The AFM Probe - Fundamentals, Selection, and Applications

Transcription

1 The AFM Probe - Fundamentals, Selection, and Applications

2 Introduction Appropriate selection of AFM probe can be one of the most important decisions to be made towards a successful AFM experiment As there are many probes to chose from, this selection may appear daunting The purpose of this webinar is to enable the AFM researcher to select an AFM probe like an expert.

3 Outline Probes overview Key metrics for probe choice Applications based probe decisions Very High Resolution Imaging Quantitative Nanomechanical Fast Scanning in Air and in Fluid Biological Applications and Force Spectroscopy Nano Electrical KPFM Nano Optical - TERS Probe Preparation and Cleaning Bruker AFM Website Conclusion

4 Basic Operation of the SPM: Simplified Schematic AFM Probe Bruker Confidential 4

5 3: Forces commensurate with atomic bonds. 2: Sensitivity on the scale of atoms 1: Apex on size scale of atoms Bruker Confidential 5

6 1: The Probe Apex: The critical factor for determining AFM resolution The microscope maker s rule: you can t visualize anything smaller than what you are interrogating it with. This is true for photons, electrons, and AFM and why the Apex size and geometry are so important. RTESPA OTESPA NCHV-A TESP-HAR TESP-SS CDR50C MCNT-500 VITA-DM-GLA-1 DPT10 MLCT (Dec 13) 10/28/2013 6

7 2: AFM Sensitivity: From the cantilever s perspesctive Simple explanation of optical lever: smaller cantilevers deflect the spot more, creating more signal: However, its not so simple. Optics and detection electronics come into play: But the same scaling holds true, shorter levers create greater signal. See Fukuma and Jarvis, RSI, 77 (2006) But there is a tradeoff... 10/28/2013 Bruker Confidential 7

8 3: Spring Constant Forces must be commensurate with surface As you continue to reduce length, the spring constant (k) of the lever increases to the 3 rd power. To maintain low tip sample forces the thickness of the cantilever has to be scaled at the same rate. This is fundamentally why AFM cantilevers are small. L must be short enough to get good signal 200um or less T must then be thin enough to apply a force soft compared to surface & tip See brukerprobes.com for to see probes ranging from: L - less than 20um to greater than 300um t less than 0.2um to greater than 7um Sarid, Scanning Force Microscopy, ISBN , Oxford University Press 10/28/2013 Bruker Confidential 8

9 Cantilever Dynamics and Beyond For many modes resonant frequency also plays a critical role in the AFM s speed and resolution. The probe s frequency scales with geometry and in general a higher frequency is better. So additionally, one cannot simply scale length and thickness together for many applications. As cantilevers get smaller, tip mass and cantilever drag are become very important and add significant complexity to cantilever design. SCANASYST-AIR-HR 10/28/2013 Bruker Confidential 9

10 Point of Order... AFM ers will routinely use relative terms expecting them to be interpreted in the context of the Application, not absolutely. Cantilever Coatings both simply called coatings Front (Tip) Side Coatings: Used for electrical/conductive measurements Backside Coatings: Used to increase laser reflectivity. These are generally prefered, but they can cause thermal drift (bimetallic effect) or contamination in biological. Spring Constant normal levers span 4-orders of magnitude In Bio Applications: Soft < 0.05 N/m, Hard >.5 N/m In PeakForce Tapping: Soft < 0.1N/m, Hard > 20 N/m In Tapping Mode: Soft < 5 N/M, Hard > 50 N/m Resonant Frequency normal levers span Fluid Tapping: low < 5kHz, high > 50kHz Air Tapping: low < 20kHz, high > 500kHz 10/28/2013 Bruker Confidential 10

11 VERY HIGH RESOLUTION IMAGING 10/28/2013 Bruker Confidential 11

12 AFM probes for very high resolution TappingMode imaging of Mica in Water Localize interaction with very small amplitude (<2nm) Localize interaction with very sharp tip May need to cherry pick probe Preserve tip: moderate spring constant (1-10 N/m) Use the maximum Amplitude Setpoint possible (reducing peak force) Recommended probes ScanAsyst-Fluid+ or FastScan B

