ga El About Ubuntu Sometimes it is useful to take a screenshot from the current screen This can be done quite straightforwardly using the standard Print Screen key for the whole double monitor desktop or the key combination Alt Print Screen to make a screenshot of only the active software window as in Windows software A dialog appears showing a miniature version of the screenshot with options to set the name and location to save the file The default format is ong which uses lossless compression and can be opened in most software like jpg In the terminal console window the screenshot can be started directly from a command Furthermore a small pro gram to take screenshots can be open under Accessories Take screenshot This has the advantage that more advanced options can be added e g a time delay The basic command is gnome screenshot To get the list of different advanced options enter this gnome screenshot help gnome screenshot 9 1 3 Use JPK scripts with Linux console There are several JPK scripts which can be applied to the data Splitforcefile for instance separates a force file into trace and retrace curve Splitmapfile splits force maps into separate force files There are two scripts to convert force files of the jpk force format into other file formats The script jpk force legacy export creates out files and jpk force export
63. Index Selection Clear Selection Focus NanoWizard AFM User Manual The Data Viewer has the same function as in all other imag ing modes Please read Section 5 1 2 for detailed infor mation In QI mode there are four channels available Height Height measured Adhesion and Slope These four chan nels are online calculated from the force curves Please read Section 6 5 1 for more information The Height Height measured as well as Slope are calculated from the extend curves the adhesion channel from the retract curves The menu which appears upon right click within the Data Viewer contains basically the same options as for all imaging modes QI additionally provides the option Quantitative Image Map Single Index Selection This option allows a display of an arbitrary force curve of an pixel which can be selected by mouse click directly into the active image Sec tion 8 1 2 This is normally the current scan region Another previous image can be activated by clicking on the scan in the Quantitative imaging list Version 4 2 105 deift Instruments Nanotechnology for Life Science 8 1 2 The Quantitative Imaging Oscilloscope Quantitative Imaging Oscilloscope B x Vertical deflection Extend nN ao 8 10 14 Height measured amp smoothed nm 16 an LQ JLB Laj E St Display Analyze Horizontal axis SE x Max sl 22 a nm xmn gt
64. L second scan starts from the top position where the previous one finished SEN pra Kaes X Offset ka D Line 0 SC E Y Offset Scan Angle To start the first scan from the top of the viewer move the slider to the end position Advanced Imaging Settings here 51 1 and select the left arrow E Data Viewer Height trace Scanning from bottom to top of the Data Viewer window means that during the scan the slow scan movement is in the direction towards the cantilever substrate Slow scan direction Slow scan direction Scanning from top to bottom of the Data Viewer window means the slow scan movement is in the direction of the cantilever _ Slit scan direction Fast scan direction 1 Channel Settings View Focus Line Viewer 5 1 3 Selecting a new scan region zooming The JPK NanoWizard is equipped with state of the art hardware linearized xy piezos This means that xy positioning is very precise and that when a new scan region is selected the piezos will accurately move to the new region Selecting a new scan region and zooming in or out of images can be done by selecting an area with the mouse in the data view er or by typing the relevant offset and scan size values directly into the scan control panel Note It is not necessary to retract the cantilever from the sample when you select a new scan range The cantilever is automatically retracted from the surface during the move t
65. NA 0 35 WD 70mm SPM Software Release 4 2 09 2012 2002 2012 JPK Instruments AG all rights reserved deift Instruments Nanotechnology for Life Science Table of Contents 0 Safety instructions ANd warnings cccceccceececeeeeceeeeeeeeteeeesaueeteueessueeteeetsneetseeeas 6 0 1 Imporani SAICTY IMT OMIA ON EE 6 0 2 Warnings for the Life Science version with Optical mcroscopes 8 0 2 1 Prevention of condenser lens craeh 8 0 2 2 Prevention of Objective lens Crash 8 0 2 3 Prevention Of sample Crash 8 0 3 Other important INPON eege ee 8 0 3 1 NanoWizard 3 NanoOptics AFM SyStem c ccccccccscsesssssecesesesescecscevevevececevecevesereevevevevevavveverenereeveves 8 0 3 2 Glass DIOCK cantilever NOI TEE 9 0 3 3 Sample stage and Sample holder 9 0 3 4 Prevention of liquid spilling onto motor spindles ccccseeececseeeeeceeeeeeeeeeeecaueeeceaeeeeseueeesseeeesaaeees 9 lt 1 ee Keren Wo EE 10 See Ed i ee E EE 11 2 COMMON CUI EE 11 2 1 1 NanoWizard head and contre 11 2 1 2 BioScience and NanoScience systemS ennuennnnnennennnnnrensrrnrrnrenrnnnrrrresrnnrrrrensnnnrrrrrnsnnrrrennn 11 PRE EE e E 12 2 2 1 Location find a quiet place for the mstrument 12 2 2 2 MEMO COMME e E 13 2 2 3 AFM head Connecting eege 13 2 2 4 FO O E 14 2 3 Practical tips for using the NanoWizard c csesesecesesesescscevevececececececececsusevevevevevavavavecevenereevevevevevavvvenenenenenen 15 2
66. NanoWizard AFM User Manual Version 4 2 9 1 1 Diellet D EE 9 1 2 See e E e Bel tee TE EE 9 1 3 Use JPK scripts with LINUX console cccccecccccsseeceecceeecececausueeeeecaaueeeeeeseaeeeecaueeeessuaneeessssneeess 9 1 4 TSE account CIM ES Ure ll get aciaeetae ack adoesaceagetecs es a a EE saseeateaseanesoocues 9 1 5 NOWO dl e E 9 1 6 Bin E 9 2 Advanced SPM Software options nnannnnnenneenennnnnnennnrnsrnrnresrnrrrnsrrrrensrnrrnnnnrrnsnnrrrnnnntrrnrnrrrnnnrrrnnnrrenennneenn 9 2 1 EIERE eegen 9 2 2 EHNEN 9 2 3 foals gle grey 16 CN 9 2 4 NOH AGS OOS SINS ee suet aacuedmedsave sete deoeuclesceancesuct aaeeatesdsasesoudes 9 2 5 Pyihon and VACUO S ores toc ence oa pna cee Fuchedealgcaoe seach scitdusaensnbodasnaseasannesGachenatietsen veaeubesexeeuanaeseadeebegedteads 9 2 6 EE CNN E 9 3 Signal access module SAM setacncteceascabsoiesncnedebslactantnssedadabeins aduethcbadehaecsnteucedieebndsoens yadtaticdadsercecesenbadsbseescoheaens g4 JFR ddia LO BINNS sje ectcrce rete saree epee sve ee EEEE ARO EEEE E NanoWizard AFM User Manual Version 4 2 Ji I Nanotechnology for Life Science Instruments SU Safety instructions and warnings 0 1 Important safety information Laser safety warnings Infra red laser diode INVISIBLE LASER RADIATION AVOID EYE EXPOSURE TO DIRECT OR SCATTERED RADIATION DIODE LASER 1 mW MAX OUTPUT at 850 nm CLASS Ill LASER PRODUCT Red laser diode LASER RADIATION DO NOT STARE INTO BEAM DI
67. Nanotechnology for Life Science Instruments 9 1 5 Network settings A About Me SE To perform administration tasks such as network configuration log in Assistive Technologies as user jpkroot Bluetooth ai Broadcast Preferences Bice Use the main system menu started from the JPK icon in the taskbar Karte erger at the bottom left of the software to choose L Jpk Java 6 Policy Tool A Keyboard A j l AE Accessories gt ED Keyboard shortcuts System Preferences Network Connections el Games gt ao I Graphics gt Main Menu Ce Intemet gt Gg Messaging and VoIP Accounts Ss JPK gt Monitors m W Office gt Mouse othe N Ea Network Connections r Programming gt Gr Network Proxy SS Sound amp Video gt ef Open Dk Java 6 Policy Tool ON system Tool y E Personal File Sharing O System Tools Power Management ES Ubuntu Software Center Preferred Applications Remote Desktop Places gt oe G screensaver Administration gt Lock Screen sound Log Out lueders Help and Support Startup Applications Shut Down About GNOME A3 Ubuntu One JPK 2 GEI About Ubuntu Windows q s Network Connections x The Network Connections dialog contains all the settings for the S Wired Wireless Mobile Broadband s DSL network configuration Name Last Used In the first tab Wired Connections can be set up ethO or eth1 con eat n
68. ODE LASER 1 mW MAX OUTPUT at 670 nm CLASS II LASER PRODUCT Most users have a JPK NanoWizard equipped with a laser diode that emits invisible near infrared laser light wavelength 750 1000 nm Be extremely careful with such lasers since the blink reflex will not protect your eyes The NanoWizard head is provided with a tilt switch to turn off the laser automat ically when the head is in an upright position Use laser filters in optical equipment Some users have NanoWizard AFMs containing a class 2 laser diode with emission in the visible red part of the spectrum This may be harmful to your eyes Do not stare into the laser beam The emitted wavelength of the laser diode is 670 nm with an emitted power of 0 5 milliwatts Even with the glass block detached the emitted power of the laser remains below 1 milliwatt Thus the laser belongs to laser class 2 Additional notice for users of the Life Science version Users with the NanoWizard in the Life Science version must have a laser filter in the optical micro scope to prevent the laser shining into the users eyes The laser filter is fitted by JPK on installation and fixed in the housing of the binocular tube The filter in the top of the AFM head is also provided for your safety Do not remove the filters in the AFM head or optical microscope Note also that the laser beam will be present in the optical path through the side ports of the optical microscope Do not stare into
69. Programmers ISBN 073571119 recommended for those with experience of Java programming Advanced The Jython console is opened via Advanced JPK SPM Jython console E Open Script JPE SPM Jython Console Real Time Scan Logging Settings The Jython console can be used to run pre programmed macros from JPK so a detailed knowledge of Jython is not required for many operations gt To load the full set of macros provided enter the following command Advanced System Status F JPK SPM Jython console JPython 2 1 on javal 3 0 gt gt gt from jpktools import macros Type copyright or credits for more information gt gt gt from com jpk tools import macros This command imports the file macros py from the directory com jpk tools The currently loaded modules can be dis played using the command gt gt gt dirt Any macros copied to the file spmmacros py in the users home directory are automatically loaded when the Jython console is opened Note the first is part of the name and files starting with a are hidden files in Linux There are many macros available for different applications if you have a particular request please contact us on sup port jpk com and we may be able to supply a macro for your needs Only a few examples are listed here for the more commonly used macros NanoWizard AFM User Manual Version 4 2 149 Ji f f Nanotechnology for Life Scie
70. Run o DirectDrive Step 1 of 2 ES Next Cancel Automatic cantilever tuning For Automatic cantilever tuning the values of the drive and gains are chosen by the software to try and achieve the Target Amplitude that is set ja AC Feedback Mode Wizard Select a value of the Target amplitude default is 1 V Setup AC Feedback Mode ERES ERES Lajla Lea ES 140 150 DO 120 100 Click the infinity symbol to run continuously re peatedly perform sweep curves Click Full Range Y for a spectrum overview Lock in amplitude mV oa CH SE LI CH Gap aseyd ul 207 Red curve frequency Blue curve phase wkl Nf em 200 0 120 140 160 180 200 Frequency kHz Drive Ampl Drive Freq Start Freq End Freq Phase Shift Lockin Low Step 1 of 2 a Next Cancel NanoWizard AFM User Manual Version 4 2 41 Ji F f Nanotechnology for Life Science Instruments fg AC Feedback Mode Wizard Setup AC Feedback Mode with DirectDrive Laj aj Lej I JE Gap seyd ul 9307 E w ke 3 a E Ki S T x CO EI 282 0 282 5 283 0 284 0 284 5 285 0 285 5 283 5 Frequency kHz Drive Ampl Drive Freq Start Freq End Freq Phase Shift Lockin Low Manual Automatic Run a DirectDrive Step 1 of 2 a Next Cancel
71. Setup pull down menu to manage scan repetitions The default is set to Infinite Scans If Single Scans is enabled the system stops scanning after one scan and rests in idle mode Autosave Scan Repetitions Single Scans EN Laser Alignment Infinite Scans 2 Range V During scanning there is the possibility to select Stop After Current Scan to prevent the system from scanning further images after the current scan Stop After Current Scan 22 NanoWizard AFM User Manual Version 4 2 3 2 4 The Data Viewer The Data Viewer is designed for displaying data collected in Imaging modes It is also useful in many other measure ment modes for displaying locations of point measurments e g for Force Spectroscopy mode or line measurements e g for Manipulation mode relative to previously scanned images or optical images Therefore the Data Viewers are available in all measurement modes f Data Viewer Height trace The Data Viewer displays images that are currently being scanned plus old scans from stored images in the Scan List see Section 3 2 8 Multiple windows can be opened to show data from different channels or with different settings The Data Viewer also allows many scan controls to be ap plied graphically using the right mouse click menu For more details see Section 5 1 2 The controls on the bar at the bottom of the data viewer set the display options and are described briefly below Chan
72. TTL Output Pin 11 Initial Level Final Level LEI TTL Level Segment Pin 1 back Pin 1 back a Pin 5 Line Clock Pin 6 Pixel Clock Ramp Settings Baseline na l F Z Closed Loop _ Continue from previo e TTL Output Pin 6 Pixel Clock Coat ever D 2 Start Option Final Level r 14 TTL Level Segment NanoWizard AFM User Manual The TTL Output lists all available TTL Outputs Pin 3 Pin 6 can be used to switch between high and low state and to control TTL pulses All other available Pins are limited to a level change Pin 1 back and Pin 2 back can be addressed at the back of the controller The other Pins are located at the signal access module The Style LEVEL offers a switch between low and high state Therefore the Initial Level and the Final Level can be set at the TTL Output marked in red These values define the level state at the beginning and end of a force distance curve The TTL Level Segment itself leads to a change to the chosen Target Level The Target Level can be HIGH LOW or TOGGLE The current level state will be detected and compared with the Target Level In cases that the Target Level HIGH LOW is already reached there is no level change The TOGGLE option leads to a level change independently of the current level state Version 4 2 125 Ji F f Nanotechnology for Life Science Instruments Ramp Settings Baseline
73. The force constant bridges the gap between contact and non contact cantilevers IC mode is also pos sible with these cantilevers but it may not be Stable in air because of the surface adhesion Full Range Y Full Range X o 10 20 30 40 50 60 70 80 90 100 Select Frequency Range Frequency kHz Frequency Selection For the choice of cantilever it is important to match the spring constant to the particular sample Hard cantilevers i e cantilevers with a high spring constant can indent both soft and hard surface material thus reducing the contrast and potentially leading to artifacts from having the imaging force too high Soft cantilevers may not be able to indent either harder or softer features on the surface In general it is better to start with normal contact mode cantilevers spring constant around 0 2 N m and try another cantilever with a higher stiffness if there is no surface contrast NanoWizard AFM User Manual Version 4 2 Ji F f Nanotechnology for Life Science Instruments 5 7 3 Typical starting values Feedback Control For the imaging the Setpoint value is the average vertical deflection so the feedback gains settings is therefore similar to contact mode The oscillation of the cantilever provides extra amplitude and phase channels to observe differ ences in mechanical properties of the sample but is not used for feedback sl coja A high setpoint corresponds to a high tra
74. Time Motion Time Acceleration gpm d X movement e next pixel 1 If Use the same rate for extend and retract is enabled the Retract Sample Rate automatically updates if the Extend Sample Rate is changed and vice versa Extend Retract Speed Z Length QI Control Panel and Extend Retract Time depend on each other Update Time or Speed helps either to maintain the duration of a segment Constant Duration if the speed or z length is changed or to maintain the speed Constant Speed if the duration or z length is changed The Sample Rate determines the number of collected pixels per second It should be sufficiently high to detect all features within the force curves Reducing the Sample Rate helps to reduce the amount of data The relevance of Add Retract Motion Time Acceleration Next Line Delay and Next Line Retract is explained by the drawings below The Baseline function automatically corrects for vertical drift Please read Section 6 2 1 This sketch shows a plot of the piezo height green and x movement of the piezo cantilever blue over time during a QI measurement The round caps in the height signal between two force curves represent schematically the x movement from pixel to pixel The shape of these caps can be adjusted by Additional Retract Motion Time and Acceleration Reducing these values in creases the scan rate Generally the scan rate concerning the x direction is limited by the height contrast of the sampl
75. ackground Pattern If an image has been made with the same cantilever it is easy to select particular points within a scanned image v Show Manipulation Pattern Select New Scan Region Rotate Zoom Shift Measure Distance The coordinates of the points are shown in the X Y posi tion panel of the Spectroscopy Control window The coordinates are relative to the center of the 100 x 100 um scanner range not necessarily the current scan region Select Cro Of Spectroscopy Point Selection Clear Selection Focus 84 NanoWizard AFM User Manual Version 4 2 xX Y Position 14 923 15 834 21 823 26 119 Delete Clear New E E E Force Scan Repetitions X O Bingle O Number of Scans Infinite Go through XY Position list Repeat mode Repeat each point Force spectroscopy points can be activated by clicking on them in the X Y Posi tion section of the control panel The selected spectroscopy point is shown in green in the Data Viewer and is highlighted in the X Y Position list Double click on the table entry to type new values to set an absolute position By default the position of the tip is only changed when another point in the list is selected Run will repeat the movement indefinitely at the current point To move automatically from point to point use the Force Scan Repetition settings Either Single scans an arbitrary Number of Scans or Infinite scans can be r
76. ade or if the environmental changes are small For each Baseline measurement the Vertical deflection value is measured at the start of the force curve the furthest point from the sample The Relative Setpoint is now used as the change in Vertical deflection PI e on P wW M oF o d Baseline 263 7 mv In this example the baseline is being adjusted for each spectroscopy curve e Baseline 8 gt The Baseline from the last curve has been measured as 263 7 mV So if the Relative Setpoint is set as 0 2 V then the force curve will use the Vertical deflection value of 0 4637 as the turnaround point 82 NanoWizard AFM User Manual Version 4 2 6 2 2 Timing settings Extend Time Extend Time controls the speed of the movement on both the extend and re Ext Delay tract parts of the force spectroscopy experiment as they are the same in Basic Retr Delay force spectroscopy mode oz Jee The Retracted Delay is a waiting time at the retracted position far from the 2048 H2 surface e g to allow the sample to recover or to hold stretched molecules between the tip and surface This is always at constant height Z position Note that the Retracted Delay takes place between consecutive force curves and is not shown in the Force Time Oscilloscope or saved with the data Sample Rate The Extended Delay is a waiting time at the most extended position near the surface e g to wait for sample molecules to adsorb to the tip
77. after the administration tasks are finished Close 9 1 5 2 Static IP address allocation For static IP address allocation Connection name Auto etho Z Connect automatically In the IPv4 Setting panel Wired 802 1x Security IPv4 Settings IPv6 Settings Method Manual S ee Select the method Manual si nemaak ante oe aii Choose Add to set up the Addresses Now the IP address Netmask and Gateway address from your net ae work administrator should be entered in the relevant text fields Search domains The required DNS Servers or Search Domains have to be added from your network administrator Routes l Close the dialog with Apply to save the settings and return to the 7 Available to all users _ Cancel Apply Network Connections dialog NanoWizard AFM User Manual Version 4 2 143 Ji F f Nanotechnology for Life Science Instruments b Network Connections In the Network Connections dialog on the first tab Connections S Wired Wireless f Mobile Broadband VPN 3 DSL check that Ethernet connection is still selected Name Last Used Add Auto etho Sam Click Close to save the new settings and exit the Network Settings es dialog lete Remember to log out of the jpkroot account after the administration tasks are finished Close 9 1 6 Timer A simple electronic timer can sometimes be useful during experiments First this
78. all available TTL outputs TTL Control Pin 1 back and Pin 2 back are at the rear side of the controller Sub D 25 TTL Output Pin 1 back the signal access module Pin 1 and Pin 2 are level changer TTL Input female pin assignment These two Pins can be controlled with and without Style Level All other Pins can be accessed via the Digital out Sub D 15 female pin as signment at the signal access module Pin 2 back Style Pin 3 Pin 6 are able to change between high and low state and send puls e es The Style can be switched between LEVEL and PULSE Pin 3 ene Pin 6 offers furthermore the option to start a pixel clock via the TTL Control ES Panel During imaging and force spectroscopy a TTL pulse is sent at each aig tip position corresponding to a pixel in Style Pin 9 Pin 11 and Pin 13 Pin15 are able to change the Level and can be Level accessed at the signal access module Pin 1 back e The Style LEVEL offers the possibility to switch between high and low state Style LEVEL E Whenever the Level button is pressed a level change is done eae The Style PULSE offers the possibility to set pulses Two sorts of pulses are Style available The pulse time can be controlled Level Pulse Time iu IW Triggex Pulse After the button Trigger Pulse is pushed a pulse with the defined pulse time is created TTL signal pulses can be synchronized with AFM measurements via TTL cloc
79. also be chosen from old force mapping scans but the map must be selected in the Map List If no data is seen in the map oscilloscope then check that a map is selected in the Map List the selected map is outlined in green Display All Y Max M Y Min sl A e14 Uoi3elap ILIU A Color extend red Color retract red dark Show Current 7 Show Index Index Selection Index Comparison l Save Analyzed 6 7 2 The Force Mapping Control panel Force Mapping Control LIB Basic Advanced Rel Setpoint v 0 4 v Baseline na Adjust Baseline 1 z Length movement Ext Delay Retr Delay Delay Mode Sample Rate Z Closed Loop L Grid Square Pixels Fast Slow x Offset Y Offset Fast Points Slow Points Grid Angle Position AS Position Y NanoWizard AFM User Manual The Force Mapping Control panel contains the main settings for this mode The grid can be shown or hidden during the experiment The area for the Force Mapping experiment can be updated using the Data Viewer window as for normal images see Section 5 1 3 Tabs for Basic or Advanced mode offer different control settings for the force scan movement These Basic settings are explained in Section 0 For starting with Force Spectroscopy Maps Basic offers an easy tool to perform force Maps including variable approach delay and retra
80. an be used for the calibration manual ly transferring the images to the correct folder on the AFM comput er See Section 8 2 5 for the manual calibration procedure Once the image collection is started all the images will be saved automatically Therefore it is important to adjust the illumination and focus see Section 8 2 1 before starting mg Optical Image Calibration Size of the calibration area Width v Lon d Height v 100 0 The initial panel allows the user to select the Size of the calibration area default full piezo range This should be pm reduced for high magnification objectives The calibration region is always centered within the xy piezo range The Data directory for the calibration images and data is Data directory home kate jpkdata optcal 2006 11 06 16 07 07 e automatically generated with a date time stamp This can Automatic image acquisition also be edited Automatic image acquisition can be used for cameras with the JUnicam software This should be switched off for external cameras with separate software see Section 8 2 5 for manual calibration Click Continue to start image collection NanoWizard AFM User Manual Version 4 2 111 Ji f f Nanotechnology for Life Science Instruments Optical Image Calibration Collect Images Filename o optcal 00 jpg 35 00 35 00 1 optcal 01 jpg 17 50 35 00 2 optcal 02 jpg 0 00 3
81. ance peak is greater The phase contrast also depends on any static offset between the drive amplitude and cantilever response so the offset can be changed as described below to bring the phase in line with the amplitude resonance Phase 5 6 2 Adjusting the phase shift to improve phase contrast To get the best quality phase images the phase curve generally has to be shifted using the Cantilever Tuning win dow as explained below The Phase Shift is used to compensate for the uninteresting contributions to the phase difference between drive amplitude and the measured cantilever lock in amplitude so the images show the best con trast for the part that depends on the tip sample interaction The plots of the lock in amplitude red and the lock in phase blue are displayed for each value of the Phase shift 74 NanoWizard AFM User Manual Version 4 2 0 9 0 8 0 7 0 6 60 0 5 Lock in amplitude V 63p sseyd ul 307 0 4 0 3 0 2 100 295 4 295 6 295 8 296 0 296 2 296 4 Frequency kHz Initial phase and amplitude Phase shift is 0 deg 0 9 80 0 8 0 7 0 6 0 5 Lock in amplitude V Bap aseyd ul y307 0 4 10 10 295 4 295 6 295 8 296 0 296 2 296 4 Frequency KHz Enter 180 deg and Run Result bad inflection point does not hit drive frequency 80 KS 2 eee et 60 40 Lock in amplitude V 100 295 4 295 6 295 8 296 0 296 2 296 4 Frequency KHz Enter
82. and path the cur rently selected path is always displayed in green Manipulation Point Selection The mouse function can be changed with the right click menu to change the view in the Data Viewer Clear Selection Focus Channel Settings view d m E Data Viewer Height trace When a point in the path is selected in the Position list then it is shown highlighted as a green dot in the Data Viewer The selected point may be moved around using the mouse click and drag x Y Position 2 063 2 9 Channel Settings View Focus Line Viewer Delete Clear New 132 NanoWizard AFM User Manual Version 4 2 EI Data Viewer Height trace The single point is moved and the lines connecting it with the adjacent points can now be seen clearly x Y Position 2 405 2 342 Delete Clear New Channel Settings View Focus Line Viewer f Data Viewer Height trace More than one path can be drawn this example shows three paths A path can be selected from the pull down menu in the panel to the left of the screen The currently selected path is displayed in green and other paths are displayed in yellow Note for drawing straight lines press control key Ctrl and define start and end position Path 3 gt P Delete P Clear f New X Y Position Nr X in um Yi
83. and updated whenever this is changed For many applications such as electrochemistry or conductive AFM then another signal such as the current is used as an In put Channel There is a built in Voltammetry Oscilloscope for these applications using Axis 4 as the default input NanoWizard AFM User Manual Version 4 2 147 deift Instruments Channel Setup External channel Inputs Internal Outputs Testing Internal channel Nanotechnology for Life Science Enable Gated Photon Counter 1 Photon counter 1 a Gated Photon Counter 2 Photon counter 2 a Height sensor Height measured High Resolution 17 High Resolution O High Speed 13 High Speed 1 I High Speed 14 High Speed 2 A High Speed 15 High Speed 3 CJ High Speed 16 High Speed 4 F Precision 1 Vertical deflection Precision 1 Precision 1 Precision 2 Lateral deflection Precision 2 Precision 2 il Precision 3 Precision 3 a Precision 4 Photo Sum O Precision 4 Precision 4 Fi Precision 5 Tunnel Current EI Precision 5 Precision 5 Precision 6 Precision 6 id Precision 7 Precision 7 Il Precision 8 Precision 8 A Precision 9 Precision 9 L Precision 10 Precision 10 II Precision 11 Precision 11 EI Precision 12 Precision 12 a Approximate scan file size 1572 86 kb Approximate scan memory size 12582 91 kb X Voltage Output Settings Output Channels Axis 4 AxisS Axis G Axis 7 Axis 8 A External Hardware KPM or CAFM module Ou
84. ans e g for zooming into particular features on the cells the speed can be increased When scanning a relatively flat hard sample in air then the scan speed can often be increased to 2 3 Hz although note that the feedback gain values should also be increased so that the feedback reacts quickly enough For scanning small flat areas in contact mode for high resolution imaging for example when imaging bacteriorhodopsin protein crystals the scan rate should be increased further usually in the range 4 8 Hz Note that the full range of values to control the speed of the tip over the surface are found in the Advanced Imaging Settings panel It is useful to open the Oscilloscope window during scanning see Section 5 4 1 to show the trace and retrace data together On regions where the height of the sample increases the difference between the setpoint and the measured value of the feedback channel can become quite large so the feedback follows the surface even if the gains are too low although high forces may be applied if the gains are too low On regions where the height of the sample is de creasing the difference between the setpoint and the measured value of the feedback channel is limited by the free state of the cantilever and the feedback responds more slowly to the changes This leads to a difference between the uphill and downhill parts of the scan and this can be seen clearly by comparing trace and retrace Regions where the height i
85. are will recognize any sequentially named images in a basic graphics format TIF 8 bit JPG BMP or PNG Note that if a time series is used in the fluorescence camera soft ware it is often possible to export all the images automatically index Filename X um ue with a certain format and sequential names The files can be o O1 jpg 50 00 50 00 i A SS pone transferred to the AFM computer in any normal way for example 4 05 ip9 50 00 50 00 using an ethernet connection or USB device WinSCP is a useful 5 06 jpg 50 00 25 00 Ge 25 00 228 00 free tool that can help make the transfer convenient 8 09 jpg 25 00 25 00 9 10 jpg 50 00 25 00 e aT 30 00 0 00 Once there are suitable files in the Data directory the SPM soft 12 13 jpg 0 00 0 00 i i i 13 E 25 00 0 00 ware will automatically shows the names in the list The list dis PS b T ice Zeg SSC play is updated every time a new position is chosen with the 17 18 jpg 0 00 25 00 18 19 jpg 25 00 25 00 mouse or arrow keys 19 20 jpg 50 00 25 00 20 21 jpg 50 00 50 00 21 22 jpg 25 00 50 00 f i 23 jpg 0 00 50 00 If the file path is correct and the files are in the correct format the 23 24 jpg 25 00 50 00 SSES SH TRG 222r names will appear in the Filename column and scrolling through continue cancel the list will display each calibration image in the top panel From this point the procedure for selecting the tip reference position and calculati