13 AFM probes for very high resolution PeakForce QNM of Polydiacetylene (PDA) in Air Height Stiffness Adhesion 0.5 nm 1.4 nm Peak Force Tapping provides much higher resolution than TappingMode in air & provides property maps Localize interaction with very sharp tip May need to cherry pick probe tip Preserve tip: low spring constant <1N/m & setpoint <500pN Recommended probes lattice defect ScanAsyst-Fluid+ or FastScan C b A.Enkelmann, Adv.Pol. Sci., 1984, 63, 91

14 AFM probes for very high resolution PeakForce QNM for sub-nm resolution calcite in liquid Height 2 nm DMTModulus channel provides a qualitative stiffness map Alternate rows of atoms have significantly increased contact stiffness, rows switch over step Need moderate spring constant for modulus contrast Localize interaction with very sharp tip May need to cherry pick probe tip Preserve tip: moderate spring constant <5N/m & setpoint <5nN Stiffness Recommended probe ScanAsyst-Fluid+ or FastScan B Data collected in collaboration with Dr. Daniel Ebeling, U. Maryland

15 AFM probes for very high resolution PeakForce Tapping to Resolve the DNA Double Helix PeakForce Tapping images reveal corrugation corresponding to widths of major and minor grooves MAJOR MINOR DNA loosely bound to mica surface (~1mM NiCl2) to minimize conformational effects Challenge for high-resolution imaging requires very low force and high sensitivity Short cantilever provides sensitivity Low spring constant, setpoint protect tip and sample from damage Recommended Probe FastScan-Dx: 18um length, k~0.25n/m 10/28/2013 Bruker Nano Surfaces Division 15

16 AFM probes for very high resolution Guidelines for probe selection Resolution determined by localization of interaction Smaller tips resolve smaller features without influence from neighboring structures Peak Force Tapping mode localizes interaction by default while Tapping Mode requires small amplitudes to localize force Lateral forces can quickly damage the tip, leading to larger interaction areas Shorter (<125um) probes are recommended Shorter cantilevers are more sensitive, higher frequency, and have smaller viscous background in liquid Spring constant should be selected to avoid tip and sample damage Keep peak force low (typically <500pN) Larger spring constants are often more stable, especially in TappingMode (less concern about adhesion), so very soft (<0.5N/m) cantilevers are not recommended Consider cantilever resonant frequency (f0) For Peak Force Tapping, resonant frequency f0>10*modulation frequency For TappingMode performance is usually better f0>10khz 10/28/

17 QUANTITATIVE NANOMECHANICAL MEASUREMENTS 10/28/2013 Bruker Confidential 17

18 AFM Quantitative Nanomechanics (QNM) Calculate sample properties directly from force curves Probe selection for Nanomechanical Measurement key parameters Tip shape (R and/or half angle) Spring Constant (k) Resonant Frequency Length Coating Remember: Quantitative force measurement requires calibration of spring constant and deflection sensitivity. Modulus also requires calibration of tip shape. Nominal values are not accurate. ~ (ii) 10/28/

19 AFM probes for QNM High resolution PF-QNM of a heat-sealed bag DMTModulus (a) Barrier layer Nylon Strength & gas impermeability Tie layer ULDPE Preserves layer adhesion Sealant layer Metallocene PE/LDPE blend Adheres to itself when heated (b) (c) (a) Study of interphase between Tie and Sealant layers requires high resolution and good modulus contrast Sharp tip (R<25nm) needed to provide resolution to separately measure individual lamella Moderate spring constant (k~2-10n/m) needed for good match to material stiffness ~ MPa Backside coating to reduce optical interference (b) Recommended probe: TAP150A also known as MPP (k~5n/m, R~8nm, Al backside coating) 10/28/

20 AFM probes for QNM Expanding PeakForce QNM to softer samples Cells & gels Tip half angle ~18 Tip radius ~20nm Real AFM tip Real AFM tips are not a simple sphere or cone Choose the deformation model (Hertz/DMT (spherical) or Sneddon (conical)) that best represents your tip shape at a given deformation depth By choosing spring constant, tip shape, and model carefully, the widest range of properties can be studied 10/28/

21 AFM probes for QNM Guidelines for probe selection Tip shape (end radius, half angle) determines resolution Larger tips: shape matches models better and are more stable Smaller tips: resolve smaller features without influence from neighboring structures Spring constant and tip shape: select to match sample stiffness Most important for modulus & deformation measurements Resonant frequency >10*modulation freq. to avoid cantilever resonance Not generally an issue in air, but can be a problem in liquid with soft cantilevers Shorter is usually better More sensitive, higher freq, and have smaller viscous background Longer cantilevers are easier to align and less prone to optical interference 10/28/

22 AFM probes for QNM PeakForce QNM and Force Volume of Agarose gels Sneddon model works well over the biologically relevant kpa- MPa range when coupled with soft cantilever in liquid Relatively blunt tip provides better repeatability and is more gentle for very soft samples Soft cantilever (k~2-10n/m) needed for good match to material stiffness ~10-500kPa Backside coating to improve reflectivity of silicon nitride cantilever Similar results obtained with PF- QNM and FV at ramp rates from 1Hz to 500Hz Recommended probe: A stiffer probe would be better here MLCT-E (k~0.1n/m, R~20nm, half angle ~18deg, Ti/Au backside coating) 10/28/

23 AFM probes for QNM PeakForce QNM of live cells on glass bottom Petri dish Spring constant should match modulus Ecell=kPa << Eglass=GPa MLCT-D has k ~0.03N/m Good for Ecell (see cell structures) Too small for Eglass (out of range) Glass Surface Tip Shape should match Fit Model MLCT has pyramidal tip Indentation on soft cell 100nm Indentation too deep for DMT (sphere) Sneddon better modulus estimation Recommended probe for cell: MLCT-D (k~0.03n/m, R~20nm, half angle~35deg, Ti/Au backside coating) DMT Modulus = 50kPa Sneddon Modulus = 37kPa Modulus image of B16 mouse carcinoma cell. Force curves show fit for DMT and Sneddon modulus. Images obtained on the BioScope Catalyst AFM in PeakForce QNM using MLCT-D probes in fluid. 10/28/ Bruker Nano Surfaces Division

24 FAST SCANNING IN AIR 10/28/2013 Bruker Confidential 24

25 Why Fast Scanning SPM? Fast Scanning allows real-time study of dynamic processes at the Nanoscale Allow rapid collection of thousands of images for meaningful statistics Collect large (~25MPixel) images in reasonable time for examination of both fine detail and superstructure in a single image

26 AFM probes for Fast Scanning in air Guidelines for probe selection Consider cantilever resonant frequency (f0) and quality factor (Q) For TappingMode, cantilever bandwidth is approximately f0/q Cantilever Q depends on the fluid environment (air, liquid) and on the length of the tip For Peak Force Tapping, resonant frequency f0>10*modulation frequency and modulation frequency should be high enough to have at least one tap per pixel Shorter (<50um) probes are recommended Shorter cantilevers are higher frequency, have smaller viscous background in liquid, and are more sensitive Resolution determined by localization of interaction Smaller tips resolve smaller features without influence from neighboring structures Peak Force Tapping mode localizes interaction by default while Tapping Mode requires small amplitudes to localize force Lateral forces can quickly damage the tip, leading to larger interaction areas Spring constant & setpoint should be selected to avoid tip and sample damage Keep peak force as low as possible, but larger forces may be required for good tracking Larger spring constants are often more stable, especially in TappingMode (less concern about adhesion), so very soft (<0.5N/m) cantilevers are not recommended Bottom line PeakForce Tapping is not very fast in air. For Tapping Mode, the best probe is the FastScan A! 10/28/

27 Image Specifications: Size: 2.2um ScanRate: 100Hz Pixels:256 x 256 TipV: 440um/s Frame Rate: 2.5s Real Time Video Duration: ~4min Sample Courtesy: Dr. Jamie Hobbs 10/28/2013 University Bruker of Sheffield Nano Confidential 27