86. at the cantilever is mounted in the AFM head at a 10 degree angle to the sample Sometimes when the fit range has been selected a red curve is not seen and this warning message may occur This means that the frequency peak or choice of fit range was not appropriate and either the measurement or the fit must be repeated A Function fit did not converge Please try again OK Note caution must be used when fitting values for the spring constant and the 50 50 fit must be compared with the curve to check it is appropriate The example here shows a bad fit curve that would not give a warning message but where the fit algorithm has found another peak that does not represent the cantilever resonant frequency average pm Hz LA CH LA Le ZH cud ep wu In this case you must select a better fit range 0 0 I0 153 20 25 30 35 40 45 50 55 Frequency kHz 7 3 3 Using thermal noise to calibrate soft cantilevers in fluid The most accurate spring constant determination using the thermal noise method requires that the calibration is per formed in air However this will not always be optimal The thermal noise detection method can also be used in fluid but there may be additional complications particularly with very soft cantilevers NanoWizard AFM User Manual Version 4 2 101 Ji F f Nanotechnology for Life Science Instruments Si es The spectrum to the left is representative of a soft 0 02 N m cantile ver
87. automatic or manual calibration the final image is automatically calibrated and imported into the SPM software It will appear in the back ground of all Data Viewer windows and in the Old Scans section of the Scan List In the Scan List the image behaves like AFM im ages i e the image can be displayed or hidden by clicking on the Show toggle Clicking the Remove button will remove the image from the Data Viewer An AFM scan area can now be selected directly in the optical image However as the initial selection of the tip location may be slightly incorrect it may be necessary to shift the optical image slightly to cor rect for the actual tip position Start an AFM scan low resolution 256 x 256 pix els is sufficient Select the optical image in the Scan List The optical image is now highlighted in green in the Scan List and plotted on top of all the images within the Data Viewer Click the right mouse button within the Data Viewer and select Shift Optical Image This will make the optical image transparent The semi transparent optical image can now be shifted using click and drag with the mouse Align the features of the two images To go back to the normal view of the AFM image click the right mouse button in the Data Viewer and deselect Shift Optical Image then go to the Scan List and click on the current scan After shifting the optical image should be resaved This will adjust the original calibration file to inc
88. back side of the cantilever the side that is usually hidden when lying face up on a gel pack Both sides of the cantilever should be the same shiny dark grey color If one side is a bright silver color this is usually the aluminium coating and the cantilever should not be used in liquid Silicon nitride cantilevers are almost always supplied with a gold back side coating because uncoated silicon nitride cantilevers would have almost no laser reflection In this case the gold coating is stable in water based liquids and there are no long term problems with deterioration of the coating There is however an increased temperature and pH or ionic strength sensitivity The different surface materials react differently causing a bending seen in the Vertical deflection signal For soft silicon nitride cantilevers it is important to reduce the variations in temperature and liquid conditions especially in contact mode Sometimes it can help to work in a liquid cell rather than a free droplet for instance In intermittent contact mode the amplitude is not as directly sensitive to these changes as the deflection in contact mode 38 NanoWizard AFM User Manual Version 4 2 4 3 Mounting the sample The most important thing in AFM microscopy is to avoid unwanted movement between the tip and the sample such as vibration drift or other movements due to unstable mounting of the sample Therefore the sample must be properly mounted in order to reduce vibra
89. cantilever is scanned The cantilever tip moves vertically towards and away from the surface As the tip is pushed against the sample surface elasticity values can be measured for indenta tion Moving away from the surface adhesion can be measured or the response can be measured for material or molecules stretched between tip and surface The absolute forces can be measured if the cantilever is calibrated i e the spring constant has been measured see Section 7 3 A simple way to become familiar with the Force Spectroscopy features is to perform force distance curves on a clean glass slide or mica surface in air using a contact mode cantilever with a moderate spring constant around 0 1 0 5 N m These samples are hydrophilic so the tip and sample are usually covered with a thin layer of water When the two surfaces are brought close together the water layers can form a capillary neck and there is strong attractive force These classic force distance curves show the attractive adhesion as the surfaces come together or are separated and also the strong repulsion as the tip is pushed against the hard surface 6 1 Overview of Force Spectroscopy mode 6 1 1 Introduction to the force spectroscopy settings t Force Spectroscopy wi Select Force Spectroscopy mode in the Measurement Mode drop down box On the left side of the main SPM window the Scan Control panel is replaced by the Spectroscopy Control panel with the parameters to control the t
90. ced Force Settings yp Calibration Manager 96 NanoWizard AFM User Manual Version 4 2 mw Calibration Manager Height ee enn Weg When the Calibration manager Lateral deflection UNKNOWN opens the latest force distance T curve is automatically loaded ma Accept value 4 The trace value of the Vertical Deflection is plotted in red and retrace in blue The full curve can be displayed in the plot window using Full Range X and Full Range Y A EIS uomaa gh ge E a ES a e KA EU a See It is often helpful to zoom into the Full Range X Select X Range steep repulsive part of the curve using Select X Range 0 8 0 5 0 0 0 5 1 0 1 5 2 0 2 5 Height measured and smoothed um Manual Sensitivity Selection Select Fit Range Only the steep repulsive part of the force 1 d curve is used for the sensitivity measure 1 Sensitivity Spring Constant gt 0 4 0 4 UNKNOWN ment A larger scale can help with choosing O nmiv vans ENET RE SS a good sensitivity value For Select X range AEPA Accept Value the new range is defined by clicking and Ses dragging in the plot area with the mouse 0 2 v2 2 Once the display is convenient for the par 2 2 ticular force curve use Select Fit Range H 0 4 DA S i and click and drag on the plot to set a region Bi E for a linear fit of
91. cience Instruments 7 Calibration 7 1 Height calibration 7 1 1 Calibration procedure Every z piezo needs calibrating This is necessary because the relationship between the applied voltage and the piezo length is not linear and suffers from hysteresis Users with z linearization have a separate Height measured channel which does not suffer from these problems Even so on flat samples it can be helpful to image with a calibrated z piezo using the channel Height instead of the Height measured channel For height calibration you need a stand ard sample with features of a known height These can often be purchased from cantilever manufacturers a range of different step heights are available It is recommended that a single user performs the calibration experiment and gives the calibration value to the others Due to aging of the piezo material the z calibration should be repeated around every 6 months First the calibration sample should be scanned and the step heights measured using the channel Height These values can then be used to calculate the scaling factor for the calibration Known SE of the sample Multiplier Measured height of the sample Note it is important to know the calibration used to make the scan of the calibration grid as described above Gen erally the image height channel will include the old calibration so the Multiplier value above will be a correction factor value should be around 1 that should be used to
92. cked these values are all displayed in Volts but the sensitivity es 83 00 Jnmfviluse it value is still stored for use later and saved with the files Note use it must be ois la PeT ticked to proceed to the thermal noise measurement of the cantilever spring con Stant as it is important to know the deflection in nm for this measurement S MA If for some reason you want to discard the measurement e g after changing the cantilever UNKNOWN can be reselected and the values will be disregarded Typical sensitivity values are in the range 30 100 nm V If the value lies significantly outside this range it may be worth checking that everything is set up alright see Section 4 2 for details of adjusting the optical detection path The Sensitivity measurement is the first stage in the thermal noise measurement of the cantilever Spring Constant 7 3 Spring constant calibration thermal noise measurement 7 3 1 General information Most suppliers of cantilevers deliver data sheets with their cantilevers that state the approximate spring constant Usually this has been calculated from the cantilever shape length width thickness The spring constant is very sen sitive to the thickness of the cantilever however and these quoted values are usually not very reliable The range of values quoted by the supplier should give some idea of the variability but generally the spring constant is not known within a factor of 2 or 3 98 NanoWizard
93. cking force like contact mode imaging Start with a setpoint of 1 V The higher the setpoint is the smaller the drive ampli E i tude should be to avoid artifacts Start with a drive amplitude of 0 1 V Drive Freq Phase shift The image quality can be optimized by adjusting the drive amplitude changing the force modulation drive amplitude during the imaging will influence the image quality of both the height and the amplitude images As a rule of thumb increasing the drive amplitude will lead to a bigger contrast in the amplitude image However sometimes a too high drive amplitude will lead to more artifacts Note that when the drive amplitude is changed it also might be necessary to readjust the IGain and PGain values This paper is one of the first where force modulation was presented it can be used as a reference for finding further papers and information about force modulation mode Using force modulation to image surface elasticities with the atomic force microscope P Maivald H J Butt C B Prat er B Drake J A Gurley V B Elings and P K Hansma Nanotechnology 2 103 105 1991 78 NanoWizard AFM User Manual Version 4 2 Sp Force Spectroscopy The Force Spectroscopy mode performs force distance measurements In imaging mode the force is held constant while the cantilever is scanned laterally over the surface In force spectroscopy measurements the lateral position is set at a fixed point and the z position of the
94. cquired with AFM and light microscopy shows that features in the two types of im ages do not have exactly the same dimensions In the case of images acquired with the JPK NanoWizard one can be sure that the dimensions are correct due to the precise positioning enabled by the use of linearized piezos The differ ence between the two images is due to aberrations arising from the lenses and mirrors of the light microscope With this in mind the DirectOverlay software uses the automatically recognized cantilever position to map the optical image and calibrate it before importing the calibrated image into the SPM software This optional feature can be used for online comparison of AFM and light microscopy images during imaging or for precise offline overlays 8 2 1 Image focus for optimal tip location Recognition of the cantilever position is used in the optical image calibration procedure This has a strong advantage that the transformation between optical image pixel coordinates and AFM scan coordinates is calculated using only the cantilever images so the colocalization of the optical and AFM sample features is independent information The only NanoWizard AFM User Manual Version 4 2 109 Ji f f Nanotechnology for Life Science Instruments exception is for the final shift of the optical image to correct for the unknown cantilever tip position All the magnifica tion rotation stretching and nonlinearity however are calculated solel
95. creates text dump files To run a script open the Linux terminal window 138 NanoWizard AFM User Manual Version 4 2 The terminal window automatically opens the user specific jokdata directory To apply a script the directory containing the script must be selected cd opt jpkspm bin and finally the script scriptname and the corresponding data which are to be processed by the script along with the storage directory are to be specified jokdata directory filename The script then finally processes the file and stores the new file in the same folder In case that all force files of the file name root Force jok force located in the directory jpkdata Test are to be converted into out files the corresponding com mands are to be entered thetmann pecis fopt jpkspm bin File Edit View Terminal Tabs Help tneumann pcec13 cd opt jpkspm bin A tneumann pce13 opt jpkspm bins j pk force legacy export jpkdata Test Force jpk forc If all jok force files of a directory e g Test are to be converted either jpk force i e without any filename root is to be entered i e all files of the jok force type are processed or only the directory containing the files is specified with no par ticular reference to files Wheumann pecia optipkepm pin File Edit View Terminal Tabs Help tn umann pce13 cd opt jpkspm bin tneumann pcc13 opt jpkspm bins jpk force legacy
96. ct settings More complex force maps can be performed under Advanced Different types of force segments can be strung together The Advanced mode is an available Extension and not in cluded in the basic software version These Advanced mode settings are ex plained in Section 8 Grid The scan region can also be set here using the Fast and Slow axis sizes and offsets as for images Usually however the region is set using the mouse in the Data Viewer as for image scans The number of points in the grid can also be set The minimum size is 8 x 8 spectroscopy points and the maximum size is 256 x 256 spectroscopy points Rectangular grids and grids with different spacing in the fast and slow scan direc tions are also allowed depending whether Square Pixels is enabled The total number amount should be considered in limiting the file size i e not just the grid points but also the number of data points per force curve Version 4 2 91 Ji F f Nanotechnology for Life Science Instruments 6 7 3 The Force Map Oscilloscope Display Analyze Horizontal axis Full Range X Autoscale XY Use linearizing A Range Vertical axis chi heehee chee Channel Vertical deflection e Display Y Max Y Min Full Range Color extend red Color retract red dark Loi Show Current C Show Index Index Selection Index Comparison Save Force Scan Save Analyzed In the Force Map Oscilloscope spectroscopy
97. cted syringe settings are used for the conversion from distance to volume Custom settings are saved Pump control H more than one pump is installed Syringe 3 then the option Couple pumps can i e used to Start and Stop tiow trom a gt be used to start and stop flow from all E ro ro the pumps simultaneously This is el alol release a d zl a lsen vf a EEN Wiehe inject Stop all the same volume should be injected with one pump and withdrawn using another pump The pump speeds are adjusted so that the total volume is constant WEEN g The Preferences settings are inter Stepsize for flow rates zl 60 00 a wen face controls that are used for all the Timebase for displaying flow rates minutes v pumps E configuration File Browser File Edit View Go Bcokmarks Help The settings for the pump control are Ss E a saved in Reload lome Computer Searck a 100 Q viewas icons lt nome user jpkdata configuration e E along with the general display set IPQuUI Userproperties ipgui user frame pumps properties sprrqgui user tings for SPM and IP The connec properties properties tion and syringe settings are remem B B bered as long as these files are not spmgui user frarne syringes properties properties deleted or overwritten by a new soft ware installation 5 items Free space 337 4 GB 4 6 3 Pump control for Aladdin pumps For Aladdin pumps the configuration has to perfor
98. ctive lens holder must not be rotated Note The revolver with objectives can only be rotated if it is completely retracted This is particularly important if the optical microscope uses any automatic move ment of the stage or objectives Please ensure that the automatic switching be tween objectives makes a retract before the rotation 0 2 3 Prevention of sample crash Be careful with objectives that have a short working distance When the objective is moved upwards it may lift up the sample and damage the cantilever The sample holder has a groove on the left side that helps to observe the objective sample distance during the movement 0 3 Other important information 0 3 1 NanoWizard 3 NanoOptics AFM system This manual covers the functionality of the NanoWizard 3 AFM standard system Customers with one of the Nan oWizard 3 NanoOptics version receive an additional manual explaining the handling and safety information Please read carefully this manual If you have any problems with your system please ask JPK for assistance 49 30 5331 12070 8 NanoWizard AFM User Manual Version 4 2 0 3 2 Glass block cantilever holder Note The flat top and bottom of the glass block are optically polished glass Only place these planes on soft tissue paper JPK recommends Kimberly Clark Kimwipes Lite 200 green box Do not touch the optical surfaces with twee zers or any other material that could scratch the surface and do not store
99. d AFM User Manual Version 4 2 8 4 TTL Control TTL transistor transistor logic devices are wildly used in lots of electronic applications and can be used to synchro nize the NanoWizard AFM with external hardware components like cameras and spectrometers A particular character istic of TTL signals is that they can switch between a low state below 1 V for a digital 0 and a high state above typi cally 3 3 V for a digital 1 in a high switching speed This is called level change A combination of a fast switch between low and high state leads to a pulse segment 8 4 1 Hardware configuration The Vortis controller unit for the JPK NanoWizard 3 can be equipped with and without a signal access module SAM The full range of TTL control elements can only be used with the signal access module E ee EN D JPK NanoWizard controller without SAM NanoWizard controller with SAM TTL Signal without SAM The access to the TTL signal is given at the back of the controller via a Sub D 25 female pin assignment marked in green The standard Vortis controller without signal access module offers the possibly to use two TTL outputs marked in red a 00000000000E90 000000000008 25 Sub D 25 female 14 Extension mixed signal Sub D 25 female pin assignment Pin Assignment 1 2 TTL level change Note The TTL outouts Pin 1 2 can switch between high and low state level changer but there is no possibility to g
100. d Change Add Delete Manage Groups Advanced Settings Help f Close The Create New User Editor lets you set up the particular options for Create a new user Short Name Q Short name must consist of gt lower case letters from the English alphabet gt digits gt any of the characters and _ the new account The first tab panel creates the name for a user account The Name will be the log in name for the new account Cancel Change User Password x Li Changing user password for test The Password section is very important Please choose secure Set password by hand passwords especially if the computer will be connected to the inter pen nem Confirmation Generate random password Password set to O Don t ask for password on login oa 2 140 NanoWizard AFM User Manual Version 4 2 Change Advanced User Settings x Changing advanced settings for JPK jpktest Advanced Wl Access external storage devices automatically dl Administer the system W Configure printers Connect to Internet using a modem C Connect to wireless and ethernet networks Monitor system logs C Mount userspace filesystems FUSE Send and receive faxes Cearc Groups settings x E SE em admin Properties PK jpktest audio avahi _ Delete avahi autoipd backup Asked on login bin cdrom couchdb crontab da
101. d Z speed form each other is shown Z Extend Rate Z Extend Time z Extend Speed Retract Rate Retract Time zZ Retract Speed HEET The sample number is displayed for extend and retract segments While extending 1024 While retracting 1024 Movement after Relative Setpoint is reached There are different modes available after soectroscopy is stopped The Z Scan End a uml first is to Return to starting mode Constant height mode maintains Time including delays for standard cycle the end position of the last force curve segment Retracted piezo Time for Scan 1 05 mode and Idle mode turn the piezo into retracted and approached Start spectroscopy settings mode respectively Start Option Extend first Settings after spectroscopy stopped End Option Return to starting mode MM 6 4 Selecting spectroscopy points By default the force spectroscopy experiments are performed in the middle of the currently selected scan area It is often useful to be able to perform spectroscopy experiments at particular defined locations 6 4 1 Point selection and the Position list f Image Viewer Height trace When Force Spectroscopy mode is active points can be selected directly in the Data Viewer window Right click in the Data Viewer window and choose Spec troscopy point selection The normal left mouse button can then be used to select points in the image window where spectroscopy curves will be performed Use Absolute Limits v Show B
102. d at the top until it is finished It is then sent to the Recent Force Scan Maps section Files here are only held temporarily the number of files to hold is set in the text field If the Hold function is used the scans are sent to the Old Force Scan Maps sec tion where they will be held while the software is open At any time in the Recent or Old sections they can be saved by clicking the disk symbol If the disk symbol has a red line through it the files are not yet saved permanenily If Autosave is enabled the files are all saved By right clicking on a particular entry the file can be selected saved or displayed on the Data Viewer Files are saved in ascii format storing all the force curves ina single file with the extension map txt S Old scans can be re opened from the disk When SPM loads the map file it calculates the slope and adhesion values to redisplay a force map image 92 NanoWizard AFM User Manual Version 4 2 Saving Settings oxXx v Use one folder for all data types home lueders jpkdata Browse l Image Scans Filename root Force Scans Force Scan Maps RealTime Scans Voltammetry MicroRheology Scans Voltage Spectroscopy STM I V Spectroscopy ve map Save with Scan Channel Extend Retract Pause Folder ome lueders jpkdata Browse Height v Under the Setup menu option Saving Settings controls the default setting
103. d using right click and Select fit range Click and drag in the plot area to define the data range for fitting The curve calculated for the fit is plot ted in red so you can check that the curves agree well In this example the fit algorithm has determined the resonance frequency of the cantilever and calculated its spring constant to k 0 0927 N m This spring constant k is a property of the used cantilever and should be in the range of the nominal value that is quoted by manufacturers The tilt angle of the cantilever is important as well SPM software takes into account of the normal 10 degree tilt see Section 4 2 1 and calculates the vertical component of the spring constant as a vertical k for the experiment There fore the used vertical spring constant is k 0 0956 N m in the example given Several other values are used for the calculation they can be found in the Settings panel e Temperature note that ambient temperature differences of a few degrees do not strongly affect the spring constant calculation e Cantilever Angle if an additional tilt angle is known 100 NanoWizard AFM User Manual Version 4 2 As for the Sensitivity see Section 7 2 vertical k must be accepted before it is used by the software The round radio buttons enable the user to use or forget a spring constant measurement as for the sensitivity When UNKNOWN is selected any value in the text box is disregarded When the text b
104. dow 72 5 5 Advanced gets ego E ue eames elm eo en ee Mer a aaa ink a ee ee ee eee ee 73 5 6 intermitent contact eege 74 5 6 1 Phase CONTAS erena a e a aaa a Ea aa a a EES 74 5 6 2 Adjusting the phase shift to improve phase contrast 74 5 6 3 Variation of the drive amplitude cccceecccccccesseeecceeeseeeeceeeseeeessauseeeesaaueeeeeseuseeesssaueeeessaneeessnsagss 76 5 7 Force modulation Bu Tee 76 5 7 1 ENEE We d EE 76 5 7 2 Off resonance cantilever tUNING ccccccseccccccsessececceeeeeececeeeeceeeseaeeeeesaeaueeeeseaeeeeseuaaeeeessaaeeeeessaaass 77 5 7 3 Kelte Bir lad ale Kr WE EE 78 US EE 6 een Renee Re Amen eR Lee eo en ec eee iene ee ne ee ve ae 79 6 1 Overview of Force Spectroscopy mode 79 6 1 1 Introduction to the force spectroscopy settimgs 79 6 1 2 The Spectroscopy Oeclloscope oaeen ipe iire i aar a a r R ipin adaueini 80 6 1 3 Force Time Oscilloscope ee 81 6 2 Basic force spectroscopy MOOS race cincnccesccisanecensicdadcnusesnceudeddusendcesesaendatueacdeastnebsannadiucondaesesseudenenaesudensuctasdasnnidns 81 6 2 1 EE EH 82 6 2 2 NII SE MUNG EE 83 6 2 3 ZNO SEG OOD EE 83 6 3 Advanced telen E 83 6 4 Selecting SPECtrOSCOPY POINS REENEN 84 6 4 1 Point selection and the Position ei 84 6 4 2 Spectroscopy Grid Manager asic deccenesnchucecunnsnrasssncncedisnsessanesessnesonueinnaedcebsadesodbinatancdaebspesonseeaneencebaadedect 85 EE 86 6 5 1 Analyze Adhesion and slope E 86 6 5 2 Force spectroscopy in A
105. drop down box This defines how the Z position of the cantilever is controlled during the measurements The list of available Feedback Modes depends on the controller type and also the software extension modules z The Measurement Mode is set with the second drop down box This defines k a imaging the kind of measurements or scans that will be made The set of available measurement modes depends on the Feedback Mode 8 2 4 2 4 2 2 4 4 2 The icons that appear after the drop down boxes depend on the choice of Measurement mode These are discussed in more detail for the individual modes 3 2 Introduction to the main controls 3 2 1 Feedback Control The Feedback Control panel always appears in the top left position in the software regardless of the measurement mode These settings define how the Z position of the cantilever is controlled during any measurements Feedback Control feedback modes there are multiple gains and setpoints NanoWizard AFM User Manual Version 4 2 The settings displayed here are explained in Chapter 5 IGain and PGain are part of the PI proportional integral feedback system The Setpoint value represents the force applied to the sample depending on the imaging mode See Section 5 2 for details on how to choose these parameters In some 21 Ji F f Nanotechnology for Life Science Instruments 3 2 2 Imaging settings Scan Control The next section of the control bar on t
106. e Leica Nikon or Olympus The AFM sample stage from JPK must match the optical microscope model The entire optical microscope and AFM can also be placed on an Note that the Life Science stages are specially shaped to fit a particular model of optical microscope and they are not interchangeable Optical microscopes from different man ufacturers have different designs and should only be used on the type of microscope they are designed for In each case the stage fits directly onto the optical microscope body with several screws These must be firmly attached to make a rigid connection between the optical microscope and the stage and to prevent unwanted drift or vibra tions The sample positioning for the Life Science stages can be manual or motorized The Life Science stages also can be removed from the optical microscope and placed directly on the anti vibration table so that the AFM can be used as a stand alone system or with TopViewOptics as for the NanoScience system NanoWizard AFM User Manual Version 4 2 11 Ji F f Nanotechnology for Life Science Instruments In the NanoScience system the JPK NanoWizard AFM is usually used on a special stage designed to sit directly on a workbench or anti vibration table CCD camera This is mainly interesting for non transparent samples where the inverted optical microscope is not suitable The sample can be observed in reflected light using the CCD camera with the TopViewO
107. e learned and used together with our software documentation on the NanoWizard computer file opt jokspm doc javadoc index htm As a next step the jython tutorial at www jython org can be useful 130 NanoWizard AFM User Manual Version 4 2 8 6 Manipulation and lithography Sg Manipulation Manipulation allows the user the flexibility to define paths over the sample surface that the tip will follow In imaging mode the tip moves back and forth EE Imaging in straight lines across the sample and in force spectroscopy the tip moves vertically over a specified point In manipulation mode the tip moves freely ae Force Spectroscopy across the surface along user specified paths Between the paths the tip Is i lifted from the surface so a series of disconnected lines can be drawn eee Force Mapping Le ch z Change to Manipulation mode using the Measurement Mode drop down Pf Manipulation box The chosen mode is bordered in green and displayed in the first line fe Voltage Spectroscopy The manipulation mode can be used for two main kinds of experiments e The tip can be used to move particles or molecules around on the surface nano manipulation e The tip can be used to draw a pattern across the sample e g scratching with a higher force nano scribing During manipulation it may be useful to calculate the applied forces to choose suitable values for manipulation of the surface or objects lying on the surface Often st