28 HIGH SPEED AFM FLUID IMAGING 10/28/

29 AFM Probes for HS-AFM Imaging in Fluid Imaging Dynamic Biological Processes Choosing a probe for high-speed imaging of molecular/cellular dynamics in TappingMode in fluid. (1 of 2) Small or Ultra-Short Cantilever length 50nm. Provides low spring constant probes with high resonance frequency (high bandwidth). Soft Cantilever/Low Spring Constant (k). Soft samples (modulus :~kpa-mpa range.) Samples weakly attached to supporting substrate. Typically Si 3 N 4 cantilevers. Some made of new material (quartz-like material). High Resonance Frequency f 80kHz. Facilitates high-resolution imaging at high scan speeds (secs/frame frames/sec). Traditional TappingMode probes have lower resonance frequency (eg. SNL-C f~13khz in fluid.) Challenging probes to design optimizing spring constant and resonance frequency. IMPORTANT: Most resonance frequencies stated by manufacturer are for operation in air. This resonance frequency will decrease 1/2 to 2/3 of this value when used in fluid. Use (fast) thermal tune to identify the correct resonance frequency of a probe in fluid. 10/28/2013 Bruker Nano Surfaces Division 29

30 AFM Probes for HS-AFM Imaging in Fluid Imaging Dynamic Biological Processes Choosing a probe for high-speed imaging of molecular/cellular dynamics in TappingMode in fluid. (2 of 2) Tip Sharpness. Small cantilevers = Force sensitivity = Imaging force = Ability to use sharper tips (w/o damage). FastScan-Dx has a silicon tip (R ~8nm, 12nm ) 11Hz 22Hz 43Hz 100nm 100nm 100nm Images of DNA origami obtained on the FastScan-Bio AFM in TappingMode using a FastScan-Dx probe in fluid (512 pixels/image). Sample courtesty of P. Rothemond and L. Qian, CalTech. 10/28/2013 Bruker Nano Surfaces Division 30

31 AFM Probes for HS-AFM Imaging in Fluid High-Speed Imaging of DNA or DNA-Protein Dynamics High-Speed TappingMode in Fluid. Short Cantilever length 50nm. Soft Cantilever k ~0.1N-0.3N/m. Stiff enough to oscillate in fluid. Too stiff damage to sample. High Resonance Frequency f 70kHz. Higher f = faster imaging speeds. Sharp tip small radius. Resolve molecular structure. 0.5fps 1.0fps Recommended AFM Probes. FastScan-Dx (l ~18µm; k ~0.25N/m; f ~110kHz; R ~8nm) 2.0fps 3.0fps High-speed imaging of Lambda Digest DNA. Parts of the DNA strand loosely bound to the surface appear to move from frameto-frame (circle). Images were obtained on the FastScan-Bio AFM in TappingMode using FastScan-Dx probes in fluid. 10/28/2013 Bruker Nano Surfaces Division 31

32 AFM Probes for HS-AFM Imaging in Fluid High-Resolution and High-Speed Imaging of Cell Membrane Dynamics High-Speed TappingMode in Fluid. Short Cantilever length 50nm. Soft Cantilever k ~0.1N-0.3N/m. Too stiff = damage to sample. High Resonance Frequency f 70kHz. Higher f = faster imaging speeds. Sharp tip smallest radius. Resolve structures on cell surface. Small cantilever = imaging force. Recommended AFM Probes. FastScan-Dx (l ~18µm; k ~0.25N/m; f ~110kHz; R ~8nm) 100nm High-speed imaging of the effects of CM15 (AmP) on the outer membrane of live E. coli cells. Ordered structures in the membrane are believed to be 2D-organized porin molecules. Images were obtained at 8 sec/frame on the FastScan-Bio AFM in TappingMode using FastScan-Dx probes in fluid. 10/28/2013 Bruker Nano Surfaces Division 32

33 BIOLOGICAL APPLICATIONS 10/28/

34 AFM Probes for Biological Samples Molecular and Live Cell Imaging Choosing a probe for imaging biomolecules or live cells in fluid. Soft Cantilever/Low Spring Constant (k) Soft samples (modulus :~kpa-mpa range.) Samples weakly attached to supporting substrate. Typically use Si 3 N 4 cantilevered probes (vs. Silicon). Gold (Au) Coated or Non-Coated Cantilever (Backside Coating). Al coating (air probes) can contaminate biological samples in fluid. Use non-coated only if absolutely necessary (see force spectroscopy section). Tip Sharpness. Sharper tips with small half angle provide high-resolution for single molecule imaging. Blunter tips with larger half angle provide low pressure needed for live cell imaging. Silicon tips tend to be sharper (smaller tip radius/half angle) than Si 3 N 4 tips. 10/28/2013 Bruker Nano Surfaces Division 34