108. e sample The movement stops when the vertical deflection reaches the Rela tive Setpoint value 4 Retraction of the cantilever while the tip is still in contact adhesion Force 5 Tip is pulled free from the surface The movement stops after a total retrac Distance tion of Z length For the extend part of the motion the surface position is not necessarily know in advance The piezo extends moving the cantilever towards the surface and data is collected continuously Once the surface position is found the data for the last Z Length of piezo movement is stored as the extend part of the curve For the next force curve the expected starting position is used from the previous curve plus an extra safety distance 6 2 1 The Baseline function Baseline In contact mode the Baseline can be used to correct for changes in the verti na Adjust Baseline never cal deflection of the cantilever over time The cantilever can bend due to changes in the environment such as temperature pH or ionic strength in the liquid When Adjust Baseline is not being used set to never then the Rela tive Setpoint corresponds to the absolute value of the Vertical deflection never To use the Baseline feature just set the interval for how often the baseline should be measured in the Adjust Baseline drop down box If 1 is used it is measured before every force curve The interval can be increased if fast spec troscopy measurements are being m
109. e Calibration images taken already or a pre existing image taken be fore the calibration using Load Image file The final option is Take snapshot This allows the user to refocus the microscope adjust the contrast and then take an image to be imported The cantilever does not obscure the field of view as it is still positioned over the final point of the calibration grid The file is saved in the calibration Data directory with a filename Snapshot When the correct image has been selected click Finish to import the calibrated image into SPM NanoWizard AFM User Manual Version 4 2 113 deift Instruments Nanotechnology for Life Science 8 2 4 Managing and adjusting imported images in SPM fa Image Viewer Current Scan Show v Scan Region Recent Scans Number of listed entries w zi enen D ortica Image Channel Settings View Focus Line Viewer E Saving settings E Image Viewer Height trace Current Scan Show Recent Scans Number of listed entries Iz 4l a Show Scan 0 Use Absolute Limits v Show Background Pattern Show Manipulation Pattern how Optical image Select New Scan Region Rotate Zoom Shift Measure Distance B Saving settings Channel Settings Select Cross Section X Y Point Selection Shift Optical Image Clear Selection Focus fa Image Viewer Height trace 114 NanoWizard AFM User Manual At the end of
110. e and the mechanical prop erties of cantilever spring constant resonance and sample stiffness slackness stickiness e g probe sample interaction Movement to next pixel Force curve Note To avoid lateral forces during imaging the z length plus the additional Retract have to be larger than the height of the expected sample feature NanoWizard AFM User Manual Version 4 2 107 Ji F f Nanotechnology for Life Science Instruments 8 1 5 QI data types and file saving software Current Quantitative Image Recent Quantitative Images Number of listed entries Show 7 ll DEE 2 Ko A E Old Quantitative Images Show Quantitative Imaging List SEK The Quantitative Imaging List icon can be found on the main shortcut bar across the top of the SPM Managing saving and removal of images using the Quantitaive Imag ing List is complementary to the Scan List described in Section 0 Open the Saving Settings menu under the Setup pull down menu Data saving works similar as described in Section 3 2 8 Choose the register card Quantitative Imaging to set the channels for file saving 108 NanoWizard AFM User Manual Version 4 2 Saving Settings for Contact Mode C Use one folder for all data types home lueders jpkdata DNA Gitter 10_1_2012 HD Gitter2 Real Time Scans Voltammetry Temperature data Quantitative Imaging Voltage Spectroscopy _S LA Spectroscopy I
111. e anti vibration table They should be loose so no tension or torsion is transmitted to the head and the cables weight is not pulling on the head The firm connection to the anti vibration table removes differential movement between the head and the stage See Section 2 2 1 for more details 2 2 4 Power On Make sure that the main power switch on the back of the computer is on then switch on the computer at the front The Linux Ubuntu system will start automatically for more information about the operating system see Section 9 1 When the computer has booted switch on the controller with the switch marked in the images The upper image shows the front of the controller for the case it is equipped with a Signal Access Module SAM such as the Vortis Advanced SPMControl Station for more information about the Signal Access Module see Section 9 3 Controller with Signal Access Module P Standard Controller e E d The lower image shows the controller without SAM such as the Vortis SPM Control Station d The blue LED on the front of the controller shows the status e Off dark no power connection or the main power switch at the back is off e Blinking on off the power is connected and the switch at the back is on but the controller is still off e On stays lit controller is switched on and can be used After switching on the Control Station or pressing the reset switch the
112. e calculated using the JPK Data Pro cessing Software R Force Spectroscopy QI mode can be chosen in the Measurement Mode drop down 5 menu Select the Quantitative Imaging option zy Imaging It Force Spectroscopy Upon changing to QI mode the corresponding data viewers Data Viewer and Quantitative Imaging Oscilloscope as well as the sf porce napping Quantitative Imaging Control panel will open Ki Manipulation fer Quantitative Imaging vm Je Voltage Spectrostopy PERC Window The Quantitative Imaging pull down menu appears in the menu bar All necessary windows concerning QI mode can be opened from can List this menu Bs Eg Uantitative Imaging List ye Quantitative Imaging Oscilloscope e Advanced Quantitative Imaging Settings Before starting a QI measurement spectroscopy and imaging settings should be adjusted corresponding to cantile ver and sample as explained in the following sections Generally to start a measurement the Cantilever needs to be in approached to the sample idle mode 104 NanoWizard AFM User Manual Version 4 2 8 1 1 Data Viewer m Data Viewer Height measured trace 6 x Channel Settings View Focus Line Viewer Relative Color Scale v Show Background Pattern Show Manipulation Pattern Select New Scan Region Rotate Zoom Shift Measure Distance Select Cross Section Quantitative Image Map Single
113. e files can be deleted in the normal file browser when SPM and DP are closed New files will automatically be created with the default values the next time SPM and DP are started NanoWizard AFM User Manual Version 4 2 25 deift Instruments Nanotechnology for Life Science 3 2 8 Managing and saving data Channel Setup inputs Internal f Outputs Testing External channel Internal channel Gated Photon Counter 1 Photon counter 1 Gated Photon Counter 2 Photon counter 2 Height sensor High Resolution 17 High Speed 13 High Speed 14 High Speed 15 High Speed 16 Precision 1 Precision 1 Precision 2 Precision 2 Precision 3 Precision 4 Precision 4 Precision 5 Precision 5 Precision 6 Precision 7 Precision 8 Precision 9 Precision 10 Precision 11 Height measured High Resolution High Speed 1 High Speed 2 High Speed 3 High Speed 4 Vertical deflection Precision 1 Lateral deflection Precision 2 Precision 3 Photo Sum Precision 4 Tunnel Current Precision 5 Precision 6 Precision 7 Precision 8 Precision 9 Precision 10 Precision 11 There are many data channels that can be acquired simultaneously dur ing AFM imaging or other measurement modes The list of available channels depends on the type of controller and also the feedback mode that is being used External input channels from other devices can also be recorded using the Signal Access Module see Section 9 3 The user can decide which channels w
114. e scanner performs a single force spectroscopy measurement at the centre of every pixel Values for slope adhesion and height are calculated automatically from each force curve see Section 6 5 and these values are used to generate the image seen in the Data Viewer The adhesion and slope information is calculated from either extend or retract segments these are shown as height trace or height retrace channels for example in the Data Viewer The movement of the tip through the points is always the same this is not the same as the trace and retrace movement for imaging The force mapping exper iment is started at index 0 bottom left hand corner and proceeds back and L esa L seed ode forth Le left to right on the first row right to left on the second etc The current spectroscopy point is marked by a yellow frame 90 NanoWizard AFM User Manual Version 4 2 Force Scan Map Oscilloscope Vertical deflection extend V 50 0 50 100 150 200 250 Height measured amp smoothed nm The force curves are also displayed individually in the Force Scan Map Oscilloscope The force curve for each index can be observed during the scanning LQ e Lej I Display Analyze Horizontal axis xMax gt 350 4 nm X Min gt 50 a nm Z channel Height meas smth Vertical axis chi ch2 Channel ch3 ch4 Vertical deflection Index points can
115. e sure there is enough safety distance for the sample or tip movement If necessary move the motors up say 10 20 um to make a large movement and then re approach before performing the optical calibration System Status User kate Sum Fary Lateral Def 0 14 V Vertical Def 640 mV Status Z Piezo Retracted Saving off Accessories fi CCD Camera DirectOverlay Optical Calibration iS Import Optical Image Pump Control FluidicsModule Temperature Controllers Voltage Output Settings 2 gt ExperimentPlanner 8 2 3 Automatic calibration To check that the instrument is in the right mode check the system status window It will indicate whether the system is in Z Piezo Retracted mode If the system is in Imaging or Idle mode in contact with the surface just click retract once to sit at the top of the piezo To start the calibration procedure select the Accessories pull down menu at the top of the screen and then click on DirectOver lay Optical calibration There are a number of panels to lead the user through the procedure If one of the JPK supported Fire wire cameras is used with the JUnicam software then the images are automatically transferred from the camera to the SPM software which is the most conven ient method Other advanced cameras which have their own software and computer can also be used Any system that can acquire cantilever images and export them as regular graphic files TIFF JPG BMP or PNG c
116. ecorded when the Run button is clicked Go through XY Position list goes through all the positions in the list If Number of Scans is active and greater than 1 there are two settings for the measure ment sequence Repeat each point makes the set number of scans at each point before moving Repeat whole list makes one force measurement at each point before moving and goes through the point list the set number of times 6 4 2 Spectroscopy Grid Manager Reset Grid Shortcut to open the Spectroscopy Grid Manager to create grids of spectroscopy points The Spectroscopy Grid Manager allows a grid of spectroscopy points to be generated automatically This is similar to performing a Mapping experiment see Section 6 7 but the points can be measured independently and the force spectroscopy files are handled separately The settings in the Spectroscopy Grid Manager have the same definitions as for selecting scan regions The values are automatically initialized from the current scan area but new values can be set anywhere within the full piezo range Update Position takes the X and Y Offset position from the current cantilever position The sizes and offsets can be typed directly in the text fields and the grid will be updated when Set Grid is clicked The spectroscopy points list in the Spectroscopy Control panel automatically updates when the grid is set and points can be added or moved as normal If any positions have been changed
117. ection Bae Note eth1 is always the Ethernet connection between the AFM con troller and the computer It is strictly recommended not to change any adjustments here Click on Edit to configure the interface properties There are two types of network configuration dynamic IP address allocation DHCP or static This is decided by the type of local network Please contact your network administrator for advice on the particular settings required for your network 142 NanoWizard AFM User Manual Version 4 2 9 1 5 1 DHCP dynamic IP address allocation For DHCP dynamic IP address allocation Connection name Auto etho Connect automatically In the IPv4 Setting panel Wired 802 1x Security IPv4 Settings IPv6 Settings Select the method Automatic DHCP Method Automatic DHCP L t DHCP Ers only Close the dialog with Apply to save the settings and return to the Manual EE Network Connections dialog Shared to other computers DHCP client ID Routes vi Available to all users Cancel Apply Network Connections x 7 n the Network Connections dialog on the first tab Connections Wired Wireless 7 Mobile Broadband VPN amp DSL check that Ethernet connection is still selected Name LastUsed Add Click Close to save the new settings and exit the Network Settings a dialog lete i Remember to log out of the jpkroot account
118. egion directly e Scroll the mouse wheel in the plot area to zoom in or out in X e Shift scrolling the mouse wheel in the plot area zooms in Y e Click and drag with the mouse wheel in the plot area to shift the displayed region of the curve e The tools Zoom and Full Range XY from the toolbar can be accessed in most oscilloscopes directly from the right mouse button menu in the plot area 24 NanoWizard AFM User Manual Version 4 2 3 2 6 Increasing and decreasing values with the increment buttons Feedback Control IGain Pain setpoint Baseline Adjust Stepsize Change step for value Setpoint 0 002 Sat The SPM software contains many text fields where values for the control parameters are shown and new values can be directly entered The increment buttons up and down arrows are used to quickly increase or de crease the value of the text field by fixed steps using the mouse To adjust the step size for each text field e g here the Setpoint right click with the mouse on one of the arrows and select Adjust Stepsize The value can then be updated Note the keyboard shortcuts page up and page down can also be used to quickly change a selected value using these increments 3 2 Personal display settings Window Tile Vertically Tile Horizontally Cascade NI Close All Frames wv Auto save Window Layout wv Avoid Opening Frames Forget Saved Window Layout Image Viewer Height trace If
119. elpful tutorials and information on basic concepts such as K hler illumination phase contrast and DIC Here are a few examples from some large optical microscope manufacturers Olympus http www olympusmicro com primer anatomy kohler html Nikon hittp www microscopyu com tutorials java kohler index html 4 1 3 2 Interference of the optical microscope with the AFM system When the optical microscope illumination is switched on during the AFM experiment there might be some perturbations visible on the AFM image On flat samples especially perturbations that appear as narrow lines visible parallel to the slow scan direction may be observed typical frequency 300HZz This is due to the rectifier in the illumination power supply Therefore it is recommended to switch off the microscope illumination during measurements of small samples such as single molecules if this interference is seen 4 2 Set up the cantilever and optical detection system 4 2 1 Mount the cantilever The cantilever is fixed to a glass block which is locked into the AFM head during scanning The optical glass is inert against most chemicals but should be protected against scratches For clean ing advice see Section 2 3 1 The cantilever is first clamped to the glass block with the cantilever spring and then held fixed in the AFM head ready for scanning The glass block can be mounted removed from the head when it is in the upright position as shown in the schematic here
120. emon dialout dip disk Manage Gas Advanced Settings fax SS floppy fend fuse games ES Help Close Users Settings jpkroot Q Change ER null Account type Change o Password Change Add i Delete Manage Groups Advanced Settings Co Ces After the creation of the user profile it is required to click on the button Advanced Setting to check that the User Privileges are correct New user accounts have not the privilege to administer the system That means that the new account will have normal user rights but not administrator privileges This is more secure particularly when the computer is connected to the internet The Contact Information is optional for a small group of users this is probably not required The Advanced Settings should not be changed It is possible to control for each user the groups settings by using the option Manage Groups Make sure that Allowed to use the JPK SPM instruments option is ON The other settings can be chosen as required depending what is installed on the instrument computer At the end click Close to save the details of the new account Click Close again to exit the Users Settings and save the current Status Remember to log out of the jpkroot account after the administration tasks are finished The new user will then be able to log on with their new account NanoWizard AFM User Manual Version 4 2 141 Ji f f
121. emove icon just clears the image from the software memory in Old Scans It does NOT remove it from the hard disk if the file has been saved However if the file has not been saved it is impossible to recover it once it has been removed Note that the more images you store in Scan List the more memory is used Fu A Files with this symbol are not yet permanently saved to the hard disk At any time in Recent Scans or Old Scans the image can be saved clicking directly on the icon or with the right mouse button on the scan name number and select ing Save data H Once the file has been saved it will be displayed with this icon The Open file icon can be used to reload old scans from former imaging sessions If the DirectOverlay option is installed optical images can be opened and will appear in the Old Scans list The Show tickbox controls which scans from the Scan List are displayed in the Data Viewer window The order that the scans are plotted in the Data Viewer is set in the Scan List Scans at the top of the list are plotted over scans further down the list The Select function can be used to bring one of the scans temporarily into the foreground by clicking on the scan number or filename The scan entry will now be outlined in green and the image is shown in the Data Viewer with green axes In Old Scans the list order can be changed using the arrows If a scan is selected it can be moved up or down through
122. emperatures but other channels can also be saved The values of all the selected channels are saved every time a new temperature value is sent from the controller Therefore if the temperature is very stable the values may only be saved occasionally The temperature is usually a direct control so that the value entered in the panel is immediately used by the temperature device lt is also possible to vary the temperature over time in a controlled way using scripts These can be used for instance to run a temperature ramp over minutes or Many different liquid cell accessories are available from JPK most can be used with perfusion either by hand or using a syringe pump Often it is convenient to control the syringe pump directly from the SPM software This is possible for several supported models such as the KD Scientific Model 200 Series as well as various models from WPI fil CCD Camera DirectOverlay Optical Calibration computer serial connection Temperature Controllers Voltage Output Settings gt ExperimentPlanner Pump control gt Start all Stop all NanoWizard AFM User Manual ae Version 4 2 The interface for controlling syringe pumps can be opened in the Accesso ries menu The pump must first be connected to the power and to the SPM Before the pumps can be controlled the communication must be set up Use Configure pumps at the begin ning to configure the hardware set tings for co
123. ence on the tip movement File name square svg Open Files of type SVG vector graphics Cancel C Image Viewer Height trace C Image Viewer Height trace Channel H Settings H View Focus H Line Viewer Channel Settings H View H Focus H Line Viewer 136 NanoWizard AFM User Manual Version 4 2 9 Advanced information 9 1 Ubuntu Linux information The Ubuntu Linux operating system is relatively intuitive to use and many features and settings can be found easily by people used to the Windows operating system Ubuntu Linux update The Ubuntu operation system is modified for the AFM operation Please note that a simple Ubuntu update is not possible Contact us for assistance if you plan to up date E mail support jpk com or call our technical support line on 49 30 5331 12545 9 1 1 Ubuntu updates When logged in as jpkroot your Ubuntu operating system will frequently remember you to make Ubuntu updates Mak ing security updates is ok but please refrain from making so called LTS updates until further notice LTS updates are clearly labeled as such in the Ubuntu update manager It may happen that SPM and IP cannot be started anymore after an LTS update was made 9 1 2 Basic tools and programs The taskbar along the lower left hand side of the desktop contains most of the shortcuts that are regularly required Internet browser usually Firefox C
124. enerate automatic pulses NanoWizard AFM User Manual Version 4 2 121 Ji f f Nanotechnology for Life Science Instruments TTL Signal with SAM The access to the TTL signal is given at the signal access module of the controller via a Sub D 15 female pin assign ment marked in green Next to the switches between high and low state automatic pulses can be generated Fur thermore TTL pulses can be synchronized with AFM measurements through pixel line and frame clocks Extension mixed signal Sub D 15 female pin assignment 8 1 oo0oo0 oo0 o0 oo0 00o o 000000 0 Pin Assignment 1 2 ground no TLL output 3 TTL level change pulse 4 TTL level change pulse frame clock 5 TTL level change pulse line clock 6 TTL level change pulse pixel clock 7 9 ground no TLL output 9 11 TTL level change 12 ground no TLL output 13 15 TTL level change 8 4 2 TTL control Panel The TTL control Panel offers the possibility of controlling and monitoring independently from the chosen mode imag ing or Force spectroscopy TTL signals Autosave scan Repetitions V Laser Alignment Range z Z Piezo Display To start the TTL control Panel select the Setup pull down menu at the Approach Parameters menu bar El Saving Settings BC Channel Setup a Advanced Feedback Settings lt TIL Control 122 NanoWizard AFM User Manual Version 4 2 The TTL control Panel offers an overview about
125. er motors Note that the leveling must be set to OFF to see the overall tilt of the curves The position of the motors is shown on the Stepper Motor window The main pair of up and down arrows on the right control all three motors together The three pairs of arrows in the center control each motor independently Increasing numbers in the display represent the approach of the AFM head towards the sample decreasing numbers represent the movement of the AFM head away from the sample The counter can be reset at any position for example they can be set to zero at the sample surface to provide a refer ence for the sample height and aid re approaching later Note however that this means they are not an absolute val ue and the motors can all read zero when the head has a significant tilt for example To obtain reference values move the head up as far as possible until all three motors are at their highest point and then zero the counters The head is now level so if all the numbers are equal the head should be level with respect to the stage Note for the NanoWizard I heads never move the motors down to their lowest point In this case the motor spindle may stick inside the head NanoWizard AFM User Manual Version 4 2 69 Ji 8 f Nanotechnology for Life Science Instruments If the AFM head has to be tilted it is better to tilt it as shown in the right image This is to avoid the cantilever spring touching the sample as t
126. ersion 4 2 2 Ji F f Nanotechnology for Life Science Instruments 3 3 2 SPM software versions About JPK NanoWizard Control If you contact JPK for assistance concerning NanoWizard dell ER the software it is helpful to know which ver DirectOverlay Control Software v 4 See mE PENNER sion of the SPM software you are using FluidicsModule SE ERGER From the main drop down menu at the top anipulation MicroRheology i iere d ET STGO of the window open Help Info to see the eieiei version number bottom left corner Piezo Response measurements Pump Control 1999 2010 JPK Inst ts AG all rights ed Jk i r i NanoWizard dees liesen for force mapping The text on the right also shows the list of FREES EAE Scanning Tunneling Microscopy A A SEANG Available Extensions This defines which Tuning Fork JPK S EE options will be available in the software peta controls will be enabled depending which Version 4 0 44 gt Instrument number VTC modules are installed L If you have a software problem it helps to have as much information as possible about the circumstances Any errors or warning messages are automatically written to a log file in the data directory user jpkdata The file name includes the date and time the software was last started e g som 2010 05 27 21 16 43 log It will help us to respond quickly to your requests if you send us the file as an email attachment 28 NanoWizard A
127. es in trace extend and retrace retrace true true Note TTL clocks are synchronizing AFM measurements and TTL Pulses A TTL clock starts when the AFM measure ment is started by pressing the RUN button Pin 6 Pixel Clock WM ae LS The Pixel Clock is the only TTL clock that can also be activated directly by eve Pulse Time Se using the TTL Control Panel Trigger Pulse 8 4 3 TTL control with Force RampDesigner TTL signal can be also used during force spectroscopy and can be controlled in an elegant way with the Force Ramp Designer The Force Ramp Designer is an available software extension and is provided together with the advanced force spectroscopy option For more information concerning Force RampDesigner see Section 8 3 Spectroscopy Control lf Advanced mode is selected in the spectroscopy control panel the Force sass Ab Ramp Designer opens automatically Basic Advanced Seton j Re Next to the common six force segments a TTL segment is available to control the TTL segments in a user defined way during force spectroscopy 124 NanoWizard AFM User Manual Version 4 2 TTL Level Pulse segment to control TTL signals within a force distance curve The TTL segment can be handled like the other 6 force segments see Section 8 3 Force RampDesigner SAA E pong Settings Baseline n a Adjust Baseline 1 Z Closed Loop Z Start Option
128. ething of a mixture between contact mode and AC mode basically contact mode with an added oscillation of the cantilever This provides qualitative data about sample stiffness alongside the normal in formation about sample topography In Force modulation mode the cantilever is modulated at a relatively low fre quency during imaging using the average Vertical Deflection as the feedback signal and Setpoint as in contact mode The cantilever is always is in contact with the sample it does not lift off the surface as in AC intermittent con tact mode The force between force and sample is continuously varied by the drive amplitude and the lock in ampli tude shows the cantilever sample response On softer features the cantilever will indent further into the sample leading to a small amplitude corresponding to a low slope in a force distance curve Hard features only allow little indentation and lead to larger deflection amplitudes corresponding to a high slope in a force distance curve In principle force modulation mode can be seen as a con tinuous force spectroscopy experiment in the repulsive regime during a contact mode imaging experiment During imaging the system effectively measures the slope of the force distance curve cantilever Amplitude at a given con tact force Setpoint over the image 6 NanoWizard AFM User Manual Version 4 2 The pair of images here show an ex ample of height and amplitude images of a polymer blend imaged
129. export jpkdata Test In case that reference to the files is used to convert files i e jok force or filenameroot jpk force is entered there is one problem that can occur if a huge batch of curves is to be processed The filename wildcards are expanded before the command is executed That means even if file extension is entered which means all files in the given directory with exactly this extension are processed the script appends one file with the complete filename after the other And since there is a limit on the allowed length of the expanded command line the script will abort if this limit is exceeded In such case the directory alone should be specified rather than the filenames pk legacy export jpkdata experiment jok force gt wildcards limitation pk legacy export jpkdata experiment gt unlimited number of files For more information about these kinds of scripts please contact support jpk com 9 1 4 User account administration The main administration account in the Ubuntu AFM systems is jpkroot The password is delivered with the AFM installation papers and this account can be used to set up other standard accounts for all the individual AFM users ff Accessories To perform administration tasks such as installing new software el Games a Computer Janitor ie Tee versions and creating new user accounts log in as jpkroot User ae Ri Hardware Drivers accounts created using
130. for instance to approach more carefully on a delicate sample or with an extra sharp canti lever reduce the gains in the approach window This can also help if the feedback rings during the approach Alternatively reduce the setpoint force decrease the setpoint in contact mode or increase it in amplitude mode To make a faster approach the gains in the approach window can be increased Alternatively the setpoint force can be increased increase the setpoint in contact mode or decrease it in intermittent contact mode Note that the gains only affect the approach Change the values in the Feedback Control panel to adjust the gains used for scanning and waiting on the surface The velocity must be adjusted in case that Approach with constant ve locity is used In this approach type it is possible to make very sensitive approach to protect the cantilever The Approach Target Height sets the z piezo height on the surface at the end of the approach The default puts the piezo in the middle of the current z range to give maximum piezo range above and below the starting value Approaching onto glass beside a cell for example the Target Height should be set around 2 3 microns to give maximum range above the glass for the cell There is also an option in the Approach Parameters to approach using a variable setpoint using the Baseline Adjust please see Section 4 5 4 To confirm a successful approach check the following settings
131. g the Vertical deflection here in blue can also be ao 81 82 s3 64 85 66 s7 plotted using the tabs on the left of the spectroscopy oscilloscope Height measured um Near the surface the vertical deflection increases as the lock in amplitude red decreases because of the damping of the cantilever oscillation The hysteresis in the amplitude curve can be seen from the vertical deflection channel to be caused by the adhesion of the tip Note the Slope and Adhesion values are calculated from the feedback channel In AC mode this is the amplitude so the Analysis function is switched off for the AC mode force curves as the numbers are not relevant Vertical deflection extend V A Dena Uomeuep JLNUA 8 0 8 1 8 2 8 3 8 4 8 5 8 6 8 7 Height measured um 6 6 Managing and saving spectroscopy curves 6 6 1 File saving The force curves produced during force spectroscopy mode are written in a compressed binary file format file names filename jpk force so they cannot be read into normal mathematical or spreadsheet processing software programs Force curves can be converted into simple text using the JPK Data Processing software either individually or as a batch for a whole folder of files Alternatively a script can be used to convert a folder of force curves to this text format After obtaining a force spectroscopy curve the data can be saved using Save Force Scan in the Spectros copy Oscilloscope Using Autosave each co
132. h make sure that the laser is properly aligned on Vertical deeler the cantilever and photodetector as described in Section 4 2 The Sum e signal should be around 1V or above and the Lateral and Vertical Deflec Lateral deflection tion should be near OV The correct imaging mode should be selected on the shortcut bar at the top of the window A suitable setpoint value should be set in the Feedback Control panel In modes using an oscillating cantilever this is chosen using the Cantilever Tuning window as described in Section 4 4 2 In Contact mode a value should be chosen depending on the vertical deflection on the photodiode and the spring constant of the cantilever A value of around 1V should be reasonable for a soft cantilever if the vertical deflection is around OV Note that in oscillating modes where amplitude is used as the feedback channel a lower setpoint value means a high er imaging force while in contact mode a lower setpoint value means a lower imaging force Click the Approach button in the icon bar to start the approach routine and you should hear the stepper motors move the NanoWizard head 46 NanoWizard AFM User Manual Version 4 2 Range gt D um 13 000 um l5 um Extended Retracted Approach Parameters Approach Type 2 Approach with feedback on Approach with constant velocity Baseline Adjust No baseline adjust Baseline update at start Dynamic baseline update
133. h the Sum in the Laser Alignment window as the adjustment screws are turned When the detector is mov ing in the right direction the Sum should gradually increase In the wrong direction the Sum will decrease When the laser spot is fully on the detector and the Sum value does not increase any further the Vertical or Lateral deflection should reach the central 0 V position Note do not over wind the screws for the detector or laser f this procedure does not work for Lateral deflection then the cantilever is probably damaged If this procedure does not work for Vertical deflection first move the detec tor positioning screws back to the middle of the range then adjust the mirror as described below and in Section 4 2 4 Note also that if the cantilever is damaged or dirty it may also prevent the reflected laser beam reaching the photodi ode If other changes do not work try exchanging the cantilever for a new one of the same type Check also for dirt or scratches on the surface of the glass block this can also interfere with the optical path Focus on the glass block surface with the top view optics or inverted optical microscope in this case optical phase contrast can be very useful to check for dirt or scratches above the cantilever 4 2 7 3 Unstable Vertical or Lateral Deflection values Sometimes the initial alignment seems successful but afterwards there are problems that the vertical or lateral deflec tion values change a lot I
134. have such g a clear linear region 0 2 0 2 S 5 3 In this case when the mouse is used to click ace o4 2 and drag on the plot area a straight line is plotted between the points black line in left rre E Ke panel The slope of this line is used for the FullRangex Select X Range pe Ke sensitivity Select a best fit straight line eege ZE through the steepest region of the repulsive Manual Sensitivity Selection Select Fit Range part of the curve Sensitivity Spring Constant When a line is drawn or fitted the slope is used to calculate the sensitivity value Bessen 3 00 D shown in the left hand part of the window here s 34 1 nm V The value is not Eesgeeeesge used immediately since the user may wish to redraw the line When you are s n a Accept Value happy with the straight line fit click Accept Value to store and be able to use the sensitivity value Sensitivity i Spring Constant O UNKNOWN A sensitivity value can be stored with the scan or force data without actually being 1 Sopp use it used directly in the SPM software to display the deflection values in units of length ee ne aaa all rather than Volts When use it M is ticked then wherever the vertical deflection is Le 5 Ad used in the software contact mode setpoint intermittent contact mode amplitude Sensitivity Spring Constant force spectroscopy axes etc the value is now displayed as nm rather than V UNKNOWN When use it is unti
135. he head approaches Not recommended Better in this direction 5 3 3 Automatic Motor Leveling As described above it is possible to adjust the AFM head manually to take account of some sample tilt by moving the three head motors independently It is also possible to calculate and carry out the movement automatically based on the tilt of a scanned image Remember though that the tilt range is rather limited by the geometry This correction is effective in correcting for small angle differences from sample mounting but not intended to apply large tilt angles to the scan head Stepper Motor Motor Leveling EE Scan List The movement should be calculated from an image that has just been scanned EE This should be selected in the Scan List and will be shown in the Motor Leveling window display Check that the correct image is selected and shown with a green Motor Leveling can be opened from the main Motor menu Recent Scans border Number of listed entries aal The tilt angle can be determined better from larger images Therefore larger scans more than 10 microns are recommended and it is better to finish the scan so that the angle is measured well in both scan directions The pixel number or reso lution is not so important so the scan can be done with fewer pixels if required for speed If very small scans are used the error will be higher and there is more chance that the angle is only local and not representative of t
136. he Conversion factor can be entered here For instance if the gain is set to 1 micro Ampere per Volt the conversion can be typed in as 1e 6 This will automatically be updated to show the current in the correct units if use it is ticked Version 4 2 E Voltage Oscilloscope EI 10 0 Display Channel Saving a The Voltammetry Oscilloscope or Sal Fs LG 2 ra Voltage Oscilloscope is opened from 9 0 ontro Sample Freq el 100 0 a e the settings panel 8 0 Time to display y 10 0 a s Start Autosave 7 0 Horizontal axis X Max M 15 v 6 0 X Min M 15 v Axis 4 E za Vertical axis If the Conversion is set and used then chl ee 4 0 Channel OFF Axis 4 will be calibrated Y Max sl 10 0 a 3 0 Y Min sl 0 0 a ee S i E This is an analogue of the Real Time i Scan see Section 9 2 2 designed to i plot channels against each other as a 0 0 function of time 10 E 0 5 10 15 Ss EAN SE 9 2 5 Python and macros There is a very good online tutorial on www python org Within very short time this language can be learned and used together with our software documentation on the AFM computer file opt jokspm doc javadoc index html As a next step the jython tutorial at www jython org can be useful For a deeper insight these two books can be rec ommended Markus Lutz Programming Python ISBN 1565921976 Robert W Bill Jython for Java
137. he colorizer settings Channel Trace Trace si Other channels apart from the height can also be displayed The channels available depend on the imaging mode and on the Channel Setup which can be selected from the main Setup menu Each channel can be displayed as either trace or retrace Settings Leveling Limit Multiplier ___ Offset ER Leveling The image can be leveled by subtracting the background from each scan line none linear quad ratic GI order cubic 3 or quadratic 4 The average value or curve is subtracted from each line inde pendenily the higher the order the more small features in the image will be highlighted This only affects the online display the full image information is always saved for later analysis 58 NanoWizard AFM User Manual Version 4 2 Limit Allows the user to vary the color scale settings through a choice between Statistics or Min Max Statistics Sigma is the statistical width of the image range of data values This is the unit for the automatic colorizer settings o Multiplier is the factor that is applied to scale the width o of the color scale Offset gives a shift in the centre position Min Max Auto Manual The lowest and highest values are read The lowest and highest values can be automatically set manually View Angle Degrees a l mm 180 135 90 45 0 45 90 135 180 The display can zoom into parts of the image in the viewer using the
138. he controller is switched to OFF and then connect the black power supply cable for the controller Connect the head cables to the NanoWiz ard head see below and the network cable to the lower network plug of the computer Now the controller can be switched on Note that the power switch for the controller is on the front of the controller box special unused The other connections are not necessary for standard use Note Please avoid storage of heavy or electric equipment on the controller for an optimal performance 2 2 0 AFM head connections In the picture above of the back of the controller box two cables can be seen which connect to the AFM head Please do not strain these cables NanoWizard AFM User Manual Version 4 2 13 Ji f f Nanotechnology for Life Science Instruments On the right hand side of the AFM head are the two sockets for the controller cables Check the plugs and sockets carefully there is only one way to connect them Do not force the connectors Note Always check that the controller is switched OFF before connecting or disconnecting the AFM head Note that the placement of the head cables can be very important for taking high resolution images Try to eliminate all sources of vibration from the AFM head Vibrations can be transmitted via air via the table and via the two cables which connect the AFM head with the controller We recommend taping the two head cables to fix them to th
139. he left of the software depends on the measurement mode Imaging is the default measurement mode and the Scan Control settings are described here Scan Control mja e Line 0 Fast The icons in the upper part of the window are introduced briefly below and further discussed in Section 5 1 1 The parameters displayed in the lower part of the Scan Control window are also explained in Section 5 1 1 They are used to change the scan size and position offset the speed of the tip over the surface and the number of pixels slow captured in the images Line Rate Pixels x Offset Y Offset The Advanced Imaging Settings panel can also be opened directly from here for more advanced imaging parameters See Section 5 5 for more infor mation Scan Angle Advanced Imaging Settings Open a new Data Viewer window see Section 5 1 2 The zoom symbol activates a new scan area that has been drawn in the Data Viewer using the right mouse button option Select new scan region See Section 5 1 3 Switch between normal image scanning and Line Scanning mode In Line Scanning mode one scan line is scanned repeatedly rather than moving to the next line to form an image See Section 5 1 1 Switch between normal image scanning and Outline scanning mode to get an overview of the sample area using an optical microscope or top view optics See Section 5 1 1 OO H L 3 2 3 Scan Repetitions Choose Scan Repetitions in the
140. he movement Check the calculated motor movements to see that they are sensible before clicking Start leveling Always try to avoid the stepper motors reaching the end of their range Once Start leveling is clicked the movement will start first the motors moving away from the surface followed by the motors moving towards the surface After the movement re approach to the surface to start imaging The Oscilloscope icon in the shortcut bar opens the oscilloscope window directly It can also be opened from the pull down menu View in the menu bar The Oscilloscope window can be used to display the present line scan of several channels simultaneously Both trace and retrace information can be displayed together which can help with optimizing the imaging settings see Section 5 2 for example Up to four channels can be displayed in the same window all with both trace and retrace Any chan nel can be displayed so long as it is switched on in the Channel Setup see Section 3 2 8 The Line Viewer that is located in the Data Viewer window see Section 5 1 2 gives a simplified oscilloscope func tion In the Line Viewer the leveling settings are taken from the Data Viewer but in the Oscilloscope the leveling can be set independently The same scan lines may look different in the oscilloscope and line viewer if different leveling settings are chosen DI Oscilloscope Height trace nm P P 0 5 dE 2 5 2 0 Offset um
141. he whole surface After the independent movement of the motors the center position will change laterally so the tip will not land on exactly the same region of the sample So it is important to measure the large scale tilt of the sample and not some local feature We e Saving Settings 0 NanoWizard AFM User Manual Version 4 2 LI D x Motor Leveling Channel Trace Height measured Trace h a Average over whole image Select points for tilt calculation Calculate Motor Leveling Channel Trace Height measured Trace x PaA d Average over whole image 2 Select points for tilt calculation Nr X in um 6 624 Y in um 12 278 0 0 Heights in um 9 963 10 797 0 0010 3 736 5 095 0 0010 WINIH O 0 411 4 234 0 0010 Delete j Clear P Calculate i NanoWizard AFM User Manual Sr KI The Motor Leveling display shows the currently selected image from the Scan List The Height measured channel is generally used as this has the best height accuracy and the display here always shows the display leveling off so it may look different in the Data Viewer depending on the display settings there The default setting for the angle calculation is Average over whole image This is appropriate where the sur face is very flat for example single molecules on mica and the large scale heig
142. he window which is convenient for laser alignment during setup The minimize icon returns the window to its smallest size for normal operation Es If there are any problems check that the laser is switched on in the main SPM icon toolbar If not toggle the laser icon If the laser is on in the software but the red diode is off on the AFM head then the laser safety tilt switch is activated This switches off the laser when the head is upright to protect the operator When the head is placed on the three motor feet the laser will switch on again NanoWizard AFM User Manual Version 4 2 33 Ji F f Nanotechnology for Life Science Instruments 4 2 3 Adjust the laser beam onto the end of the cantilever Use the optical microscope or TopViewOptics to view the cantilever with the CCD camera The IR laser spot can not be seen with the human eye but the CCD camera is sensitive to a wider range of wavelengths If an inverted optical microscope is used a low magnification objective is recommended e g 10x or 20x to give a large field of view and make it easier to find the laser spot Decreasing the illumination can help to find the laser spot as scattered light even far from the focus is visible If the laser soot can not be seen in the optical microscope check for any safety or fluorescence filters in the optical path that may be cutting IR wavelengths The optical microscope image here shows the laser spot in the field of view b
143. his Vertical Deflection value is usually displayed in Volts and is the difference in voltage between the different sections of the photodiode Often this value is sufficient since the conversion from Volts to an actual deflection distance is relatively linear across the center of the photodiode The deflection value in Volts can be used for all the normal AFM modes and applications imaging force spectroscopy etc For some measurements however it is important to know the deflection of the cantilever in units of length The most common case of this is for calibrated force measurements when both the deflection and spring constant of the cantilever must be known This may be for force spectroscopy to measure the forces of adhesion or mechanical properties of the sample or it may be for contact mode imaging to know the exact interaction forces during imaging In intermittent contact mode this conversion from Volts to nm can also be important if it is necessary to know the physical size of the oscillation amplitude The conversion factor from Volts to nm for the deflection of the cantilever is usually called the sensitivity It depends on the type of cantilever and how it is mounted so the measurements must be repeated each time a cantilever is mounted or re mounted The values will usually be similar however for identical cantilevers mounted a similar way The sensitivity is measured by doing a force curve on a hard surface and looking at the deflect
144. hould be placed centrally between the two grooves with the cantilever arm over the polished glass part close to the inclined edge The cantilever itself should be in the centre of the glass block do not move the cantilever substrate chip too far forwards When the cantilever is in the right position grip the bottom loops of the spring firmly with a pair of tweezers and squeeze to lift the front part Slide the spring into the groove and release the loops to clamp the cantilever firmly Do not touch the optical surfaces with tweezers The spring may gradually become weaker and must be replaced occasionally Several springs are delivered with new instruments more can be ordered from JPK if they need replacing or are lost The glass block is held in the AFM head with a locking mechanism The notches in the glass block must be lined up with the metal tabs on the AFM head to place the glass block into the AFM head The glass block should then be turned by 90 degrees into position Make sure that the cantilever and spring are on the left 32 NanoWizard AFM User Manual Version 4 2 The glass block can now be locked in position with the two finger grips on the rim of the steel disk Using the thumb Lea and index finger turn the knobs slightly to the left and a small click should be felt as the spring locks 4 DES Summary when the microscope is in its upright position the cantilever spring must be parallel to the desk The gro
145. ht differences are dominated by the sample tilt In this case proceed to the next step with Calculate For samples where there is a clearly defined back ground that should be used for calculation and features that should be ignored this is not appropriate For the image shown it is better to choose Select points for tilt correction If Select points for tilt correction has been chosen click in the image display to set points Three points is the minimum but it is better to use rather more points than needed to ensure there is a good average Choose points all over the region of the background level of the image The listed points will appear in the table below Delete deletes the currently selected point Clear deletes all the points in the list When the points are set reasonably click Calculate to proceed to the next step Version 4 2 71 deift Instruments Nanotechnology for Life Science i Molor Leveling A C Safety Height 50 09 a Jum Ta level the scanner the stepper motors will be meved by the Following amounts Mozor A 0 50 mm Mozor B 0 49 mm Molo C 0 22 mm Start leweling cancel 5 4 Tools for monitoring scanning 5 4 1 The Oscilloscope window E Once the background level has been set from an image the calculated stepper motor movements are shown The Safety Height is added to all the stepper motors and should be the final height above the surface at the end of t
146. ibrations to the AFM head Therefore it is recommended that the cables are taped firmly to the vibration isolation table Simple adhesive tape can be used wide masking tape for marking areas for home decorating is particularly suitable because the wide tape sticks well but the adhesive is designed to not leave a residue so the cables do not become sticky 12 NanoWizard AFM User Manual Version 4 2 2 2 2 Controller connections The NanoWizard 3 is equipped with a controller and PC that are connected via Ethernet For the PC as well as for the controller standard power cables for the correct volt age 230 or 115V depending on the country can be used There are several USB sockets on the computer to connect keyboard mouse the JPK motorized precision stage and any other USB devices Firewire cameras con nect directly with the Firewire IEEE1394 connection see Section 4 1 1 for more information on the CCD camera The lower of the two network plugs is dedicated for connection with the controller There are two monitor cables which plug directly into the graphics card The RS232 sockets connect temperature devices with the system F 7 d The connections for the controller are found on the back of the controller box The power supply socket of the controller is equipped with a fuse which depends on the particular equip ment Please ask JPK for assistance if required email support jpk com First check that t
147. id Angle sel ola deg Advanced Settings 106 The Quantitative Imaging Control panel on the left side contains all main settings for running a QI measurement The Setpoint Z Length Extend and Retract Time can be directly typed into the corresponding text fields They should be optimized correspond ing to the sample The Scan Control Grid panel defines the position and resolution of the scan area see Section 5 1 3 Under Advanced Settings even more parameters which can be adjust ed are available Section 8 1 4 NanoWizard AFM User Manual Version 4 2 8 1 4 Advanced Settings Open the Advanced Settings window using the Quantitative Imaging pull down menu in the main menu list or by clicking the shortcut button in the Quantitative Imaging Control panel The standard settings meet the requirements of a wide range of samples However very delicate and challenging samples need further adjustment of scanning parameters 2 Advanced Settings Extend and retract rates Use the same rate for extend and retract Extend Sample Rate Extend Time Extend Speed Retract Sample Rate Retract Time Acceleration Baseline Adjust Baseline Next Line Delay Next Line Retract e 100 0 amp inm Number of collected pixels While extending 50 While retracting 50 Time for Image 11 15 min z EN Additional Retract Height 2 z length
148. iff cantilevers may be used in which case the thermal noise calculation is not appropriate but the spring constant supplied by the manufacturer should be sufficient for a general estimate of the applied forces Typical forces used in lt 1nNin any case nano manipulation lt 500 pN to move molecules in case of H bonds between molecule and its support 100 pN to move molecules 5 nN depending on the material Other manipulation experiments can involve applying a voltage to the tip e g local oxidation or using a functionalized tip to create some surface modification along the manipulation paths In this case the manipulation force will probably be kept to a minimum as for imaging since some other mechanism will create the surface pattern or modification NanoWizard AFM User Manual Version 4 2 131 deift Instruments 8 6 1 Paths and points Manipulation Control Velocity m s setpoint hd V IGain v a Hz PGain v al Pan o O Delete Clear New x Position E Delete Clear New f Data Viewer Height trace Use Absolute Limits Nanotechnology for Life Science In the SPM software the whole set of movements for the manipulation is divided into paths and points Each path is a set of x y points and the tip moves from one point to the next with a constant Velocity over the sample surface During the movement along each path the Setpoint is held at the value in the Mani
149. ight differences without any leveling procedure being applied to the scan lines The height range being covered can also be seen on the Z Range piezo display as below If there is a significant sample tilt it may be better to remove this first by adjusting the stepper motors see Section 5 3 2 before reducing the Z Range Note that you may then be scanning a different area of the sample unless you can find the same location again optically NanoWizard AFM User Manual Version 4 2 67 Ji F f Nanotechnology for Life Science Instruments The Z Range can be set from the Setup menu on the main pull down menu bar and can only be changed if the tip is retracted from the surface Autosave scan Repetitions ir Cantilever Tuning e e 15ym max range There are seven different z ranges available that can be fz Z Piezo Display SEH selected 15 10 7 5 5 3 1 5 or 0 75 um Approach Parameters 7 oum lia Saving Settings 2 Hm Always start with a larger Z Range for samples with an 5 Channel Setup aan unknown topography range It can then be reduced it if needed according to the sample image The approach will also be faster for a larger z range then the range can be decreased at the surface just before imaging AYScanners 1 5m TTL Control 0 75um max resolution Exit The z range panel can be seen at the bottom left of the user interface and the active z range is marked in this panel as the white part of
150. ilever which can be immersed in water Perform your experiments in a dry atmosphere Do not pour any fluid over your AFM head The metal cover of the AFM head is made of aluminum Be careful not to allow any contact with any aggressive acid or base The black membrane that protects the AFM head from underneath is not stable against benzene and alcohol Do not allow aggressive fluids such as acids and bases to come in contact with the polymer NanoWizard AFM User Manual Version 4 2 Ji F f Nanotechnology for Life Science Instruments 0 2 Warnings for the Life Science version with optical microscopes 0 2 1 Prevention of condenser lens crash Be careful not to hit the AFM head when lowering the condenser illumination optics of the optical microscope This is especially important for users of the NanoWizard microscope and particularly when the motors are extended and the head is higher than normal If the condenser is going to hit the head lower the head on the motors first Always lower the condenser carefully 0 2 2 Prevention of objective lens crash Do not allow the objectives to crash into the microscope stage The maximum load of the objective revolver should be three objectives arranged in a 120 degree arrangement as shown in the pictures here Please check that the specific lenses used fit in this conformation It is possible that some lenses with an extra large width may have to be used alone or the obje
151. ilever is far from the sample surface so usually the distance is initially reduced using the stepper motors This fast correction is generally NanoWizard AFM User Manual Version 4 2 45 Ji F f Nanotechnology for Life Science Instruments known as the coarse approach since there is no check on the cantilever signal This can also be useful to move the cantilever into the range of the optical microscope objective in order to see the cantilever and laser spot for alignment Ta The motor icon in the shortcut menu bar opens the Stepper Motor window ZS Stepper Motor 25 The coarse step size can be set in the Step Position 99 099 um paray per Motor window The Up arrow moves the f Coarse Step Size head and cantilever away from the sample Motor A 117 um and the Down arrow moves the head and Motor B 74 1 um cantilever towards the sample Motor C 118 um Y Warning If in doubt about the tip sample separation start with relatively small step sizes During the coarse approach watch the movement of the glass block towards the sample Do not crash the glass block and cantilever into the sample The motors can also be moved independently see Section 5 3 for details to remove any overall tilt between the sample and the AFM head 4 5 2 Automatic approach The automatic approach makes small steps and checks at each step to see if the sample surface is within range H Laser Alignment 20 Before trying to approac
152. ill be collected during scanning The channel setup can be selected in Setup gt Channel Setup T T E S E Precision 12 Precision 12 Approximate scan file size 1572 86 kb Approximate scan memory size 12582 91 kb Saving Settings for Contact Mode D se one folder for all data types nome tneumann jpkdata fibroblasts_living 2011 12 08 3 Temperature data Quantitative Imaging Voltage Spectroscopy STM I V Spectroscopy Image Scans Force Scans Force Scan Maps Real Time Scans Voltammetry Save with Scan Trace Retrace Filename root Folder 5 living 2011 12 08 3 Auto save also applies to incomplete scans ertical deflection a ateral deflection O position Tip Probe position Tip no probe entry i E no name entry Comment no comment entry Height measured A Photo Sum apacitive sensor X position apacitive sensor Y position Missing Channel The Saving settings window is used to define the default file locations filenames and text comments for the different types of data files that can be collected using the SPM software The settings here also control the default list of data channels that will be saved in different measurement modes This is opened by choosing Setup Saving settings from the main menu Once a channel is enabled in the Channel Setup the Saving Settings panel can be used to specify whether it should be saved in data files from different measu
153. in air There are three visible peaks in this spectrum corresponding to the resonance at around 4 kHz and the first and second harmonic average pm Hz ZH sud Beep wW 0 10 20 30 40 50 60 70 Frequency kHz Select Fit Range Run D Reset g Ze When the same cantilever is immersed in a fluid each peak is damped 200 200 reducing both amplitude and frequency The resonance of the cantile ver is now extremely low and it can be difficult to fit the curve properly At lower frequencies these curves are also more susceptible to noise In this case the user may wish to fit the second peak or first harmonic a i to get a better fit However if this is done a correction factor must be o used In this case the spring constant would be correction factor spring constant determined using the fit in the SPM software average pm Hz ZH zud ewp Wu Frequency kHz Select Fit Range Run w Reset Fit Values Settings Please note that for the higher peaks overtones the correction factor Temperature Ke is not 1 0 Cantilever Angle 10 For further information about correction factors see the NanoWizard Handbook use correction factor Correction Factor For rectangular cantilevers the correction factors have been described by Butt and Jaschke Butt and Jaschke 1995 Nanotechnology 6 1 7 And for triangular MLCT type cantilevers by Stark et al Sta