35 AFM Probes for Molecular Imaging Contact Mode Imaging of Membrane Proteins Contact Mode Imaging in Fluid. Very Soft Cantilever low k. sample distortion/damage. Gold Coated (backside) Cantilever. Non-coated only if necessary (drift). Sharp tip small radius. Resolve molecular structure. 20nm Recommended AFM Probes. MSNL-C (k ~0.01N/m; R ~2nm) SNL-D (k ~0.06N/m; R ~2nm) 10nm 10nm Images of aquaporin OmpF (top) and bacteriorhodopsin (bottom) obtained on the MultiMode AFM operated in Contact Mode using a SNL-A probe in fluid. Image courtesy of D. Mueller, ETH D-BSSE, Basel. 10/28/2013 Bruker Nano Surfaces Division 35

36 AFM Probes for Molecular Imaging PeakForce Tapping Mode Imaging of DNA PeakForce Tapping in Fluid. Soft Cantilever intermediate k. Stiff enough to oscillate in fluid. Gold Coated (backside) Cantilever. Sharp tip small radius. Resolve molecular structure. Recommended AFM Probes. ScanAsyst Fluid+ (k ~0.7N/m; R ~2nm) SNL-A (k ~0.56N/m; R ~2nm) Image of plasmid DNA obtained on the MultiMode AFM operated in PeakForce Tapping Mode using a ScanAsyst Fluid+ probe in fluid. Image courtesy of A. Pyne and B. Hoogenboom, UCL, London. 10/28/2013 Bruker Nano Surfaces Division 36

37 AFM Probes for Live Cell Imaging Contact Mode Imaging of Live Cells Contact Mode Imaging in Fluid. Very Soft Cantilever low k. Sample distortion/damage. Gold Coated (backside) Cantilever. Non-coated only if necessary (drift). Blunt tip larger tip radius. Low pressure won t damage cell. Recommended AFM Probes. MLCT-C (k~ 0.01N/m; R ~20nm) DNP-A (k~ 0.06N/m; R ~20nm) OBL (k ~ N/m; R ~30nm) 45 m Image of live endothelial cells obtained on the BioScope Catalyst AFM operated in Contact Mode using an MLCT-C probe in fluid. 10/28/2013 Bruker Nano Surfaces Division 37

38 AFM Probes for Live Cell Imaging PeakForce Tapping Mode Imaging Live Cells PeakForce Tapping in Fluid. Soft Cantilever intermediate k. Stiff enough to oscillate in fluid. Gold Coated (backside) Cantilever. Sharp tip small radius. Resolve molecular structure. Recommended AFM Probes. DNP-A (k~ 0.56N/m; R ~20nm) MLCT-F (k~ 0.6N/m; R ~ 20nm) ScanAsyst Fluid (k~ 0.7N/m; R ~20nm) 5 m Sneddon Modulus data overlaid on 3D topography image of live E. coli cells. Dividing cell (right) has lower modulus than single cell (left). A single flagellum is also observed. Imaging obtained on the BioScope Catalyst AFM operated in PeakForce Tapping Mode using a DNP-A probe in fluid. 10/28/2013 Bruker Nano Surfaces Division 38

39 FORCE SPECTROSCOPY 10/28/2013 Bruker Confidential 39

40 AFM Probes for Force Spectroscopy Localized Measurements of Modulus, Molecular Unfolding, and Binding Interactions. Choosing a probe for single point force measurements of modulus (pushing) and unfolding/unbinding (pulling). (1 of 3) Cantilever Spring Constant (k). Modulus/Indentation based on expected sample modulus (kpa MPa = softer probes; MPa GPa = stiffer probes). Unfolding/Unbinding based on forces being measured (pn = softer probes; nn = stiffer probes). Measured cantilever deflection should be within 2-3V of noncontact deflection value. Probe too stiff = deflection too small (below noise floor cannot measure). Probe too soft = deflection too large (photodetector response non-linear at large deflections). Important to calculate deflection sensitivity and spring constant (thermal tune). Nominal values are not accurate. Note: For very soft probes sometimes easier to calculate k in air and then re-measure the deflection sensitivity in fluid (k does not change). 10/28/2013 Bruker Nano Surfaces Division 40

41 AFM Probes for Force Spectroscopy Localized Measurements of Modulus, Molecular Unfolding, and Binding Interactions. Choosing a probe for single point force measurements of modulus (pushing) and unfolding/unbinding (pulling). (2 of 3) Coated or Non-Coated Cantilever (Backside Coating). Al coating (air probes). Au coating (fluid probes) Al can contaminate biological samples. Non-coated (most probes are coated) Backside coating can introduce drift, especially in fluid or at elevated temp (bimetallic effect). Use non-coated cantilever only if absolutely necessary (can introduce other issues) Beware of optical interference with non-coated probes (observed in baseline of force curves). Also ensure adequate sum signal (force sensitivity) typically want >1.5V. Optical interference causes regular pattern in force curve. 10/28/2013 Bruker Nano Surfaces Division 41

42 AFM Probes for Force Spectroscopy Localized Measurements of Modulus, Molecular Unfolding, and Binding Interactions. Choosing a probe for single point force measurements of modulus (pushing) and unfolding/unbinding (pulling). (3 of 3) Tip Shape. Very important for modulus measurements. Based on modulus fit model (indentation) eg. DMT/Hertz = sphere; Sneddon = cone. Tip Sharpness. Modulus/Indentation blunt/dull probes minimize pressure (tip or sample damage). Unfolding/Unbinding larger R increases chances of molecule attached to end of tip. Tip Functionalization. Modification of tip with chemical group (eg. CH 3 /COOH) or molecules/ligands. Cleaning probe is first step in functionalization process (refer to previous slides). See AN#130 Common Approaches to Tip Functionalization for AFM-Based Molecular Recognition Measurements. 10/28/2013 Bruker Nano Surfaces Division 42

43 AFM Probes for Force Spectroscopy Optically Targeted Cell Modulus Measurements Modulus Measurements (in fluid). Soft Cantilever k ~ N/m. Cells have low modulus (kpa). Gold Coated (backside) Cantilever. Non-coated if drift issues. Conical or Spherical Tip. Soft cells = indentation depth. Sneddon = conical, Hertz = spherical. Blunt Tip (or Spherical) larger radius. Won t damage cell membrane. Recommended AFM Probes. Spherical (k ~ N/m; R ~50nm-5µm) MLCT-C (k ~0.01N/m; R ~20nm) Force curves performed on live endothelial cells fluorescently labeled with a membrane potential indicating dye (bis-oxonol). Cells were found to be stiffer in hyperpolarized state. Studies conducted on the BioScope Catalyst AFM using spherical probes (k ~0.01N/m) in fluid. Images courtesy of H. Oberleithner, Munster, Germany. 10/28/2013 Bruker Nano Surfaces Division 43

44 AFM Probes for Force Spectroscopy Single Molecule (Un)Folding Studies Molecular Unfolding Studies (in fluid). Soft Cantilever k ~ N/m. Forces measured are in pn range. Gold Coated (backside) Cantilever. Non-coated if drift issues. Blunt Tip larger radius (R). >R = chances of catching molecules. Tip Functionalization. Au-coated tips if molecule has thiol groups. Recommended AFM Probes. MLCT-C (k ~0.01N/m; R ~20nm) MLCT-D (k ~0.03N/m; R ~20nm) DNP-D (k ~0.03N/m; R ~20nm) AFM pulling/unfolding curve for a single titin 8-mer construct. The retract portion of the curve show the classic sawtooth pattern with ~200pN force required to unfold each of the 8 repeated subunits. Force curves obtained on the MultiMode AFM using a MLCT-C probe in fluid. 10/28/2013 Bruker Nano Surfaces Division 44

45 KELVIN PROBE FORCE MICROSCOPY (KPFM) 10/28/2013 Bruker Confidential 45

46 KPFM Modes and Probe Selection Guide AN140-RevA1-PeakForce_KPFM-AppNote 10/28/2013 Bruker Nano Surfaces Division 46

47 Spatial Resolution Geometry L l W L The tip needs to be sharp Tip height and cantilever width, length: Taller, Higher aspect ratio Smaller Cantilever 10/28/2013 Bruker Nano Surfaces Division 47