154. ines the integrator and the gain parameter PGain the proportional amplifier Feedback Control ex Both gains can be adjusted at any time including during imaging and can be set independently from each other The numbers can be entered directly in the text field or the value can be changed in increments by clicking on the arrows Note the approach gains are set separately in the approach window In the simplest explanation the gains control the reaction speed of the feedback loop If the feedback control is too slow the z scanner always has a delayed reaction to the topography If the feedback control is too fast the z scanner tends to overcorrect and may start oscillating As a general rule it is best to increase the gains as much as possible Using higher gains means the feedback reacts more quickly and the maximum force is reduced The cantilever tip follows the surface more accurately producing a better topography and the tip reacts quickly to any change of height preventing damage to the tip or the sample It is also often possible to reduce the setpoint imaging force when higher gains are used If the gains are set too high how ever the feedback may oscillate and they will need to be decreased again In general the gains will usually need to be higher for Faster scanning increasing the Line Rate Larger scan region faster tip speed over the surface in microns second Samples with a large topography range more z height
155. ion i e there is no corre pri lation to the surface determined by the precedent approach or F ex tend segment Velocity 8 3 2 Advanced Spectroscopy Control Spectroscopy Control The Advanced panel in the Spectroscopy Control window provides general ep ES settings that are valid for all segments YF a ESSIEN Advanced Settings preferences allow to type the force curve resolution as Sample rate in Hertz or as Sample number in data points per segment Corresponding a ly editable text fields appear in the force curve segments O Synchronize sample rates 2 movement Constant Duration k It is also possible to Synchronize sample rates along all force segments in order to keep the bandwidth constant Z Speed Z Length and Duration see extend retract segments depend on each other Z movement helps to either maintain the duration of a segment Constant Duration if Z Speed or Z length is changed or to maintain the Z Speed Constant Speed if Duration or Z length is changed Settings after spectroscopy stopped Return to starting mode Return to starting mode constant height mode There are different modes available after spectroscopy is stopped The first is EE to Return to starting mode Constant height mode maintains the end posi Idle mode approached tion of the last force curve segment Retracted piezo mode and Idle mode turn the piezo into retracted and approached mode respectively 120 NanoWizar
156. ion of the cantilever when it is in hard contact with the sample In this region of the force curve the movement of the piezo should corre spond with the deflection of the cantilever since the tip and sample move together The plot of the deflection versus distance should have a slope of 1 in this region and the piezo movement is used to calibrate the measured deflection These force curves in hard contact with the surface have the potential to damage the tip however so this procedure is often carried out at the end of an imaging experiment Ti Force Spectroscopy To measure the cantilever sensitivity it is necessary to first perform a force distance experiment on a hard clean surface such as glass or E Force Spectroscopy mica ES Imaging Select Force Spectroscopy make sure that z linearization is ena ak Force Mapping bled e Approach the cantilever onto the hard surface using contact Force Spectroscopy mode ES Force scan series List e Perform single or multiple force distance curves as in Chap bc Force Spectroscopy Oscilloscope ter 6 t S S Force Time Oscilloscope l a H The height calibration menu can be found in Force Spectroscopy Calibration Manager The panel Vertical Deflection should be selected by clicking on the far left of the window The tab Sensitivity should be displayed by default If the sensitivity has not yet been measured the Sensitivity value is set as UNKNOWN e Advan
157. ion rate If the data is collected very quickly the performance may start to suffer Check that only the required channels are being read open the Channel Setup to optimize the high speed performance The data collection for the real time oscilloscope is started with Start The data can be saved if the Autosave button is selected before the Start is clicked Version 4 2 145 Ji 8 f Nanotechnology for Life Science Instruments Saving Settings Siei The settings for saving data can be chosen using Setup Saving Set tings and choosing the tab for real time scans Here the file name sav ing directory and channels to save can all be selected The date and time is added to the Filename root so the files are named by default as v Use one folder for all data types home lueders jpkdata Browse Image Scans Force Scans Force Scan Maps Real Time Scans Voltammetry MicroRheology Scans Voltage Spectroscopy STM I V Spectroscopy ve Filename root real Save with Scan M Folder ome lueders jpkdata Browse Wertical deflection M Lateral deflection Lock in amplitude Lock in phase X position Tip V position Tip Error signal Excitation frequency Time Lock in amplitude 2 Lock in phase 2 Lock in amplitude 3 Lock in phase 3 Output amplification Photon counter 1 Photon counter 2 Tunnel Current Logarithmic Input Height meas
158. ip movement Spectroscopy Control y If A HE V F Rr Z Lamm l IThe top part of the panel has shortcut icons for several Basic f Advanced useful settings which are described below Rel Setpoint el 0 400 v SE Dem Tabs for Basic or Advanced mode offer different control settings for the force scan Adjust Baselinel1 movement These Basic settings are explained in Section 0 and provide an easy Z Length way to perform force curves including variable approach delay and retract settings Extend Time ane The Advanced mode is an optional software extension and not included in the AC Lelay i i f Retr Delay basic software version These Advanced mode settings are explained in Section E Constant Farce 8 3 and allow more complex force scans Different types of force segments can be BEE a Hz freely combined such as length segments relative force movement force clamp Z Closed Loop O and delays Custom settings can also be saved and reloaded ER The lower part of the panel has the list of X Y positions where the force curves will be performed These can be selected by hand or set automatically as a grid see Section 6 4 Alternatively the force experiment can be controlled as Force Mapping see Section 6 7 Hr x in Um Yin um Delete Clear New NanoWizard AFM User Manual Version 4 2 79 Ji F i Nanotechnology for Life Science Instruments y Shortcut to open the Calibration Manager window
159. is active should be stored also in this folder Version 4 2 115 deift Instruments 8 2 5 Manual calibration Nanotechnology for Life Science For some optical applications the user may wish to use a different CCD camera or an alternate imaging device If the imaging apparatus can acquire images of the cantilever and then provide these images as TIFF JPG or PNG files then the calibration procedure can still be used however the acquisition of images for the calibration can no longer be automatically acquired ms Optical Image Calibration Size of the calibration area Width e 100 0 a Am Height e 100 0 a um Pata gegen JY ome kate jpkdata optcal 2006 11 06 16 07 07 E Automatic image acquisition mg Optical Image Calibration Collect Images Index Filename DO JO 0 P OM 10 LI 12 13 14 LS 16 17 18 19 20 21 22 23 24 25 00 0 00 25 00 50 00 50 00 25 00 0 00 25 00 50 00 50 00 25 00 0 00 25 00 50 00 50 00 25 00 0 00 25 00 50 00 50 00 25 00 0 00 25 00 50 00 50 00 50 00 50 00 50 00 25 00 25 00 25 00 25 00 25 00 0 00 0 00 0 00 0 00 0 00 25 00 25 00 25 00 25 00 25 00 50 00 50 00 50 00 50 00 50 00 Y um Please specify filenames for all images 116 Cancel For the manual calibration procedure using external image acquisition software Automatic image acquisition must be deselected
160. ists The first list includes the Control structures for putting the repetition rate and conditions to the experiment The other lists concern the software and hardware features such as the Imaging TemperatureController Fluidics Module and the Spectroscopy features Control structures Control structures while loop while loop introduces a while conditioned loop for loop for loop sets the number of repetitions for the subsequent commands if else statement EE ENT if else statement introduces an if else condition Display text Wait a defined time sets a defined delay stop script Display text displays a specified text Stop script stops the script These commands are usually combined with scripts to execute a specified ex periment Alignment Alignment Channels reads in specified photodiode channels Photo diode aligns the photo diode to a set value Retract Retract retracts the scanner full or piezo only Approach Approach approaches the scanner full or piezo only Get piezo height Set Piezo Height TemperatureController Temperature Controllers Set Temperature setTemperature sets the temperature for a specified controller waitForTemperature Wait for Temperature introduces a waiting loop for the set temperature to be reached Spectroscopy XY Position XY Position select scanning positiom start scanning and walk through pattern Start scanning and walk through pattern start scanning Star
161. ivered with the latest version of the JPK software The software programs SPM is devel oped in house by JPK The information in this chapter provides an overview of the main components of the SPM soft ware and links to find more specific information on particular features from the menus or shortcut icons 3 1 1 Starting the program Double click the SPM icon on the desktop and the program will start 3 1 2 Software overview JPK NanoWizard Control 1 Menubar Pa a 2 Toolbar 3 Feedback Control 4 Scan Control Setup Motors Accessories DataViewer Advanced Imaging Window Gei Al ru Allee Feedback Control IGain Help Contact Mode i X a Imaging Jel x Data Viewer Height trace PGain Scan Control SI Line 0 v v v FEH e ER 10 0 amp um Line Rate 5 Z Piezo and System Status display Pixels X Offset Y Offset Scan Angle 512x512 g oa iz v 0 a bh A um j a Advanced Imaging Settings Z Range D um Extended 15 000 um System Status Sum 5 34 V Vertical Def 0 272 V Lateral Def Status 0 15 V Scanner Retracted Channel Settings View Focus Line Viewer 7 Information and status bar The graphic here shows the initial software state The next sections of this
162. ks Three different sorts of TTL clocks are available Pin 6 Pixel clock a TTL pulse is generated for each pixel trace and or retrace Pin 5 Line clock a TTL pulse is generated for every line trace and or retrace Pin 4 Frame clock a TTL pulse is generated for every frame Note The general Pulse settings have to be set at the TTL Control Panel Therefore the Style PULSE has to be cho sen and the Pulse time has to be defined For activation of the TTL clocks the JPK SPM Jython Console has to be used NanoWizard AFM User Manual Version 4 2 123 Ji F f Nanotechnology for Life Science Instruments Advanced All clocks can be activated by using the Jython console Open Script JFK SPM Jython Console k Real Time Scan Logging Settings The Jython console can be opened via Advanced JPK SPM Jython console The Jython console can be used to run pre programmed macros like TTL Advanced System Status clocks from JPK so a detailed knowledge of Jython is not required for the operations To activate TTL clocks the following commands have to be used Pin 6 Pixel clock dspManager setPixelClockEnabled true true Pin 5 Line clock dspManager setLineClockEnabled true true Pin 4 Frame clock dspManager setFrameClockEnabled true For a Line clock as well as a Pixel clock a pulse routine can be defined e Pulse only in trace extend true false e Pulse only in retrace retrace false true or e Puls
163. later if required The files are saved in subfolders named for the filter results The typical case is that two sub folders are created with the names 1 and 0 where the accepted and not accepted force curves are saved If there is a folder unclassified then there were unexpected results from the filter fit did not converge missing chan nels etc Open one of these curves in the Data Processing software and apply the saved process file to see what the problem was Please note that Reset just sets the counters in the filter panel to zero It does not create new folders or change the saved data NanoWizard AFM User Manual Version 4 2 89 Ji JI Nanotechnology for Life Science Instruments 6 7 Force Mapping 6 7 1 Introduction to Force Mapping The Force Mapping feature is an extension of the Force Spectroscopy mode Please read the first part of Chapter 6 to have an overview of the basic Force Spectroscopy features before starting w Force Mapping E Select Force Mapping mode from the Measurement Mode drop down box The tip movement in Force Mapping is similar to normal force spectroscopy but the measurements are performed over a grid and the force curves are analyzed to give a value at each point which creates a number of Force Mapping images TT Image Viewer Slope retrace See ee EI Data Viewer Height trace Although the display in force mapping mode normally shows a grid of filled points th
164. libration window and the Filename will appear in the list These files are automatically saved in the Data directory The X um and Y um values correspond to the cantilever position where 0 0 is the center of the X Y range While the images are still being collected there is a mes sage at the bottom of the panel Once all the images have been collected click Continue The next step is to locate the cantilever tip position in one reference image Select an image from the list and enable the Reference tick box The image will be displayed in the top panel Click in the image on the point corresponding to the tip location A white square will appear in the image indicating the desig nated tip location Note that the display panel showing the image can be enlarged to make this step more precise When the tip location has been selected in this image a Pixel X and Pixel Y value will appear for the chosen point When the selected tip location in the reference image is reasonable click on Calibrate at the bottom of the panel 112 NanoWizard AFM User Manual Version 4 2 mg Optical Image Calibration Locate Tip Positions Reference Pixel X O el E O O O O O O E O O El O El O El O O El O Pa O O Optical Image Calibration Select Image to Load G Median image Calibration image v Load image file Take snapshot Please select an image to load Pixel Y o bi III E a
165. lude the offset that was applied during the manual ad justment of the image position Version 4 2 Select Optical Calibration File and Image Calibration file ate jpkdata optcal 2006 11 06 16 07 07 optcal shifted Image file Image Viewer home kate jpkdata optcal 2006 11 06 16 07 07 fl2 jpg Cancel Current Scan Show Show Scan Region Recent Scans Number of listed entries Iz Ale v remove J optical image v remove J optica image Channel Settings View Focus Line Viewer TS Saving settings Additional images can be imported using the same optical calibration file with the offset that was ap plied if the optical image had to be shifted By clicking on File at the top of the screen and then Import Optical Image the user can select the appropriate calibration file and image If more than one optical image is in the Scan List then they will generally both cover the whole dis played area The only visible image will be the one at the top of the list The up and down arrows in the Scan List can be used to change the order of the images Note do not open too many optical images at once as the software may slow down Additionally to import an updated optical image the Snapshot function can be used First select an optical image in the Scan List to define the optical calibration
166. lue to fit in the scan region Note the SVG files must contain only Only outline elements have been imparted outline objects Other elements e g font objects filled or patterned objects will be ignored by the import filter 6 The SVG file contained parts not understood by the importer such as bitmap images or fant objects Text may be used but must first be converted by the vector graphics software so that it is stored as a set of the outline paths rather than as characters in a font The exact export option will depend on the vector graphics software but should be in the Text menu and be described as something like Convert text to outline or Convert to outline path EI Data Viewer Height trace This example shows one of the example manipulation paths which can be found under opt jpokspm data manipulation svg There are several basic examples supplied with the JPK SPM software such as arrays of lines or points and simple geometrical shapes These can also be modified in SPM and resaved Paths or patterns can also be created using standard vector graphics software such as Corel Draw or Illustrator Free software SEIS zone JL ve d Focus d une Viewer is available to create vector graphics on www inkscape org 134 NanoWizard AFM User Manual Version 4 2 8 6 3 Simple manipulation examples 8 6 3 1 Lithography For test purposes a nano scribing experiment can be performed onto the polycarbonate pol
167. m manually using the pump display see WPI manual instructions WPI supports a dual pumps control cable to run the pumps manually Please remove the dual pump cable to support the pumps with the JPK pump control software NanoWizard AFM User Manual Version 4 2 53 Ji f f Nanotechnology for Life Science Instruments Pump Control gt Start all a min Stop all Ee fa gt a inject Name Serial number Standard flow rate he RS 232 Units Volume Dispensed Pumping Direction Motor Indicator Indicators Indicator Indicator Operating fm mmm wm mmm mmm am iOm min hr Withdraw OPumping a dus ania cs nsec Prereee rere i iii titi iit titi iii TP Program Direction Functions Pumping Direction Key Aladdin syringe pump is connected with the PC Therefore the serial number is displayed blue circles However in that example the syringe window displayed a com munication ERROR red circle For error fixing hold the DIAME TER SETUP button 1 for 3 4 seconds In that case the first parameter PF is displayed After 2 seconds the next parameter will be visible Standard setting PF Power failure mode 0 AL Alarm mode 0 TTL display TTL external TTL bB keypad lockout 0 Ad RS 232 pump network address 01 it is important to set the address on 01 If Ad is displayed next value is baud rate 1920 RS 232 Units Volume Dispensed Pumping Directio
168. mage Scans Force Scans Force Scan Maps Save with Scan Channel Extend Retract Pause ome lueders jpkdata _ Browse Height Vertical deflection Lateral deflection Approximate image file size X position Tip Approximate data size Y position Tip Error signal Axis 4 no probe entry Precision 5 Precision 6 Photon counter 1 Photon counter 2 High Speed 2 High Speed 3 High Speed 4 Precision 3 Precision 7 Precision 8 Precision 9 Precision 10 Precision 11 Precision 12 High Speed 1 High Resolution Comment Axis 5 no comment entry Axis 6 Axis 7 Axis 8 Capacitive sensor X position Capacitive sensor Y position Height measured Photo Sum no name entry Channel Image Image Extend Retract Height measured Height Adhesion Slope Select all Select all Missing Channel QI mode provides two data types The raw files containing all force curves are saved as jpk qi data files The force curve channels Height Vertical Deflection and Height measured are essential for data collection and are saved by default Other channels can be enabled if required The mere image files containing the results of online data analysis Height Height measured Adhesion and Slope are saved as jpk qi image files The corresponding channels for saving can be found in the lower part of the Saving Settings window 8 2 DirectOverlay importing calibrated optical images The direct comparison of images a
169. mages also appear in the Old Scans section of the scan list NanoWizard AFM User Manual Version 4 2 Current Scan Show Si Hold Current Scan Recent Scans Number of listed entries Iz al Show 7 a E Ah da Old Scans t 2 Show C Saving settings Scan List elt Current Scan Show ell Hold Current Scan Recent Scans Number of listed entries Y a a Show P a Old Scans Ej ejej j Show v A s X Scan Save Data vy Show wv Select Focus Remove Li Saving settings s Scan List DN Current Scan Show Recent Scans Number of listed entries el 4l a Show v Leg Hold Scan 14 gt S Old Scans a O Show RE wll PAX optical image G i E Saving settings Current Scan Show Hold Current Scan Recent Scans Number of listed entries Iz al Show AY Old Scans A E y a E Scan 6 E Saving settings Scan List He Current Scan Show Hold Current Scan Recent Scans Number of listed entries zl Al a Show iz S Old Scans Ff t y Show D X Scan 6 E d Saving settings Click Hold to store a good scan The images are displayed with a thumbnail image and the scan number x Note The R
170. manual introduce some of the software options and controls that can be seen in this screenclip This chapter provides quick overviews of the functions more detailed instructions are given in the referenced sections later in the manual that describe approaching imaging and the particular types of experiment that are possible 1 2 The menu bar runs along the top of the screen the pull down menu options are listed in Section 3 1 3 The icon toolbar provides shortcuts for some of the most commonly used options as listed in Section 3 1 4 The Feedback Control panel can be seen on the left side of the screen Here the feedback parameters can be set these are briefly introduced in Section 3 2 2 and more thoroughly discussed in Section 5 2 The Scan Control can be seen on the left side of the screen during imaging mode These settings are briefly introduced in Section 3 2 2 and more thoroughly discussed in Section 5 1 NanoWizard AFM User Manual Version 4 2 17 Ji F f Nanotechnology for Life Science Instruments 5 The Z Range Z piezo display and System Status panels display information about the current state of the AFM system such as the current piezo position and whether the instrument is approached on the surface 6 The Data Viewer window displays the scan data either from the current scan or from previous data files The options for setting the data and display settings are introduced in Section 3 2 3 7 The statu
171. mode is contact mode where the direct bending of the cantilever is used as the feedback signal In this case the Vertical Deflection value is used as the feedback channel The other main group of feedback modes all use some kind of cantilever oscillation where the resonance properties of the cantilever are used for the feedback signal The feedback channel could for instance be Amplitude Phase Fre quency or some combination of different feedback loops can be used For the NanoWizard and II the standard version of this type of mode is called Intermittent Contact mode For the NanoWizard 3 the simplest version of this type of mode is called AC mode There are many more options for the NanoWlzard 3 feedback modes 4 4 1 Contact Mode To image in contact mode first attach a contact mode soft cantilever to the glass Contact mode e l l block and align the laser beam as described above Contact mode is the default feed back mode when the SPM program is launched 4 4 2 AC Mode pose Mode To perform experiments in intermittent contact mode AC mode the correct option should be selected from the feedback mode list in the shortcut bar Contact mvae Force Modulation Mode Cantilever Choose e AC Mode for oscillating the cantilever in air A non contact cantilever is usually used in this case generally 40 NanoWizard AFM User Manual Version 4 2 a stiff cantilever with a high resonant frequency 200 400kHz
172. modify the old calibration Imaging The height calibration menu can be found in Imaging Calibration Manager In i Open Saved Image the Calibration Manager the three main panels available are Height Vertical De S scan List flection and Lateral Deflection Choose Height Advanced Imaging Settings Hover Mode Settings Old calibrations can be selected from the dropdown box here showing DEFAULT k Oscilloscope ki Spectrum Analyzer zm Calibration Manager m Calibration Manager Height Vertical deflection Lateral deflection shown in the main panel A calibration The details of the selected file are DEFAULT Open Calibration file can be opened using Open Cali bration which will allow you to find a Filename etc opt jpkspm config 3 0 1 instruments C6 calibrations default cal PER ane calibration file saved on disk Comment Original JPK calibration for device Cer Generally values are updated using Edit Conversion to create a new file from the old one Edit Conversion 94 NanoWizard AFM User Manual Version 4 2 m Edit Conversion In Edit Conversion the details can be EE diese changed Enter the new value of Multiplier Multiplier Unit Note the value of Multiplier here should Comment always be around 0 6 If it is significantly Original JPK calibration for device C6 different from 0 6 probably the calculation was wrong If the Multiplier value is aro
173. mplete force distance curve is saved automatically All parts of a force curve trace and retrace segments as well as time delays can be saved in the jpk force force curve format The only part of a force curve that is not saved is the retracted pause between one curve and the next Under the Setup menu option Saving Settings defines the settings for all file formats Choose the Force scans tab and the name directory and channels can be set for saved force curves Saving Settings Use one folder for all data types home lueders jpkdata Browse f Image Scans Force Scans Force Scan Maps Real Time Scans Voltammetry MicroRheology Scans Voltage Spectroscopy f STM I V Spectroscopy Filename root force save Save with Scan Channel Extend Retract Pause Folder home lueders jpkdata i Browse Height Ca Vertical deflection 2 Filename Timestam S P Lateral deflection m E D DE no name entry Y position Tip Error signal O a i Probe Lock in amplitude 2 no probe entry Lock in phase 2 Lock in amplitude 3 Lock in phase 3 Output amplification ia a DW NanoWizard AFM User Manual Version 4 2 87 deift Instruments Nanotechnology for Life Science Extend and Retract of Height and Vertical deflection channels are required data for collection Other channels can be enabled if required for the experiment Note during force scanning the keyboard shortcu
174. mplitude can also have an effect on the imaging conditions The drive ampli tude is the value of the alternating voltage that is applied to the piezo which drives the cantilever oscillation The drive amplitude is not related to the drive frequency For a higher drive amplitude the oscillation amplitude of the cantilever is also increased which can be helpful on some sticky samples The force applied to the sample depends on the relationship between the setpoint and the drive amplitude If the set point is not changed then the force applied to the sample is increased when the drive amplitude is increased There fore it is recommended to increase the setpoint manually while increasing the drive amplitude Additional hint In the cantilever tuning window the magnitude of the lock in amplitude Vornm S displayed in Volts The cantilever must be calibrated to get the amplitude displayed in nanometers The sensitivity of the cantilever has to be determined See Section 7 2 for details about sensitivity calibration 5 7 Force modulation mode Contact Mode Inthe software Force modulation is another feedback mode like contact or AC modes which can be used for imaging samples such as polymer blends to show materials with Contact Mode different stiffness In the user interface Force modulation mode can be found in the drop down menu at the top along with contact and AC mode 5 7 1 Material contrast AC Made Force modulation mode is som
175. n Motor Indicator Indicators Indicator Indicator Operating emm o Dispensed be mmm mmm mmm mmm am o ml min be mmm mmm mmm mmm mmm mmm mmm mmm mmm mmm wf Phase Function Program Direction Functions Pumping Direction Key If the pump network address is not activated via Addr or Ad it can be that dUAL or rECP is displayed instead of Addr or Ad 01 54 NanoWizard AFM User Manual Version 4 2 In that case click on DIAMETER 1 and if the Parameter rECP or dUAL is displayed than activate the very left button 2 green circle until Addr is visible After 2 seconds the Addr will be switch to Ad 00 If so click on button 3 blue circle and select Ad 01 In case of faulty operations it can be useful to reset the pump In that case press the right most arrow key 3 dh blue circle while turning ON the pump The display will show RESET However it this case the default settings are active After Reset you have to set the Address to 01 NanoWizard AFM User Manual Version 4 2 55 deift Instruments 5 Imaging 5 1 Imaging settings RUN Nanotechnology for Life Science After a successful approach the Run icon in the top shortcut menu bar will be active When Run is clicked the scanner will start moving By default Run launches an infinite series of scans If only a single scan is desired this can be changed under the main menu Options To stop the scan click Run again or click the retract b
176. n air there may be a systematic change as the cantilever approaches a surface because of electrostatic effects This can be corrected by just adjusting the laser again to the centre of the detector In liquid there are more complicated problems because the deflection of the cantilever is sensitive to many environ mental effects If the vertical deflection shows a large jump or sudden large changes then always check the cantilever optically first One common problem is that of air bubbles sticking to the cantilever particularly if the buffer or medium contains a high protein concentration e g cell medium with added serum FCS In experiments with cells or large objects sometimes material from the sample can adsorb to the cantilever causing a change in deflection One specific problem in liquid is when aluminium coated silicon cantilevers are used Soft silicon cantilevers can usu ally be bought either with or without aluminium coating on the back side The metal coating is used to increase the reflected laser signal and is fine for use in air but it is not stable in liquid The coating may corrode or even peel off the cantilever completely causing unstable changes in the vertical deflection and sum values JPK does not recom mend using aluminium coated cantilevers for any experiments in liquid It is important to check the product codes when ordering silicon cantilevers since it is possible to order the coated ones without realizing it Check the
177. n be programmed from the SPM software A direct digital synthesizer DDS is also provided NanoWizard AFM User Manual Version 4 2 151 Ji F f Nanotechnology for Life Science Instruments The AXES connectors output the analog signals DAC that drive the xyz piezos These signals are wired internally to the NanoWizard 3 head the outputs can be used for monitoring or feedback purposes These channels have a modulation input that can be activated using the tip button to the right The tip buttons switch the modulation inputs in a three way cyclic manner indicated by an LED off no modulation default red internal modulation do not use or ask JPK for assistance green external modulation the signal provided on the Mod In input is added to the internal software controlled signal For example a signal from a function generator can be used to move a scanner The ANALOG IN connectors can be used to modify the internally wired channels to read in externally supplied analog signals instead Like the AXES connectors these connectors have tip button to switch between the internal signal channel LED off de fault and the externally supplied one LED green Note that in this case the channel data read into the software for this channel will be overwritten by the external signal A total of twelve 18 bit channels 1 12 sampled at 800 kHz Precision four 16 bit channels 13 16 sampled at 60 MH
178. n um Channel Settings View Focus Line viewer 0 34 415 46 06 l 33 046 44 691 Delete Clear New The buttons Delete Clear and New at the top apply to the whole paths Delete removes the currently selected path and Clear removes all stored paths The buttons Delete Clear and New at the bottom apply to individual points within the selected path Delete removes the currently selected point and Clear removes all the points in the current path mam After the manipulation paths have been defined clicking Run performs the entire manipulation movement all set paths once after which the cantilever is left in the retracted position If an optical view of the cantilever is available then the movement of the cantilever can be observed during the experiment The current point is shown in red in the Data Viewer during the movement NanoWizard AFM User Manual Version 4 2 133 Ji F f Nanotechnology for Life Science Instruments 8 6 2 Importing and exporting scalable vector graphics files Manipulation Often manipulation patterns are required that have fixed shapes or distances Manipulation Pattern Manager and are more complicated than is practical by hand drawing Ce Import Manipulation Pattern In the main menu the option Manipulation Import Manipulation Pattern can be used to import a pre saved file the format used is scalable vector graphics file svg Import Pattern for Background
179. na EI Adjust Baseline 1 ye eee The Style PULSE offers the possibility to set zZ Closed Loo p OI Pin 6 Pixel Clock pulses The Pulse Level and the Pulse Time can be set at the TTL Output marked in red Z Start Option continue from previo TTL Output Pin 6 Pixel Clock Pulse Level D Pulse Time KL 17 90 a 8 5 ExperimentPlanner 8 5 1 The concept For unattended experiment performance the ExperimentPlanner allows simple programming of complex experi ments Commands steering different parts of the software and hardware can be selected and stitched together to cus tom design experiments To familiarize the user with the concept of the ExperimentPlanner the NanoWizard comes with several exemplary scripts which can be found under jpokuser jpkdata filename py With those exemplary scripts the user gets to know what typically marks an experiment and how it should be de signed The use of loops in a script a crucial part of an experiment where scans are repeatedly performed becomes clear by looking at those scripts Due to the command selection characteristics no or little programming experience is needed to design an experiment 8 5 2 Planning and running experiments Accessories fi CCD Camera DirectOverlay Optical Calibration iS Import Optical Image Pump Control 7 FluidicsModule To start the ExperimentPlanner select the Accessories pull down menu at the top
180. nce Instruments Fake approach There is a macro to fake a successful approach so the instrument goes into approached mode regardless of the approach setpoint or current position gt gt gt instrument fakeSuccessfulApproach 1 this command also can be abbreviated by gt gt gt macros fake Save a frequency sweep The data from a frequency sweep can be saved as a text file Note open the Jython console and import the macros before performing the sweep Perform the sweep and then save it using the command in the Jython console gt gt gt macros saveSweep filename test dat The sweep time t and number of pixels n can be set before performing the sweep or the default values used gt gt gt macros set_sweep_time t gt gt gt macros set_sweep_pixels n 9 2 6 JPK scripts Advanced Scripting for your own applications is also available and the window can be CIE Open Script opened using Advanced Open script This enables Jython programs to JPK SPM Jython Console run on a separate thread Contact support jpk com if you have a particular ki Real Time Scan request or question Logging Settings There are many pre programmed scripts that can be used for special experi Advanced System Status ments ee asss After selecting Open script from the File menu a panel of the files in the B Ee E ForceGrid py B ead scripts folder IS dis
181. ncreases on the trace scan line correspond to regions where the height decreases on the retrace scan line 66 NanoWizard AFM User Manual Version 4 2 k When the gains are not high enough for the scan speed and topography the scan lines tend to trail as the tip follows the surface downwards and the trace and retrace lines do not agree with one another a an a mai tu ce height um In this example the trace and retrace curves of the height channel are dis played On parts of the lines where the topography height is increasing the line follows the surface corrugations On parts of the lines where the height is decreasing the tip follows a smooth downwards curve and does not follow Ch 1 tra wn 3451y ase the surface details is Increasing the I gain leads to better congruence of trace and retrace curves 67 7 d Decreasing the scan rate also leads to better congruence of trace and re trace a a A e Ba a 331 T YD When the feedback parameters are set appropriately for the scan speed and topography the trace and retrace lines both follow the surface topography and there are only small differences between them Ch 1 trace height um a a d L Si h a oa N W sa gt N Ww DU 3454 5 3 Controlling the piezo and stepper motors 5 3 1 Reducing Z Range for higher resolution To optimize the range and resolution the working range of the cantilever piezo can be set to different
182. nd allow the relative setpoint for relative force scanning to be maintained more accurately over time specifyPoint R 150 NanoWizard AFM User Manual Version 4 2 Continue When the parameters have been set usually it is necessary to click Continue to update parameters or begin to acquire data Usually start and stop from the script not using the Run button 9 3 Signal access module SAM The controller unit for the JPK NanoWizard 3 can be equipped with a so called signal access module SAM The SAM allows electronic access or modification to most of the analog signal channels that appear in the software both input and output In addition sev eral digital input and output chan EA Ip nels are provided ei P NanoWizard controller without SAM NanoWizard controller with SAM mm ep em ANALOG IN Precision Precision Precision High Speed OR WER WER We CR WR SR Wa CR DACH MOS Wa GR Den Wen Wen Wen Axis3 In DIGITAL Wo High Resolution Q E 2 d 4 OO OF OREO gt POWER In Out The SAM is divided into several logical blocks The MONITOR connectors are analog channels that directly output the unprocessed electronic signals com ing from the NanoWizard 3 head These can be used to monitor signals by means of external equipment and employed for feedback triggering etc The ANALOG OUT connectors provide a few analog output DAC channels that ca
183. nd ie Sh VNU DEE 108 8 2 DirectOverlay importing calibrated Optical images nnnssnesnnnnnneenserrrnnrressrtrttrrestrnttnrtnnrnrnrnnnennnn renee 109 8 2 1 Image focus for Optimal tip location cceeccccseeeeeseeeeceeeeeeceeeeeeceueeeeeuaeeesaaueessaeeeeseeeessaeeeesaeees 109 8 2 2 Coarse alignment with Optical image ccccccssseeeececeeeeeeeeeeeeceeeeseeeeceeceeeeessaeaeeeeeesseeeaeeeeeeeeaaas 110 8 2 3 Ae Eier ee AN OMNI EE 111 8 2 4 Managing and adjusting imported images in GM 114 8 2 5 Ee BR ANNO AU OM EE 116 8 2 6 e RTE 117 8 3 Advanced spectroscopy mode and Force Hamplesgner 117 8 3 1 PND 1S WMI Soars occ adem ie E E E 120 8 3 2 Advanced Spectroscopy Control 120 ERR HE OM e EEN 121 8 4 1 Hardware Confguraton mne 121 8 4 2 RE ele NEEN 122 8 4 3 TTL control with Force RampDesSigner 0 ccccccssecccseeeeeeceeeeeceeeeecseeeeeesaeeeessaeeeseneeessaeeeesaaeees 124 8 5 EIER gereest Eege eet Geer 126 8 5 1 DM TNO AG CO E 126 8 5 2 Planning and running experiments sssesseseneesrreesrrersrtrrsrrrrsrrrrsrrrrsrrrrnrtrrrenrrennrrenrnen nnee 126 8 6 Manipulation and Itbograpby nnen nne 131 8 6 1 Pano and Beie UE 132 8 6 2 Importing and exporting scalable vector graphics files cceeeccceeceeeeeeeeeeeeeeeeeeeeeeeeeseeeeseeeeas 134 8 6 3 Simple manipulation examples EE 135 8 6 4 Background Beet EE 136 9 Advanced information EE 137 9 1 Ubuntu Linux information EE 137 IV
184. nel Settings View Focus Line Viewer Channel Settings View Focus Line viewer Many data channels can be collected and saved simultaneously creating multiple images in one scan The available channels depend on the imaging mode and also which channels are switched on in the Channel Setup see Section 3 2 8 Channels can be displayed in either the trace left to right in the normal view or retrace right to left scan direction The display Settings control the pre processing of the displayed data and also how the numerical values are converted to a color image The controls here can be used to rotate and zoom into parts of the image within the viewer Neither func tion affects the scanning angle of the cantilever only the data display The display settings are conerted to scan settings if a scan region is selected and confirmed with the zoom magnifying glass icon Rescales the currently scanned or selected image to the borders of the data viewer Displays the current line scan for each fast scan line as it is acquired The channel and leveling settlings are used as for the main data viewer image There are some useful mouse shortcuts for changing the Data Viewer display region directly e Scroll the mouse wheel in the image plot area to zoom in or out of that location e Click and drag with the mouse wheel in the image plot area to shift the view region NanoWizard AFM User Manual Version 4 2 23 IP MK
185. ng the temperature from the SPM software The list of installed Temperature Controllers is found in the Ac cessories menu When the temperature controller is opened the tempera ture is displayed and controlled from the SPM software The temperature data can also be saved during the course of an experiment 50 NanoWizard AFM User Manual Version 4 2 Saving Settings BIS l Use one folder for all data types nome rachel jpkdata Browse Image Scans Real Time Scans Force Scans Force Scan Maps Temperature data Select temperature controller BioCell temperature controller Filename root Temperature DataSave Temperature sensors home rachel jpkdata Browse Folder Control lt K Sample lt Setpoint Extra channels to save alongside temperature Height Height measured Vertical deflection Lateral deflection Auxiliary channel 1 Auxiliary channel 3 Error signal IW Time Advanced aa Je Open Script IPK SPM Jython Console ki Real Time Scan Logging Settings overnight Advanced System Status 4 6 2 Pump control for syringe pumps The Temperature data tab in the Saving Settings panel provides the settings for the temperature saving The saving data usually includes the Con trol and Sample t
186. ng the values from the other files is the same as described in Section 8 2 3 for the automatic calibration See Section 0 for information on using the optical images in SPM The only difference with an external camera is that the Snapshot function cannot be used as this is dependent on JUnicam The user must remember to transfer the files to the LINUX computer and then they can be imported as described above using File Import Optical Image 8 2 6 Offline Direct Overlay Optical images can be opened in the JPK Data Processing DP program to allow offline overlays and the export of calibrated optical images that correspond to particular AFM scans The JPK Image Processing program is not de signed to allow full image processing capabilities for the optical images instead functioning as a first step in offline image processing See the separate DP Manual for details on importing and managing optical images in DP 8 3 Advanced spectroscopy mode and Force RampDesigner Spectroscopy Control lf Advanced mode in the spectroscopy mode is selected in the spectroscopy p nE AR control panel the Force Ramp Designer opens automatically For more in er Re i ee se formation concerning Force Spectroscopy see Section 6 asic Vance eg F Rel Gerpom NanoWizard AFM User Manual Version 4 2 117 J f f Nanotechnology for Life Science Instruments Force Ramp Designer File Description NN NMNMMAM ll lm Gees Z
187. nt is quite small Do not force the mirror 36 NanoWizard AFM User Manual Version 4 2 Prism Laser Adjustable Photodetector mirror Cantilever Prism Laser _ Adjustable Photodetector mirror Cantilever The mirror helps to correct for the difference in angle of the optical path when the cantilever is immersed in air or liquid Adjustments need to be made when changing from air to liquid operation or back again A small change in the mirror angle leads to a relatively large change in the optical path so the mirror is only used for coarse adjustment Once the Sum is reasonable the detector positioning screws can be used for fine alignment to maximize the Sum and center the spot See Section 4 2 4 for more details The screw movement for the mirror adjustment is very stiff This is to prevent drift in the system turning carefully will move the mirror the small distance needed 4 2 7 Troubleshooting alignment problems E Laser Alignment Vertical Def 10 11 V Lateral Def 4 48 V sum 0 04V If there are problems aligning the optical system as described above first check that the Sum value is reasonable If Sum is near O V then the optical system is not properly aligned and Vertical Def and Lateral Def values are not reliable The maximum Sum value de pends on the type of cantilever both the shape and whether there is any metal coating on the back side Values vary between ar
188. ntrol 51 deift Instruments Nanotechnology for Life Science Status Connection failed Check connection Add syringe pump KD Scientific Model 200 Series R5232 dev ttyS1 b Active Syringe pumps KD Scientific Model 200 Series Status Connection failed Check connection A The Pump wilh adress 00 dic nel aiswer Info Address and baud ete tar be coriored using the meau on Fe pump Each pump needs a dite ent address using the address 0 is discouraged Suggested va wes are For WPl punps basdrate 19200 address 1 3 For KD pumps baud ate 9690 address 1 5 Please also chec lt woether you selected Fe rght serial port feo z4 Configure Pumps Active Syringe pumps KD Scientific Model 200 Series Name Syringe 2 Pump control oooo min KJE EJEJ syringe 3 gt Start all sl 440 2 440 2 a jimin l a itm Preferences 52 NanoWizard AFM User Manual Select the model to be used from the list under Add syringe pump Select the serial port in the next com bo box from the list of available ports Click Add to try the connec tion A new syringe pump will be added in the Active Syringe Pumps section even if the settings are not yet correct Check connection can be used to try the settings or auto to try some default settings Some of the com munication settings should be first read from the syringe pump itself such as the Baud Rate and Address
189. ny of the advanced functions of the signal access module please contact JPK at sup port jpk com or call 49 30 5331 12070 for assistance 9 4 JPK data formats The SPM software generates images with a jpk file extension JPK files are a form of the tif format They contain one thumbnail image data for each channel that has been stored and a list of scan parameters A more detailed description about the jpk files can be found on under _ opt jpkspm doc TifSpec sxw NanoWizard AFM User Manual Version 4 2 153 Ji I Nanotechnology for Life Science Instruments This sxw file can be opened with OpenOffice which should already be installed on the instrument computer jpk files are designed to be read by the JPK Image Processing software IP After opening an image in IP it can be processed and either saved in the same data format or exported as a pure image in a graphics format for example for import into a word processing or presentation software The other data formats can be managed in a similar way JPK formats Export formats Save option Save Manager in Scan List SPM Export as text Export as picture Save data or Save with JPK DP out Format jpk image format force force map image format jpk force force spectroscopy jpk force map force map curves tnd frequency spectrum in thermal noise ascil cal height calibration file ascii File contains Full collected data scan parameters Limited inf
190. o keep the peak of the resonance curve below 2 V lock in amplitude aS VI O CH 150 2 Lock in amplitude mv A aseyd ul 4207 Lock in amplitude mV Gap eseyd ul 907 100 4 5 50 6 300 0 300 5 301 0 301 5 302 0 302 5 125 0125 5126 0126 5127 0127 5128 0128 5129 0129 5130 0 Frequency kHz Frequency kHz Well shaped resonance curve good signal to noise ratio Critical signal to noise ratio approx 4 1 350 Possible reasons for badly shaped resonance curves e Contamination of the tip with adsorbed dirt Lock in amplitude ri A aseyd ul 3307 e Cantilever or tip physically damaged e Chip of the cantilever not tightly fixed to the glass 100 z block 0 492 493 494 495 496 497 498 Frequency kHz e The tip is making contact with the sample during the cantilever tuning Badly shaped resonance curve 4 4 3 Using intermittent contact mode in liquid Load an appropriate cantilever spring constant around 0 3 N m often a silicon nitride triangular cantilever see our User Handbook for more details and use the steps above to align the laser detection path For optimal conditions the oscillation of the cantilever should be adjusted when the tip is relatively close to the surface Therefore we recommend approaching the surface in contact mode then going to piezo retracted mode by clicking the retract icon only once to 44 NanoWizard AFM User Manual Version 4 2 set up the parameters for the intermitten
191. o the new scan region A new region can not be activated 60 NanoWizard AFM User Manual Version 4 2 during a scan however Run must be toggled to allow the new region to be set f Data Viewer Height trace f Data Viewer Height trace Channel Settings View Focus Line Viewer f Data Viewer Height trace Channel Settings View Focus Line Viewer bsol imi e sa To select a new scan area right click v Show Background Pattern wv Show Manipulation Pattern with the mouse and choose Select new scan region Click and drag in Zoom the Data Viewer to draw a red box a defining the new scan region Measure Distance Select Cross Section Clear Selection Focus Nothing happens immediately when a new region is drawn The region can be redrawn as many times as required until the region is correct then it must be activated pm L AN l SSC To activate the new scan region click the zoom icon in the Scan Control panel The new scan area should now be surrounded by yellow axes The old scan image is still shown in the background with the pixels shown with the real size Start the new scan with Run It is also possible to select a new scan region completely outside the previous image To zoom in or out Select View in the Data Viewer control and adjust Scale Select Zoom in the right mouse button menu click and drag Scroll the mouse wheel
192. ock and the cantilever are immersed in liquid the optical path of the laser beam reflected from the tip is changed because of the difference in refractive index between air and the liquid Therefore you will need to correct the optical path with the adjustment mirror when you immerse the cantilever in liquid See Section 4 2 4 for more details Tip When the system was set up for experiments in air and should now be adjusted for liquid turn the mirror a few degrees to the right Turn it to the left when you want to scan in air after an experiment in liquid For the NanoWizard 1 the mirror adjustment is on the front of the AFM head in a relatively deep recess The screw is marked by a red circle on the image here Insert a small screwdriver carefully in the black plastic housing and turn very gently The full turning angle of the screw is only approximately 100 degrees with stoppers at both ends Do not over wind the screw On the NanoWizard ll and NanoWizard 3 head the mirror is adjusted using a screw that looks like the laser and detector positioning screws This is the cen tral screw at the front of the head marked by a red circle in the image There is a release mechanism so that the positioning screw is not permanently in contact with the mirror to minimize drift Push carefully on the finger grip and turn gently to make contact It is easy to feel when the mechanism slots into place and the mirror can be turned Again the range of moveme
193. onsole terminal window Here many different commands can be entered directly Open a file browser for local folders on this computer Open a file browser for remote folders over the network NanoWizard AFM User Manual Version 4 2 137 Ji F f Nanotechnology for Life Science Instruments NewFolder File Browser SIE File Edit View Go Bookmarks Help In all the file browser windows there is an option on the a 0 BB we 8 ue amp right hand side View as icons Placesv x lt D lueders jpkdata NewFolder B lueders Seeman If this is selected then a thumbnail preview of each file Gemen is shown The system can recognise normal image aiii files and also our jpk format So this is useful for oe having a quick preview of which images are in a Soe certain folder B Downloads 1 item Free space 5 1 GB a Programming SS Sound amp Video rie System Tools Places to open a file browser E Accessories gt el Games gt N i i ye JP The JPK logo starts the main menu system for the Ubuntu operating G Internet gt system ven gt rae i At the bottom there are some commonly used options gt gt gt System to find settings for user Preferences or system Administration Ubuntu Software Center Places gt e e Sts oc TT In the other menu options are program lists and small tools or accessories Administration gt Lock Screen Log Out lueders
194. onstant UNKNOWN N m Fit Values Settings Thaw Mes D ma A ona K na ertical K n a Accept Value Full Range Y Full Range X Select X Range Sensitivity UNKOWN Spring Constant Fit Values Settings 10 03 kHz G Q 2 036x10 A 366 4 fm VvHz K 0 0927 N m ertical K 95 57 mN m Accept Value A Full Range Y Full Range X Select X Range average pm Hz average pm Hz Nanotechnology for Life Science 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 2 4 6 B 10 S125 TA 16 IB 20 22 Frequency kHz Run co Reset 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0 2 4 6 B 10 32 14 16 18 20 22 Frequency KHz Select Fit Range Run oo Reset ZH wd elep WwW ZH sud ewen y The result should be a frequency spec trum like one shown in the figure to the left Click Full Range Y to see the whole spectrum The spectrum should show a peak at the cantilever reso nance frequency It is possible to save the frequency spectrum clicking the right mouse button and selecting Save Data are saved in ascii format as tnd file If necessary zoom into the peak by right clicking in the plot area and choosing Set X range The new range is defined using click and drag The resonance must be fit with a Lo rentz curve The range for the fit can be define
195. ormation spe Single image without and calibration conversions cific to export format additional information Is reload in SPM IP Yes possible except ASCII formats tnd or dat File can be read by No for most JPK formats general software e g PowerPoint Yes ASCII formats out md dat File can be read by Some e g jpk SPIP AFM processing software 154 NanoWizard AFM User Manual Version 4 2 Note All trademarked names mentioned in this manual remain the exclusive property of their respective owners JPK Instruments AG Bouchestrasse 12 12435 Berlin Germany Tel 49 30 5331 12070 Fax 49 30 5331 22555 support jpk com www jpk com JPK DOCO0011 All rights reserved NanoWizard AFM User Manual Version 4 2
196. ound 1 5 V for non coated silicon cantilevers and around 5V for gold coated silicon nitride cantilevers If the Sum is significantly below 1 V then there is generally a problem and the system should definitely be readjusted 4 2 7 1 Not able to align laser onto cantilever Check first that the laser beam is correctly aligned on the canti lever as in the image here If it is not possible to move the laser onto the cantilever then probably the position of the cantilever or glass block is wrong Check the cantilever position on the glass block it should be placed centrally between the two grooves with the cantilever arm over the polished glass part close to the inclined edge as shown here If the cantilever is far forwards or to one side this Za x x may cause alignment problems Try repositioning the end of the cantilever nearer the centre of the glass block Make sure the spring and cantilever are on the left as you look at the AFM head from the front If they are on the other side the optical alignment will not be successful NanoWizard AFM User Manual Version 4 2 37 Ji I Nanotechnology for Life Science Instruments 4 2 7 2 Not able to align detector with laser reflection Sometimes the Sum value is reasonable but Vertical or Lateral deflection are at the 10 V position In this case turning the detector adjustment screws may not change the value because they stay at the saturated end position If so watc
197. ove in the glass block the cantilever spring must be on the left 4 2 2 Setting up the laser detection system As the tip of the cantilever is scanned across the sample its motion is detected by a laser beam that must be focused onto the cantilever The angle of the reflected beam from the cantilever is detected by a four segment photodiode The reflected laser spot must be in the centre of the detector to give maximum sensitivity for imaging and force control The adjustment of the laser beam on the top of the cantilever and on the photodiode must be repeated each time a new cantilever is mounted in the glass block since the cantilever will always be in a slightly different position Open the SPM software and click the icon on the shortcut bar to open the Laser Alignment window DR Laser Alignment Est Vertical deflection 0 12 V Da Lateral deflection g The Laser Alignment window gives a graphical representation of the detector signals for the adjustment process The Sum value is the total signal from all four quadrants of the detector If the laser beam does not fall onto the detector the Sum will be 0 V and no red spot is displayed If the reflected beam reaches the detector then the position is repre sented by the red spot The actual values of the vertical and lateral deflection are also given in Volts The shortcut icons allow the user to toggle quickly between a large and small view The maximize icon enlarges t
198. ox is selected the calculated spring constant value can be used with Accept Value or another number entered manually If for some reason you want to discard the measurement after changing the cantilever for example UNKNOWN can be reselected and the values will not be used If the fit is reasonable as in the above example click Accept value and activate the tick button M Use it The spring constant is now applied in the rest of the software In the Spectroscopy window the y axis vertical deflection will now be displayed in units of force nN In Contact mode the value of the Setpoint is also displayed as a force Besides k the spring constant the fitting algorithm also calculates some other useful values f is the resonance frequency Q is the quality factor Q factor A is the integrated amplitude of the resonance Typical values for the Q factor are a few hundred in air and around 1 3 in liquid Note that there are differences in the way the spring constant or k is calculated or measured which are beyond the scope of this manual to discuss in detail One difference is between the dynamic value calculated here using the thermal noise and the static value such as when a large range force curve is performed Another difference is be tween the spring constant quoted by manufacturers which is measured or calculated perpendicular to the cantilever and the value Spring Constant calculated here which takes account of the fact th
199. p Welocit K 436 73 S a i e f E SS r EEZ Lal the tip velocity for particular settings or as an alternative way to set the scan ime for Image 23 min l speed The total time for the image is also displayed calculated for the overall set of parameters NanoWizard AFM User Manual Version 4 2 73 Ji F f Nanotechnology for Life Science Instruments 5 6 Intermittent contact mode 5 6 1 Phase contrast During an AFM experiment in intermittent contact mode the cantilever is driven at a specific resonance frequency Depending on the conditions of the sample interaction there will be some phase shift between the drive amplitude ap plied to the piezos and the lock in amplitude measured from the detector Most contributions to this phase shift are independent of the sample position and will not change over a scanned image The part of the phase shift that is due to the tip sample interaction can show contrast between different parts of the sample This phase shift can be meas ured and displayed in a phase image The height and phase images are collected simultaneously Height Sometimes in the phase images two different components within a topograph ically flat sample can be distinguished as in the example shown here seo The quality of phase images is generally strongly influenced by varying the setpoint Often the phase contrast is lower in intermittent contact mode imaging in liquid since the width of the reson
200. ped here OK J Cancel NanoWizard AFM User Manual Version 4 2 119 Ji F f Nanotechnology for Life Science Instruments 8 3 1 Ramp Settings Ramp Settings Baseline n Z Closed Loop Z Start Option Distance from surface f Piezo approach is selected each force curve of the experiment Height Piezo retract starts in idle mode i e the cantilever is approached to the surface with Piezo approach The Ramps Settings provide automatic Baseline Adjustment Sec tion 6 2 1 Z Closed Loop Section 0 as well as different Z Start Options that define the piezo position at the beginning of the force curve Adjust Baseline Velocity Continue from previous the approach setpoint EEGEN ZE In Continue from previous mode the force curve is repeated right after the last force curve segment without any special starting position Z Start Option Piezo retract Piezo retract starts each curve in piezo retracted mode The cantilever Velocity is moved with Velocity until the first segment begins It is also possible to start each force curve from a defined Distance from surface Beginning from Height relative to the measured sur face the piezo extends with Velocity to the first force curve segment Z Start Option Height Velocity Z Start Option Absolute Z position Similar to Distance from Surface the Absolute Z position option Height P10 Jum starts from a defined but absolute piezo posit