48 Sensitivity Cantilever Spring Constant and Q Thermal Tune Higher Q (on surface) and lower k gives higher sensitivity for KPFM measurements, this is vitally important for FM- KPFM. Probe must also have clean resonance peak. Q 10/28/2013 Bruker Nano Surfaces Division 48

49 Q/k of Some Probes Types Other factors must also be accounted for PFQNE-AL f k Q Q/k Q(Engaged) Q/k(Engaged) # # # SCM-PIT f k Q Q/k Q(Engaged) Q/k(Engaged) # # # ScanAsyst-Air-HR f k Q Q/k Q(Engaged) Q/k(Engaged) # # # AC40 f k Q Q/k Q(Engaged) Q/k(Engaged) # # # /29/2013 Bruker Nano Surfaces Division 49

50 NANO OPTICAL MICROSCOPY 10/28/2013 Bruker Confidential 50

51 Tip Enhanced Raman Spectroscopy probes Application: nanochemical identification System specific: IRIS Key considerations Feedback method STM = gets closer but needs conducting sample AFM (tuning fork) = any sample, less signal Material Etched Gold yields best field enhancement Silver probes promising, but less consistent Consistent Performance TERS contrast must be large AND reproducible NearIR Deflection Laser Improved Optical Access 100X tip enhancement! See App Note 136 for more details October 28, 2013

52 TERS Probes: STM vs. AFM STM STM feedback Requires conductive substrate Optimal TERS performance 10X contrast AFM (tuning fork) - NEW 5X contrast Force feedback using tuning fork Works on any substrate Coming Soon Tuning Fork Probe Zoomed-in, showing the etched gold wire attached Prototype Tuning Fork cartridge October 28, 2013

53 Examples of TERS spectra for ChemID Methylene Blue Extremely low power Sensitivity fragile samples Nile Blue Extreme enhancement (600!) Outstanding probe performance Thiophenol No far field (shown: p/s) Sensitivity - low Raman x-section Innova-IRIS with IRIS TERS-STM probes Consistent Performance! Detecting molecular films that are undetectable in micro-raman October 28, 2013 AFM-Raman Solutions 53

54 Contrast Consistency of Bruker TERS probes Making probes consistently is a costly art to master Our testing has shown we reach >10x enhancement on average for over 80% of the tips October 28, 2013

55 PROBE PREPARATION AND CLEANING 10/28/2013 Bruker Confidential 55

56 Tip cleaning tips For specialized experiments Probe cleaning NOT a recipe for cost savings Main purpose is for functionalizing probes or eliminating contamination in liquid Only in rare circumstances (i.e. DNISP) are probes recyclable Pre-experiment probe prep options Ethanol/Isopropanol simple, good for atomic resolution UV light + DI water better for removing organics (gelpak) Plasma etching good for functionalization, oxidized tip Acidic piranha functionalization, warning:attacks metal Ultrasonication - used by some but difficult to hold tip Example of functionalized tip October 28, 2013 Please refer to App Note 44 for more information

57 A biased view of tip recovery Weighing your options Cost benefit analysis Can you change probes without disrupting your experiment? If at all possible change probes! Last ditch efforts if probe change is not practical Retract probe, run AC drive on high, run frequency sweep several times (occasionally works in fluid) Approach sample, while in contact increase feedback to cause tip to oscillate Controlled crash, using low setpoint Scan clean glass slide in contact mode Press (i.e. perform force curve) into soft sample such as BOPP Special cases Nanoident probes very expensive, durable, recoverable by making forceful indentation on Gold or Rubber. STM probes possible to make Nano-asperity w/ hard contact. Low success rate. October 28, 2013

58 BRUKER AFM PROBES WEBSITE October 29, 2013

59 Bruker AFM Probes Website: October 29, 2013

60 Bruker AFM Probes Website: October 29, 2013

61 Bruker AFM Probes Website: October 29, 2013

62 Conclusion Appropriate selection of AFM probe can be one of the most important decisions to be made towards a successful AFM experiment Though there are many probes to chose from and this selection may appear daunting the applications requirements usually point to a clear path for probe selection. The Bruker website has distilled this information for the user to help make their probe selection as the AFM experts do The Bruker applications and Probes teams are always available to assist our customers in making the best probe selection for their needs. October 29, 2013

63 October 29, 2013 Copyright Bruker Corporation. All rights reserved.