201. pically the line rate is set to values between 0 5 and 2 0 Hz Choose generally lower scan rates if e the sample is rough strongly corrugated e the scan size is high e intermittent contact is selected as the imaging mode Note that the full range of values to control the speed of the tip over the sur face are found in the Advanced Imaging Settings panel NanoWizard AFM User Manual Version 4 2 Pixels x Offset Y Offset Scan Angle 512x513 w The resolution of the image can be set with the number of Pixels Normally it is set to 512 x 512 The higher the pixel number the better the resolution for a given image size but the longer the scan will take because there will be more scan lines The total scan region of the piezo is 100 x 100 microns Within that range smaller scans can be performed in different locations The X or Y Offset can be entered directly in this text field or set using the mouse in the Data View er If the instrument is left on over a night or weekend then the scan offset should be set close to zero to reduce strain on the piezo Likewise for very prolonged scanning it is better to offset the sample than to scan a small region right on the edge of the piezo range ei o a deg The Scan Angle can also be set graphically or using the text field By default the scan angle is set to 0 degrees At values of 0 or 180 degrees the fast scan direction is perpendicular to the cantilever MRS lt gt A
202. played E CellCapture py E ForceWatch py E Multisc E Delay py E HeadLaserUpdater py B ees B DriftTestFlipFlop py E HeadTemperatureUpdater py E Ramang f B fake py B Hwrest cH1a py armas Select the correct script and click Open B force distance test py E ImageScanListenerDemo py E RTStart E ForceAutoscan py E KelvinForce py E SelectA 0 pu File Name ForceBaselineAdjust py Files of Type JPK script v Open Cancel m ScriptCenter home kate release OI yp st lt ForceBaselineAdjust py oo e s ge 00 Pago ld ForceBaselineAdjust py 3621 088006 09 25 11 57 54Z rosea 99006 09 25 11 57 54Z roga The script window will open containing a descrip tion of the script followed by the script text itself on the left and on the right a This is done as follows the zero force baseline is determined by panel where param ete rs analyzing every scansPerAdjust th force scan The furthest percentToAverage points of the approach scan are averaged to find are ente red Always read the vertical deflection value corresponding to zero force vas x i the script description for instructions Adjust the relative setpoint while force scanning to maintain a constant force There are several experimental effects e g changes in the buffer solution that can cause changes in the vertical deflection signal that are not related to force changes This script helps compensate for such changes a
203. ptical Calibration iS Import Optical Image Pump Control FluidicsModule Temperature Controllers Voltage Output Settings gt gt ExperimentPlanner X Voltage Output Settings Output Channels Axis 4 AxisS Axis 6 Axis 7 Axis amp Ce Hardware KFM or CAFM module Output type Constant Voltage Voltage sl 0 000 a v Input Channels Precision 5 Set the conversion for Precision 5 UNKNOWN voltammetry Oscilloscope This feature is started using Accessories Voltage Output Set tings The settings here allow a voltage to be sent out to a free DAC on the Signal Access Module see Section 9 3 This is useful for some electrical measurements to set a variable voltage without using an external power supply for instance for oxidation lithography conduc tive AFM or electrochemistry measurements The Output Channels settings are stored in the property file for the instrument and depend on the controller type Typically Axis 4 would be used see the channel assignment list with the control ler information for the Channel number of the BNC connection External Hardware is a switch that changes between the default 0 10V range direct from the Signal Access Module and 10V 10V range only available if the KPM box is connected in be tween this is a separate optional electronics module lf Output Type is set to Constant Voltage then the output is taken directly from the Voltage text field
204. ptics The optical magnification is generally much lower in this configuration than on an inverted optical microscope JPK TopView Optics 2 2 Assembly 2 2 1 Location find a quiet place for the instrument For highest resolution imaging and most sensitive force measurement it is important to minimize vibrations and noise from the environment of the AFM Reasonable results may be obtained even in non ideal situations but generally it is worth reducing external noise or isolating the AFM to obtain reliable results at higher resolution The most important thing is to place the AFM system in a quiet room on a solid stable base e Building vibrations are usually smallest in the basement AFM labs on higher floors may have more noise prob lems Remember the effects of lifts or other large machinery may be felt even some distance away e The temperature should also be stable ideally the ambient temperature should not change by more than 0 5 C per hour e Moving air will cause vibrations or noise so place the AFM system away from doors windows and vents Keep doors and windows shut Air conditioning can cause noise problems if it blows near the AFM system e Any loud noises or passing people can disturb the AFM experiment so small rooms with just a single instrument will generally give the best results Good vibration isolation is always required for scanning probe microscopy experiments The two AFM head cables can transmit unwanted v
205. pulation Control panel and controlled using the IGain and PGain feedback values in the Manipulation Control panel These values are independent of the normal imaging setpoint and feedback gains set in the imaging Feedback Control panel Often the setpoint for the manipulation is set at a higher force than for imaging so that objects can be moved or the surface modified During the manipulation the tip moves along the list of specified paths in order Between the paths the tip is lifted from the surface forming a series of disconnected lines The paths for manipulation can be drawn freehand in the software or loaded as a scalable vector graphics svg file created by typical vector graphics software such as Illustrator or CorelDraw The path is defined as straight lines joining the points so a curved line will have many points very close together and for a completely straight line only two points are required In Manipulation mode the default mouse action is to draw manipulation paths in the Data Viewer Click and drag with the mouse and the movement will automatically be convert ed into a set of points in the Manipulation Control panel wv Show Background Pattern wv Show Manipulation Pattern When the mouse is released then the next click and drag is Select New Scan Region Rotate Zoom Shift Measure Distance Select Cross Section saved as a new set of points in the next path The example here shows a single freeh
206. r from the sample using only the z piezo If Approach is clicked again the cantilever will return to exactly the same position because there is no mechanical movement This is good for changing settings between scans or measurements The actual distance from the sample depends on the z range and the position of the sample but it is not usually enough of a safety distance for any mechanical movement for instance to lift up the AFM head NanoWizard AFM User Manual Version 4 2 Clicking the Retract button again second and subsequent clicks means the cantilever ae a is retracted from the sample using the stepper motors The distance is the amount set in the Stepper Motor window After a motor retract the cantilever will not return to exactly the same sample location typically within a couple of microns Moving the cantilever further away from the sample is good for adjusting the sample position e g retracting 20 50 um or lifting up the AFM head e g retracting 300 500 um System Status The System Status panel at the bottom left hand corner of the software has an entry aun ue a that shows the Status of the system Here are the main options that will be shown Lateral Def 2 61 V between measurements Vertical Def 1 221 W Status Idle e Idle means it is waiting at the surface approached and ready to start a measurement e Piezo Retracted means there has only been one retract from the surface and the motors have
207. rd for automatic Motor Leveling if installed 5 3 3 Accessories Accessories fi CCD Camera Launch the CCD camera 4 1 1 DirectOverlay Optical Calibration DirectOverlay Optical Calibration in SPM 8 2 E import Optical image Import optical mage import a calibrated optical image into 0 Pump Control the data viewer 9 FluidicsModule Pump Control for syringe pumps 4 6 2 Temperature Controllers Temperature SEN 4 6 1 E Voltage Output Settings for FONNO a vonage on a DAC ExperimentPlanner opens a window for running of custom 0 gt ExperimentPlanner l ized experiments 18 NanoWizard AFM User Manual Version 4 2 Vertical deflection Lateral deflection Error signal Height measured Colortable Scan List Quantitative Imaging List Advanced Open Script JPK SPM Jython Console Real Time Scan Logging Settings Advanced System Status Data Viewer List of active channels that can be displayed in the data viewer Colortable choose the color to display the images Scan Quantitative Imaging List manage holding and sav ing of scan data Advanced Open script loads scripts to perform custom experiments JPK SPM Jython console gives a direct command line for running macros etc Open the Real time oscilloscope window Manage the Logging Settings Open the Advanced System Status window Some pull down menus depend on the choice of Measurement mode iG Open Saved Image ia Scan List
208. rement modes The more channels that are saved the larger the data files 26 NanoWizard AFM User Manual Version 4 2 The Filename root is a prefix for the filename the time and date are added to automatically generate file names This is convenient if the computer clock is set at the correct time Files are saved by default in the chosen Folder which can be set separately for each different data type in each tab The option Use one folder for all data types at the top is a useful shortcut to save all types of data in one location Browse can be used to select an existing directory or to create a new directory In addition to the data channels it is possible to add comments such as notes on the sample or the probe into the stored file Lei Autosave ON Autosave OFF To avoid losing any images the Autosave function can be activated in the main icon toolbar There are two options for images either Autosave saves only the full completed images or it saves every partial image even if only one data line is collected This is controlled by the tickbox option Auto save applies also to incomplete images in the Saving Settings window Interesting partial scans can always be saved manually from the scan list 3 3 Software versions and updates The software programs for online control SPM and data or Data Processing DP are developed by JPK If you have any problems or suggestions for improvement please send an email
209. rk et al 2001 Ultramicroscopy 86 207 215 0 817 Generally used Used when first reso Example correction factors for rectangular 2 0 251 nance frequency is too cantilevers from Butt and Jaschke 1995 low 0 0863 Not generally used Note that these correction factors are only valid when the laser spot is positioned on the cantilever tip The correction factors and sensitivities are changed if the laser spot is moved towards the cantilever chip Especially for higher modes Ge and higher peaks the calculated spring constant is changing drastically by moving the laser spot along the canti lever When using very soft cantilevers in liquid the determination of the spring constant can be problematic The best way is to calibrate such cantilevers air In case that the cantilever is coated with substances like proteins which must not dehydrate either higher modes of the thermal noise spectrum in combination with corresponding correction factors can 102 NanoWizard AFM User Manual Version 4 2 be used as mentioned above But then be aware that the determined spring constant can deviate drastically with changing laser spot position Another way could be to determine the spring constant of the clean cantilever in air using the resonance peak In air the resonance peak should be high enough to be clearly distinguishable from the low fre quency noise Note the spring constant value and coat the cantilever e g with the protein of intere
210. rt 8 2 2 Coarse alignment with optical image The Direct Overlay feature is enabled by the linearized x and y piezos As such the calibration procedure can be car ried out on an area up to 100 um x 100 um the piezo range of the NanoWizard The optical image is extrapolated to 50 larger than the calibration area i e maximal region 150 um x 150 um When using higher magnification objec tives 63x and 100x it will be necessary to reduce the size of the area to be calibrated below 100 um since part of the AFM region may be outside the optical field of view To ensure that the appropriate area is calibrated the user should initially make a coarse alignment of the area to be scanned using the AFM with an appropriate area within the optical field of view Approach to the surface as normal and use the optical microscope to find the region of interest for scanning If the JPK FireWire camera is being used start JUnicam by clicking on the camera icon If another camera e g specialized fluorescence camera with separate software is being used start its software to get a live image of the optical field of view The Outline tool can be helpful to show the scan area and move either the sample or the cantilever until the scan area is within the interesting region of the optical image 110 NanoWizard AFM User Manual Version 4 2 The safety distance between the tip and the surface depends on the approach Target A Height see Section 4 5 Mak
211. rter than the steep straight part of the force curve The display of the fit line can be turned on or off using the Show fit tick boxes in the Analyze panel The fit lines give a quick guide to show whether the Fit length is set appropriately if there is a significant difference between the line and the force curve the Fit length should generally be reduced The Adhesion is measured as a single value the difference between the lowest data point and the free end of the curve Note that this analysis is not valid for more complex force curves n general force curve analysis depends strongly on the type of sample or interaction being studied and particular models should be chosen and fitted separately for the particular type of data See the Data Processing manual for the full range of analysis which is available offline 86 NanoWizard AFM User Manual Version 4 2 6 5 2 Force spectroscopy in AC mode Typically force measurements are performed in contact mode Force distance experiments can also be performed in AC modes however measuring the amplitude or phase of the cantilever during the force spectroscopy cycle When the spectroscopy window is opened in AC mode the default channel is Lock in amplitude The amplitude of the cantilever decreases from the free value on the right as the cantilever approaches the surface towards the left Lock in amplitude extend V A pena apnyujdwe ul 7907 Other channels includin
212. rtheless most of the infor mation concerning the SPM software is relevant for all NanoWizard customers The SPM software covers all Nan oWizard applications as well as NanoWizard 3 specific modes and features as HyperDrive or MicroRheology that cannot be performed with the NanoWizard or II system Particular applications and modes need special equipment e g NanoOptics version HyperDrive cantilever holder or additional external electronics e g Piezo Response Force Microscopy These modes are optional and are covered in corresponding manuals ask JPK for information If you have any problems with your system please ask JPK for assistance 49 30 5331 12070 10 NanoWizard AFM User Manual Version 4 2 2 Installation 2 1 Components 2 1 1 NanoWizard head and controller The most important parts of your AFM system are the NanoWizard AFM head and controller Other components are different for the BioScience or Nano Science systems but the AFM head and controller are the same for both Laser filter do not remove Stepper motor housing A Feet for positioning AFM head 2 1 2 BioScience and NanoScience systems Controller with signal access module If the NanoWizard was ordered in the BioScience version with a Life Science sample stage then it can be installed on various inverted optical microscopes made by Zeiss active anti vibration table or air table depending on the type of stag
213. s for file saving in all formats Choose Approximate data size 1 37 Mb Vertical deflection v Lateral deflection the Force scan maps option to set the X position Tip Y position Tip channels for saving in force maps Error signal Lock in amplitude 2 Lock in phase 2 Lock in amplitude 3 Lock in phase 3 along with the default directory and filename Output amplification Photon counter 1 Photon counter 2 Tunnel Current Logarithmic Input Height measured Photo Sum Precision 1 Precision 2 Precision 3 Precision 4 Several formats for saving data are possible for each force map the full force spectroscopy curves or selected Precision 5 D Precision 6 Precision 7 Precision 8 Precision 9 Precision 10 Precision 11 Precision 12 High Resolution images or text values from analysis channels Missing Channel 6 7 5 ay ov Pausing force mapping Pause Force Scanning Ly Force Mapping Pause The force mapping experiment is paused with the cantilever away from the surface so that the laser deflection can be realigned No other settings can be changed until the force mapping is restarted from this panel Note that Adjust Baseline should be enabled to take account of large changes in Vertical deflection during a force mapping experiment Vertical deflection
214. s bar at the bottom of the software provides information about the most recent software actions Confirmation of file saving warnings and other information will be displayed here It is normal that this is empty when the software is first started 3 1 3 The menu bar Along the top line of the user interface you will find a pull down menu bar as shown here The table below lists the options available and gives references for more detailed explanations of their functions in imaging Setup Motors Accessories Data Wiewer Advanced Imaging Window Help Pull down menu Short explanation Details in section Setup Setup Autosave Autosave enables automatic saving of scans 3 2 8 Scan Repetitions Scan Repetitions 3 2 3 Laser Alignment Open the Laser Alignment window 4 2 2 2 Range Limit the Z Range of the z piezo for better z resolution 5 3 1 Us Z Piezo Display Open the Z Piezo Display vertical display 5 3 Approach Parameters Change Approach Parameters and initial piezo position 4 5 lel Saving Settings Saving Settings manage data saving 3 2 8 e Channel Setup Choose the data Channels that are stored to memory 3 2 8 Advanced Feedback Settings Open AC Feedback Mode Wizard window in AC Mode for 4 4 2 TTL Control Cantilever tuning Exit Ctrl Q TTL Control control and monitor TTL signals 0 Exit Close the SPM program Motor Motor Stepper Motor Control the coarse height position using the Stepper motors 5 3 2 Motor Leveling Open the wiza
215. srrrrsrrrrsrrrrstrrrrrrrrrrrrrnntrnnrrnen tenn ren nren 51 4 6 3 Pump control for Aladdin DUMPS E 53 TES Men nn Le ue BEE 56 e ALAC Se PENG E 56 5 1 1 Image properties and the Scan Control panel ccccccccccecceeeseeeceeeeseeeeeeeceeeeesseeeeeeeeeeessaaaeeeeees 56 5 1 2 TheData Viewer WINdOW ss snesensesennesrreorrrrsrrrrsrrrrrrrrrsrrrrsrrrrstrtrstrrrstrtrstrtnnrtrtrtntrrnnnren nnne n nenne 58 51 3 Selecting a new scan region GGoomimg 60 5 1 4 The A o Leg E 62 5 2 Feedback adjustment for imaging the feedback Control oanel 64 ii NanoWizard AFM User Manual Version 4 2 5 2 1 Feedback gains for imaging IGain ANd PGaiN cccccccccsseseeeceeeeecaeeeeeeeeeeseeaeaeeeeeeeeesaaeeeeeeeessaas 64 5 2 2 Simple procedure to optimize IGain ANd Pan 65 5 2 3 Scan speed and feedback adiusiment 66 5 3 Controlling the piezo ANd stepper Motors cccccsseeccccceeseeececaeueeeeecseueeeeeceeaeeeeecseaueeeessaaeeeessaaeeeeessuaeeeesesaaess 67 5 3 1 Reducing Z Range for higher resolution cccceccccseeeecceeeeeeseeeeeceaeeeeeceuseeeseneeesueeeseeeeesseneeesaeees 67 5 3 2 Independent movement of the stepper MOTOS ccccccseeeceeeceeeeeeeeceeeeeeescaeeeeeeeseueeeeesaaaeeeeseaneees 68 5 3 3 Automatic Motor Leveling cccccseccccceececceeseecceeeeeccacesseeeeecaueeecsaueeeseseeeseueeecseeeessaseessneesssass 70 5 4 Tools fOr monitoring scanmimg EEEE EEren EEE 72 5 4 1 The Oscilloscope win
216. st Determine the sensitivity anew when starting the experiment in liquid use it and type the spring constant determined in air manually NanoWizard AFM User Manual Version 4 2 103 Ji 8 f Nanotechnology for Life Science Instruments 8 Available Software Extensions The standard software version can be extended by a variety of additional features in order to enable specific meas urements These additional extensions must be purchased separately and allow a powerful extension of the capability of the standard software You can see which Available Extensions are included in your personal SPM software from the main drop down menu at the top of the window Help Info The most common Available Extensions are ex plained in this chapter Some of them are add ons or accessories to the Measurement modes but there are also addi tional Feedback Modes and Measurement Modes available 8 1 Quantitative Imaging Quantitative Imaging QI is a force spectroscopy based imaging mode Please read Chapter 6 to get an overview of the basics of force spectroscopy mode and settings Similar to Force Mapping a whole force curve is measured at every pixel of the selected sample region The main difference lies in the algorithm of the tip motion and the sample rate which both allow a higher scan velocity Finally QI mode gives quantitative data in the form of height adhesion and slope images in high resolution Additional channels can b
217. t Ctrl F can be used at any time to save the last recorded force curve The name and curve settings are all taken from the Saving Settings panel 6 6 2 Force Scan Series List E Force Scan Series List Recent Series INumber of listed entries jperies 2012 08 09 10 26 04 senes 2012 06 09 10 19 19 Seres 2012 08 09 10 1 7 56 Seres 2012 08 09 10 16 34 Preview Autosave File Filter C Autosave No Filter O Simple Filter O Advanced Filter _ox The Force Scan Series List manages the saving of force spectroscopy scans When a scan has finished the file is automatically shown in the Force Scan Series List Only a limited number of scans are held older scans are re moved automatically as new scans appear The number can be changed using Number of listed entries Note that the more scans you store in Scan List the more memory is used Files with this symbol are not yet permanently saved to the hard disk At any time the scan can be saved clicking directly on the icon or with the aon mouse button on the scan name number and selecting Save data Once the file has been saved it will be displayed with this icon S The Remove icon clears the image from the software If the scans have been saved either manually or with Autosave they are still stored on disk even if they are removed from the list either by clicking on Remove or by letting them reach the end of the list If they have
218. t Optical microscope image Prism Laser Mirror Adjustment Mirror Adjustment Detector Cantilever Screw If the laser spot can be seen on the cantilever but the Sum is close to 0 V then probably the mirror needs adjusting A small change in the mirror angle makes relatively large change in the optical path so the mirror is only used for coarse adjustment Once the Sum is reasonable the detector positioning screws can be used for fine alignment to maximize the Sum and center the spot The two Laser Alignment screenshots here show the situation before and after adjust ment of the mirror Fo Laser Alignment PS Laser Alignment Vertical Def Vertical Def 10 11 W 7 09 V Lateral Def Lateral Def cl 48 W 5 09 V O sum sum 0 04 VM 1 19 W Coo y y O h6hMmMUmUt lt SCSCCCCC SCY The mirror will not always need readjusting every time a new measurement is made The mirror helps to correct for the difference in angle of the optical path when the cantilever is immersed in air or liquid Adjustments need to be made when changing from air to liquid operation or back again Usually the mirror will not have to be readjusted for similar cantilevers except on changing from air to liquid 4 2 5 Adjust the spot onto the centre of the detector Once there is a reflection from the back of the cantilever onto the photodiode then the detector must be moved until the laser beam falls on the centre using the adjustment
219. t contact mode imaging close to the surface 1 Start with Contact mode as the feedback mode 2 Approach until the cantilever is in contact with the sample 3 Click Retract once to put the instrument in piezo retracted mode indicated in the system status panel 4 Change to Intermittent contact mode liquid as the imaging mode 5 Perform a sweep curve in the Cantilever Tuning window as described previously for AC mode in air The resonant frequency of the cantilever will be much lower in liquid because water is moved with the cantilever 6 Approach again to image as normal in intermittent contact mode Resonance frequency values of about 8 12 kHz are typical for the silicon nitride probes around 100 um long with spring constant around 0 3 N m The resonance frequency of the cantilever depends on several parameters including e The total amount of fluid and the depth of the cantilever in the liquid e The properties of the cantilever itself e The geometry of the vessel fluid cell e The viscosity of the liquid If the cantilever tuning is performed far from the surface the resonance frequency may have moved significantly by the time the cantilever reaches the surface Therefore the setpoint may need to be much lower than expected to reach the surface If the cantilever tuning has been performed far from the surface then it is recommended to retract using the piezo only by clicking the retract button once as described above to re tune
220. t saved Note that the threshold value set here is an absolute value even though the adhesion has actually a negative sign in the retract curve The Advanced Filter allows very complex filtering operations as the full po tential of the JPK Data Processing software is used The analysis must first be set up in the Data Processing software and saved as a process file Any combination of operations can be used for the analysis but the process must include one filter operation The filtering can be on the results of any opera tion or a combination of them The process must be saved in the normal way as if for batch processing this creates a file with the extension jpk proc force Please read the Data Processing manual to see in detail how a process file can be created and saved j Use Open File to select the DP process file for filtering Once the process has been selected the Process file name is shown in the panel Each force curve is processed in the background and only the results of the filter are shown Accepted is the number of files that have a filter result 1 or true Not Accepted is the number of files that a filter result O or false Unclassified is the number of files that produced an invalid filter result Total is the total number of curves saved since Reset was clicked Please note that ALL the files are saved The filtering is complex and happens in the background so it is a safeguard that the files can be recovered
221. t scanning starts the active force scan settings GUI Settings for force spectroscopy can be loaded Autosave GUI the spectroscopy GUI mode can be activated Autosave activates the autosaving mode 128 NanoWizard AFM User Manual Version 4 2 Imaging Start scanning Settings V GUI V Autosave V Fluidics Module Flush tube Inject Set Takeup Rate Set Injection Rate Optical image save image Imaging Start scanning starts the active image scan Settings for imaging can be loaded GUI the image GUI mode can be activated Autosave activates the autosaving mode Fluidics Module Flush tubes primes the tubes and ejects the liquid into the waste bin Inject directly injects into the sample chamber from specified reservoir Set Takeup Rate sets the rate for take up of liquid Set Injection Rate sets the rate for injection of liquid Optical image Save image save optical images More Help on the individual commands is given in the Experiment Planner itself When choosing a command from a list or clicking on one in the script help on the according command will appear in the help tab under the scripting window see image below In the first line the command will be quoted and parameters to be eventually set are highlighted The meaning of the parameters is explained in the upcoming lines Next inline the effect of the command is explained and an example of how to use the command is given In the lower
222. t values of 90 deg 270 deg the fast scan direction is parallel to the cantilever The icons in the Scan Control panel allow you to move around the sample surface together with the Data Viewer Open a new Data Viewer window see Section 5 1 2 The zoom symbol activates a new scan area that has been drawn in the Data Viewer using the right mouse button option Select new scan region See Section 5 1 3 Switch between normal image scanning and Line Scanning mode In Line Scanning mode one scan line is scanned repeatedly rather than moving to the next line to form an image This can be useful for adjust ing settings or for flat samples that do not show surface features on every part of the sample The scan line is set using the Line slider in the Scan Control panel mio Line 0 sl The slider displays the current scan line number and can be used to move the tip over the surface to a particular part of the sample click and drag on the slider arrow It is possible to move the slider by single steps by clicking on the background of the slider to the left or right of the handle or by activating the slider by clicking on it then using the left and right arrow keys on the keyboard Once the slider is in the cho sen position a normal scan can be started using Run or Line Scanning can be started with Run if it is selected The repeated scan lines doe not form an image so the Line Viewer Section 5 1 2 or Oscillo scope Section
223. the cantilever near the surface lt can be helpful to compare the tuning frequency peaks with the natural frequency response of the cantilever This can be done by looking at the thermal noise spectrum See Section 7 3 for more information For a simple frequency comparison the sensitivity measurement is not necessary and a typical value can just be entered in the text field without making the force spectroscopy measurements first 4 4 4 Force modulation mode Contact Mode wei In the graphical user interface Force modulation mode can be found in the drop AC Mode down menu with contact and AC mode Force modulation mode is something of a Contact Mode mixture between contact mode and intermittent contact mode and can be thought of as a kind of contact mode with an added vibration of the cantilever See Section 5 7 for more details The cantilever tuning for Force Modulation mode is similar to the normal tuning pro cedure for AC mode For imaging the setpoint value is the average vertical deflection and the feedback control is therefore similar to contact mode The oscillation of the cantilever provides extra amplitude and phase channels to observe differences in mechanical properties of the sample 4 5 Approaching 4 5 1 Coarse approach The whole NanoWizard AFM head can be raised or lowered using the three stepper motors which allows a wide range of sample heights to be measured The automatic approach routine can take a long time if the cant
224. the glass block lying on these surfaces d When deposited this way the polished optical When not in use and for cleaning in an ultrasonic planes of the glass block can be damaged bath always set the glass block on its side The glass block can be cleaned conveniently using an ultrasonic bath However please make sure that the glass block is held using a soft Support swimmer or mesh Do not ultrasonicate in a hard holder or glass beaker 0 3 3 Sample stage and sample holder Never immerse the sample holder or sample stage in liquid Do not allow the sample stage or sample holder to come into contact with acids bases or other aggressive chemicals 0 3 4 Prevention of liquid spilling onto motor spindles When the AFM head is put into an upright position after an experiment has been performed in liquid sometimes some fluid drops may fall from the glass block onto the back spindle of the AFM head This fluid may lead to corrosion it is especially important for users of the NanoWizard microscope Please remove the fluid from the glass block immediately after use with a soft piece of tissue paper NanoWizard AFM User Manual Version 4 2 9 Ji F f Nanotechnology for Life Science Instruments 1 Introduction words This manual is mainly dedicated to NanoWizard 3 customers The NanoWizard 3 hardware head controller differs from prior systems so the installation steps apply to the NanoWizard 3 system only Neve
225. the list using the arrows top up down bottom NanoWizard AFM User Manual Version 4 2 63 Ji 8 f Nanotechnology for Life Science Instruments For more display options right click on the file entry and choose from the menu dis w Show played v select Show shows hides the image in the data viewer Focus Bee Select displays green axes and selects the image for other functions such as cross section measurement or shift optical image for DirectOverlay KM Focus centers the viewer on the selected image 5 2 Feedback adjustment for imaging the feedback control panel 5 2 1 Feedback gains for imaging IGain and PGain When the feedback mode is changed in the drop down menu on the shortcut Contact Mode e toolbar see Section 4 4 the JPK software automatically updates the value of AC Mode the gains IGain and PGain in the Feedback Control panel to appropriate default values for that imaging mode Generally the initial parameters will need Force Modulation Mode optimizing however to obtain the best images of each sample IGain and PGain correspond to the integral and proportional gains for the adjustment of the height z feedback loop so the optimal value for the gains depends not only on the imaging mode but also on the sample environment air liquid etc the scan size and scan rate the choice of setpoint and the topography of the surface and the selected z range The time constant IGain determ
226. the retract curve These Full Range Y options are also available in the right click Full Range X Select X Range ce di mouse menu 100 50 0 50 100 150 200 250 l Height measured and smoothed nm Manual Sensitivity Selection Select Fit Range Sensitivity Spring Constant h 0 4 0 4 Only the i t of th hould b deiere nly the linear part of the curve should be 72 12 mc use it chosen for the sensitivity fit Check that the v Use linearizing 0 2 0 2 MEN eebe Accept Value red fit line accurately represents the steepest 5 o TEP linear region of the force curve or change g the fit region to improve the results The 5 02 02 slope of the line is automatically transferred 2 to the left hand part of the panel but is only 9 04 4 5 permanently stored once Accept Value has SS Wi been selected Full Range Y Full Range X Select X Range go pp If the force curve is very linear as in the 100 250 8 50 100 150 200 250 dt EE example here fitting should be the best way Manual Sensitivity Selection Select Fit Range to measure the sensitivity NanoWizard AFM User Manual Version 4 2 97 Ji F f Nanotechnology for Life Science Instruments a springonstant fo ue It is also possible to manually select a sensi O pm tivity using the Manual Sensitivity Selec ee S SET E tion button This may be more helpful if the 5 oo oo curve is less ideal and does not
227. the sideports of the optical microscope Switching off the laser using the software For safety reasons for both laser diodes the user can switch off the laser with the laser toggle button in the JPK SPM software if the AFM head is not in use It is not possible to turn off the laser when the canti lever is approached on the sample surface and during scanning em Laser on Laser oft Laser disabled NanoWizard AFM User Manual Version 4 2 Electric shock hazard Do not remove or open the cover of the AFM head or controller when it is plugged into the power sup ply The voltage 115 or 230V only supplied to the system may cause injury to the user Do not stick anything through the slots in the top of the AFM head or other open parts of the AFM head or controller Removal of covers or servicing parts is for trained JPK personnel only There are no user serviceable parts or components inside Ask JPK for assistance if any problem occurs Sensitive environments Our product is fully CE approved Do not use the AFM with its controller in an electromagnetically sensi tive environment Electrostatic discharge The AFM head is sensitive against electrostatic discharge Touch ground before you use it or use a ground bracelet Prevention of damage to the AFM head Do not use the AFM head or any of its components under water or any other fluid the only exception is the glass block its spring and the attached cant
228. the standard settings do not have administra DEN a tor rights this is more secure especially when the AFM computer is es EE connected to the internet Zeg Programming E Network Tools Sound amp Video Vv Vv WY VY VY MY MY MY MY MYM system Tools Bee vay we Use the main system menu started from the JPK icon in the taskbar amp Printing DEE 2 startup Disk Creator at the bottom left of the software to choose Places gt B Synaptic Package Manager Ss system Monitor pa System Testing System Administration Users and Groups Log Out lueders Help and Support GJ Time and Date Shut Down About GNOME GD Update Manager J Sepp Hutt NanoWizard AFM User Manual Version 4 2 139 Ji F f Nanotechnology for Life Science Instruments Authenticat x i i DI For security reasons you need to confirm the account password You need to authenticate to E ee So ar again when starting administration tasks An application is attempting to perform an action e that requires privileges Authentication as the super Enter the password for the jpkroot account user is required to perform this action Password for jpkroot f gt Details EN Cancel Authenticate Users Settings The dialog window Users Settings shows a list of the user accounts jpkroot l bi g Change CY vy nuit Account type Change Click on the button Add to create a new user account Passwor
229. tions and drift 4 3 1 Life Science stage The positioning screws A move the AFM head The three foot positions are marked in red The combination of a point a line and a flat surface ensures reliable positioning so that the head will always fit stably into the same position Always ensure that the feet slot firmly into posi tion The positioning screws B move the sample holder The sample is mounted on the inner sample holder D The arms of C are moved by the positioning screws and push the inner sample holder The positioning arms do not grip the sample holder tightly There is a small gap between the arms and the centre part The gap is shown here exaggerated to make it clear how the sample is pushed by the positioning arms When the sample is in the correct position the sample holder must be released by turning the positioning screws BACK one quarter turn In the life science ver sion one notes that the sample does not move during this de coupling The sam ple holder is then free from contact with the positioning arms as shown here but still held firmly by the magnetic contacts underneath The release mechanism is very important for high resolution imaging to remove the mechanical coupling between the sample and holder The standard sample holder fits biological standard samples such as petri dishes 50 mm diameter height below 10 mm and microscope slides as well as cus
230. to support jpk com SPM and DP are generally installed on an Ubuntu system but no specialist Linux knowledge is required 3 3 1 Updating the software Free software updates can be downloaded from hitp customers jpk com Customers are informed of major new re leases by email in between there may be minor updates for particular features and these can be downloaded at any time This web page is password protected please enter the login name and password The login name is your device number eg JPK00111 which you can find on the instrument and the password is provided with the AFM system Note that the website password is not the same as the root or jpkroot administrator password for the instrument computer The full install instructions are given on the website link here is just a short version To install the file on the computer log in with the administrator account jpkroot and copy the installer to a local directo ry e g to the Desktop The installer can be run from the linux console using this command sudo sh home jpkroot Desktop jpkspm xxx bin The full file path for the is required with xxx substituted for the actual release number and the jokroot password must be confirmed If you have lost or forgotten your password or have problems downloading or installing the upgrade please contact us for assistance E mail support jpk com or call our technical support line on 49 30 5331 12545 NanoWizard AFM User Manual V
231. tom made fluid cells Special sample holders are available from JPK such as the BioCell and CoverslipHolder which offer advanced performance such as temperature control perfusion etc Sample preparation information including different suitable petri dishes is given in the JPK AFM Handbook NanoWizard AFM User Manual Version 4 2 39 Ji 8 f Nanotechnology for Life Science Instruments 4 3 2 Standard stage The positioning screws A move the AFM head The three foot posi P tions are marked in red and are very similar to the Life Science stage The point a line and flat surface ensures reliable positioning so that the head will always fit stably into the same position Always ensure that the feet slot firmly into position The sample position is fixed The central magnet can be used to hold samples mounted on standard holders such as magnetic steel stubs Standard sizes are 12 mm diameter 0 9 mm thickness For imaging in air where there is less drag from the head movement samples can also be prepared on normal glass microscope slides For imaging in liquid a more firm mount is required such as the magnetic attachment O 4 4 Selection of feedback mode The feedback mode defines which signal is used for the main control of the Z position during imaging or other types of measurement The aim is usually to control the force or keep it constant during imaging for example The simplest type of feedback
232. ton down move the cross hair to choose a suitable frequency and setpoint as in the example shown here The Drive Frequency is usually chosen on the left upper rising edge of the peak The value of the Setpoint must be lower than the chosen lock in amplitude in the Cantilever Tuning window The setpoint is usually chosen around 70 80 of the lock in amplitude at that frequency although the actual numbers depend on the sample and conditions Lock in phase NanoWizard AFM User Manual Version 4 2 43 Ji F f Nanotechnology for Life Science Instruments If the setpoint is set too low as in the example here the cantilever imaging forces may be too high and damage the sample or the tip In liquid however the free amplitude may decrease between the retracted position where the cantilever tuning is performed and the surface In this case a lower value of the setpoint will be required to reach the surface It is safer to start with a higher value and then re duce it if necessary rather than risk damaging the tip or sample working Don 4 4 2 2 Oscillation amplitude and resonance curves It is important to have a good signal to noise ratio This means that the resonance curve must be well shaped and its peak must be high enough above the background noise Signal to noise at about 6 1 or better is reasonable The sig nal to noise ratio can be improved by increasing the drive amplitude but do not increase it too much Try t
233. tput type Cyclic Voltammetry K oi triangular O sinusoidal Turn round potentials Max Voltage Min Voltage Timing el 0 200 a te Melocity Number of cycles O Fixed number F 1 Infinite RUN Input Channels Precision 5 Get the conversion for Precision 5 a UNKNOWN C AN voltammetry Oscilloscope 148 NanoWizard AFM User Manual Before starting measurements check that the main Channel Setup has both the selected Input Channel and Output Channel switched on Otherwise data from these channels will not be seen in the software and hence cannot be displayed in the oscillo scopes Usually this means that Axis 4 and either Precision 5 should be selected Since the channel setup for the real time scan is handled sepa rately then the channels for real time scan capture are also im portant if the Voltammetry Oscilloscope is used see below If the settings are changed to Cyclic Voltammetry then the out put will produce a triangular saw tooth output with the settings The cycles can start and end at either the Min Voltage or the Max Voltage The time for the cycle is set implicitly through a Velocity in V s When RUN is clicked then either continuous cycles or a Fixed number of cycles will be started When RUN is not active be tween cycles the voltage stays at the Resting voltage The current voltage amplification will generally be done with some external hardware and t
234. und 1 0 then it is probably the correction factor that should be Save Calibration applied to the old calibration value Comments such as the date and user or height of the calibration grid can be entered After clicking Save Calibra tion you will be prompted for a new filename for the edited file 7 1 2 Hardware z linearization Height Measured Generally the JPK NanoWizard is equipped with a z linearization A capacitive sensor continuously measures the length of the z piezo The capacitive sensor can be read out on monitor channel 3 and is called Height measured The movement of piezo material when a voltage is applied always suffers from some nonlinearity and hysteresis Normally the height of the piezo is taken directly from the voltage applied to extend it this is the normal Height chan nel When z linearization is enabled the capacitive sensor measure the actual current height of the piezo and this value is shown in the Height measured channel The calibration of the capacitive sensor for the Height measured channel is carried out by JPK and does not normally have to be repeated by the user The calibration of the Height channel varies with the ageing of the piezo and should be repeated every six months or so see Section 7 1 1 Note that the piezo should be calibrated over the height range that will be most used for imaging so when the Height channel is used for small z ranges as discussed below it should be calibrated
235. updated whenever Reset is clicked In Dynamic baseline update it is possible to set an averaging time that adjusts the current value of the feedback channel In this case the setpoint is varied during the approach In the user interface the Setpoint value stays constant and the Approach Baseline value is updated to take account of the changes The actual value of the feedback channel being used as the current setpoint can be calculated by adding the Approach Baseline number to the Setpoint Example for contact mode If the Vertical deflection on the photodiode far from the sample was 0 52 V for example and the Setpoint in the Feedback Control panel was 1 0 V then the feedback routine would seek to achieve a difference relative setpoint of 1V between the actual deflection and the Approach Baseline value here automatically initialized to 0 52 V and Start the approach with a real deflection setpoint of 0 48 V 4 6 Environmental control for experiments 4 6 1 Temperature control and data saving Accessories fi CCD Camera DirectOverlay Optical Calibration iS Import Optical Image Pump Control FluidicsModule Temperature Controllers Voltage Output Settings 2 gt ExperimentPlanner Many different temperature control accessories are available from JPK which can be used to heat or cool the sample region The details of these accessories are covered in their separated manuals There is a general interface for controlli
236. ured v real year month day hrs min sec Note the saved data depends only on the Saving Settings not the channels that are displayed during the real time scan operation Precision 9 Precision 10 Missing Channel 9 2 3 Logging Settings Advanced The settings for the error logging can be adjusted via Advanced E Open Script Logging Settings JPK SPM Jython Console Ox The logging settings determine where relevant messages errors and warnings should be displayed Dialog Logger gt Log File information written in jpkdata log StatusBar Logger gt Dialogs information displayed in pop up dialogs gt Status Bar information appearing in the lower status bar of the SPM software Log each Message F Each logger can be adjusted separately by setting what is to be logged Warnings Log method traces and errors can give useful information about the status of the AFM and may be a Log debugging message guide to the source of a problem Log hint message Log monitored tasks Before deactivating all loggers please keep in mind that these loggers are used to track unforeseeable errors that might occur Especially the log files can be used for Log each message Log errors and warnings M Log only errors later bug fixing Log nothing 146 NanoWizard AFM User Manual Version 4 2 9 2 4 Voltage Output Settings Accessories fil CCD Camera DirectOverlay O
237. ut not yet adjusted onto the cantilever Optical path schematic AFM head adjustment Optical microscope image J Laser Adjustment Prism Laser Mirror Laser Adjustment Detector Cantilever Screws The laser position must be moved using the adjustment screws until the beam is directed onto the cantilever Note All four positioning screws have stoppers in each direction Do not over wind the screws On an optical microscope the cantilever is viewed from below while the laser beam comes from above The intensity of the spot will drop as it is adjusted onto the cantilever because the cantilever blocks some of the light Soft contact mode cantilevers can be quite transparent and the laser spot may still be seen through the cantilever 4 2 4 Adjusting the mirror for large changes in deflection Once the optical image shows that the laser is directed onto the cantilever the detection system must be adjusted so that the reflected beam falls onto the centre of the photodiode detector A mirror can be tilted for coarse adjustment of the laser path and positioning screws are used for fine adjustment The mirror and detector adjustment should only be made after the optical image of the cantilever shows the laser is aligned properly Without proper alignment of the cantilever first there is no reflection to direct onto the detector 34 NanoWizard AFM User Manual Version 4 2 Optical path schematic AFM head adjustmen
238. utton to leave the surface this can be used as a kind of emergency stop it will interrupt the movement if the scan has been started with inap propriate settings 5 1 1 Image properties and the Scan Control panel Scan Control Sjaj a Fast Slow Line Rate Pixels x Offset Offset Scan Angle Fast Slow Line Rate 56 Line 0 zA O A ziel The main controls for scanning are found in the Scan Control panel During a scan most of the options except the Line Rate and feedback set tings in the Scan Control panel become inactive The feedback parameters and scan speed can be altered during a scan but the size shape or offset of the scan can not be changed To change these settings stop the scan by toggling Run and restart with the new values Further parameters can be set using the Advanced Imaging Settings panel which can be opened through the Setup menu or directly from the Scan Con trol panel Here more complex parameters can be set such as the duty cycle or overscan or non square scan areas can be enabled See Section 5 5 for details The image size can be set using the text fields for the Fast and Slow scan sizes aS well as by using the mouse in the Data Viewer The default is for square scans so if one size is changed the other one is automatically updat ed This can be disabled in the Advanced Imaging Settings panel see Section 5 5 The Line Rate controls the speed of the scan Ty
239. values The z range that is most suitable for a particular experiment depends on the sample and type of measurement For flat samples with a height range of less than 500 nm it is recommended to select a smaller z rangein order to maximize the resolution The resolution is increased for smaller z ranges because of the 24 bit resolution of the AD converter and also the amplifier noise is lower The 24 bit resolution means pa 12 777 216 digits are available over the full selected Z range 0 9 pm per digit 0 09 pm per digit It is also important to know that the capacitive sensor measuring the length of the z piezo has two working areas for a range above 5 um and a range beyond 5 um These working areas have different amplification factors and therefor different sensitivities If the z range is reduced beyond 5 um the sensitivity and resolution will increase Note the piezo cannot retract further than the currently set Z Range Therefore if the sample topography differences are larger than the chosen Z Range the tip will not be able to move far enough away from the surface and tip or sam ple damage can result Note also that the Z range covered by the piezo during a scan includes any sample tilt as well as the roughness of the sample over a scan line Therefore even if the sample is quite flat a relatively large Z range will be needed if there is a significant tilt angle between the sample and the AFM head It is important to consider the he
240. ver package when necessary for taking out or inserting a cantilever and always in a clean environment The tips may also be damaged through inappropriate scanning conditions e Too high gain parameters Gamm and PGain may lead to a damage of the cantilever tip e Too high setpoint values in contact mode may damage the tip e Too low setpoint values in intermittent contact mode may damage the tip 2 3 3 Setting up in liquid The NanoWizard AFM is optimized for performing experiments on samples in a liquid environment The instrument is designed to protect sensitive parts of the microscope but particular care is still needed when working in liquid The glass cantilever holder is easy to clean and chemically inert and well suited to working in buffer solutions as well as in low concentration bases and acids The cantilever holder spring is made of medical steel or on request a gold coated spring can also be supplied The glass block can be immersed up to the upper edge of the groove that holds the spring This is a maximum immersion depth of approximately 4 5 mm The bottom of the AFM head is sealed against vapor to protect the scanning system from water buffer or any other liquid whose vapor could damage the scanning system Always remove the glass block immediately after your experi ment and clean it 16 NanoWizard AFM User Manual Version 4 2 3 Software overview 3 1 SPM software introduction and index The JPK NanoWizard is del
241. vides an extension to the parameters found in the Scan Control panel Scan Control CURVES eet 3 F Line 0 12 The speed for scanning is usually set in the main Scan Control panel through the Line Rate A line rate of 1Hz means that the tip makes the trace and retrace scan lines and returns to the starting position for the next scan line in a time of 1s To have a constant scan speed across the surface while collecting data points however the tip must scan a slightly larger area to give room for slowing down and changing direction The relationship between the movement of the tip used for imaging and this extra movement to change direction is normally handled using default values from the software so the user only has to choose one num ber for the Line Rate For advanced applications the user can choose particular values to control the other parts of the tip movement if desired Square image ES 20 219 um ES retrace or as a Half Scan Time the time for either the trace or retrace part of el 20 219 um the scan line The numbers are always updated to be consistent el 0 04655 around at the ends of the scan lines This is set as a percentage of the half scan D time Note that this option replaces the Duty cycle option from old versions of Overscan Time 0 0055 the software Distance of Overscan 2 08 um The Tip Velocity takes account of the physical scan size and can be used to see Ti
242. wer careful approach Decrease the approach gains especially Approach IGain and or reduce the setpoint force decrease the setpoint in contact mode or in crease it in intermittent contact mode If it is impossible to get a stable approach where the red bar covers the whole z piezo sometimes it is helpful to reduce the z range slightly for instance to 5 85 or 12 microns The feedback is too slow meaning that the approach routine is not searching the full z range This can lead to an over slow approach to false unsuccessful approaches or to the head moving too far away from the sample Increase the approach gains especially Approach IGain and or increase the setpoint force in crease the setpoint in contact mode or decrease it in intermittent contact mode Furthermore the Timeout has to be increased Retracting the tip from the sample RUN Run is used to start and stop most measurements If Run is clicked again during a Ss 48 measurement it will stop the measurement at a convenient point e g at the end of a scan line or on completion of a force curve The cantilever tip will remain approached on the surface at the starting point of the scan The Retract button can be used to retract the tip from the surface and also to interrupt a scan as a kind of emergency stop The measurement is stopped almost immediately and the tip is pulled back to the fully retracted position Clicking the Retract button once retracts the cantileve
243. y from the cantilever images The cantilever is automatically recognized from each optical image without needing to input on cantilever angle shape or magnification This requires that there is a good contrast for the optical image of the cantilever Under normal im aging conditions the optical contrast is enhanced to give the best sample image so the automatic routine could be confused by sample features and not properly recognize the cantilever Therefore it is best to optimize the illumination and contrast for the cantilever before starting the calibration This can be easily done by changing the condenser illumination settings e g in optical phase contrast change to bright field illumination as shown below or for DIC mode slide the polarizer out of the illumination pathway These changes do not affect the optical imaging path and hence the calibration is unchanged but the cantilever contrast is enhanced relative to the sample The illumination can be changed back at the end of the optical calibration procedure to give an image with good sample contrast for importing ery P i E Ce ES 2 N Phase contrast illumination Bright field illumination same region contrast is better for optical calibration A slight movement of the focus position can also be helpful Adjust the fine focus so that the tip of the cantilever is sharp The focus can be moved a few microns back to the sample level to take a sharp sample image for impo
244. ymer surface of a commer cially available compact disc In this example an intermittent contact mode cantilever with a spring constant of around 40 N m was used Imaging in intermittent contact mode did not cause any changes to the surface of the polymer but using a contact mode setpoint of around 0 3 V produced a sharp line engraved in the polymer Material from the central de pression is moved to the edges which become raised The overall topography of the scratch was a few nm Deeper lines could be produced by applying higher forces The images below show height and phase images 5 0 x 2 2 um 3 606 nm 19 88 deg slow um slow um Odeg 2 2 fast um fast um 8 6 3 2 Moving spheres The freehand drawing of manipulation paths is useful for moving objects around on the surface and switching between manipultaion and imaging modes can be used to check where the objects have been moved to and adjust the manipulation accordingly In this example 120nm diameter polymer spheres were dried onto a glass slide and manipulated using the AFM tip The imaging was performed in intermittent contact mode in air which did not disturb the spheres and manipulation could then be performed using a very small setpoint in contact mode around 0 05V higher than the free vertical deflection value This series of images show the spheres along with the manipulation paths drawn to produce the observed motion All images are 5 x 3 microns height scale
245. z High Speed and one 24 bit channel 17 sampled at 2 5 MHz High Resolution are available The DIGITAL I O connectors provide a multitude of digital TTL inputs and outputs TTL out is used to trig ger external JPK modules like the CellHesion Module or the Tip Assisted Optics module Contact JPK for assistance if required including pin assignments of the 2 D sub 15 input and output connectors The dig ital counter inputs at the 4 BNC connectors may be used for e g photon counting They have a program mable trigger level The POWER connector outputs 15 V 5V 5 V 15 V and GND on the various pins of a D sub 9 con nector Contact JPK for pin assignments or further assistance The function tip buttons F1 F4 on the far right of the SAM can be used to set individual configuration pat terns channel assignments Please contact JPK for assistance The table below summarizes the designation of the various analog inputs output on the SAM Vertical deflection unprocessed Axes 7 Out external z DAC 400 kHz 24 bit Monitor 2 Lateral deflection unprocessed Axes 8 Out Not assigned Axes 9 Exc Monitor 3 Not assigned AC excitation n 152 NanoWizard AFM User Manual Version 4 2 Axes 10 Axis Monitor 4 Photosum unprocessed Direct Digital Synthesizer DDS 120 MHz 14 Analog Out 4 bit Analog Ing Not assigned i Z piezo modulation For applications that require a

NanoWizard User Manual - Section for Nanostructure Physics

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