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Biology Applications Nanonics

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Biological applications for Nanonics SPM

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Biology Applications Nanonics

  1. 1. Nanonics Excellence In Scanning Probe Microscopy For Biological Imaging & Functional Imaging Transparent Optical & Spectral Integration for the MultiView SPM Series • MV4000 • MV2000 The Ultimate In Force Sensitivity The Next Evolution in SPMTM
  2. 2. Nanonics Excellence in Structural & Functional AFM/SPM Imaging in Biology Nanonics Imaging Ltd www.nanonics.co.il The Next Evolution in SPMTM
  3. 3. The Next Evolution In AFM Fully Integrated Platforms To Address The Simple To The Sophisticated MultiProbe MV1500 MV2000 Single Probe Series The Hydra Multiprobe
  4. 4. In General Other AFMs Have Given No Deep Thought To The Importance of Excellence In Optical Integration The Next Evolution in SPMTM Ultrasmall Z Range Scanners As Small as 6m in Z Oft Repeated Technology In Conventional AFM Probes Blocking Optical Axis Generally Worlds Apart
  5. 5. The Next Evolution In AFM 5 Nanonics Has The Only BioAFMs That Can Transparently Integrate With Any Upright Microscope Opening The World Of>> • Non transparent substrates • Applications in microbiology and virus research • Applications in food, paper or textile industry on fibers, coatings or powders in air or liquid • Tissue culture • Optical active compounds or materials studies in biosensors, capsules etc • Biomaterial studies, biofouling, And The There Is Nanonics
  6. 6. The Next Evolution In NSOM Scanners Probes & Feedback Allow For Singular Excellence in AFM And Glass Based Cantilevered Probes That Do Not Obscure The Optical Axis And Are Exclusively Available For Nanonics Customers Only & & Singular 3D Flat Scanners Feedback With Unprecedented Force Sensitivity Exclusive Glass Probe
  7. 7. The Next Evolution In NSOM Exclusive Nanonics Probes Are The Most Robust In The Industry As Proven In A SEM Their Unique Structure Allow For Unprecedented Imaging Horizons The Next Evolution in SPMTM Controlled High Pressure After Retraction 50nm 7
  8. 8. The Next Evolution In NSOM NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis Probes are also Multiprobe friendly Many Functionally Important Optically and Multiprobe Friendly Probes The Next Evolution in SPMTM
  9. 9. The Next Evolution In NSOM But Such Use Of These Exclusive Probes Is Complexed With The Ability To Use Any AFM Probe On The Market
  10. 10. The Next Evolution In NSOM Nonetheless Nanonics Systems Readily Allow For Both Tuning Fork or Beam Bounce Feedback & Any Probe Glass or Silicon Tuning Fork Feedback Advantages: • Highest force sensitivity • No feedback laser • Exact point of contact & • True non-contact Beam Bounce Feedback Advantages: • Contact mode • Mount any 3rd party probeLeading To New Directions In Research AFM Controlled Gas Delivery & Associated Kelvin Probe Alterations [Customer Publication] The Next Evolution in SPMTM
  11. 11. The Next Evolution In NSOM And The AFM Force Sensitivity Is So High That The Probes Can Come Into Contact With One Another Critical for Transport Information
  12. 12. The Next Evolution In NSOM See What Customer’s Say In Papers dx.doi.org/10.1021/nl501431y Nano Lett. 2014 The Next Evolution in NSOMTM
  13. 13. The Next Evolution In NSOM Scanners Probes & Feedback Allow For Singular Excellence in AFM And Glass Based Cantilevered Probes That Do Not Obscure The Optical Axis And Are Exclusively Available For Nanonics Customers Only & The Next Evolution in SPMT & Singular 3D Flat Scanners Feedback With Unprecedented Force Sensitivity Exclusive Glass Probe
  14. 14. The Next Evolution In NSOM The Singular 3D UltraFlat Scanner Advantages
  15. 15. The Next Evolution In NSOM Open Optical Axis Extension in Z Allow Effective Optical Sectioning Nanonics 3D Flat Scanner Design Open Optical Access From Above and Below 7mm Conventional Vertical Scanners Unlike Nanonics Have An Ultrasmall Z Range Scanners As Small as 6m in Z
  16. 16. The Next Evolution In NSOM Excellent 3D and Deep Trench Capabilities With High Aspect Ratio Glass Probes Can Be Imaged By Nanonic Due To Availability of:: • Large Z Scanning Range 85m • The Long Tip Length of 100m • The Very High 10:1 Aspect Ratio Of Nanonics Tips • Allows A Soft Touch AC Mode FIB Etched Trench
  17. 17. The Next Evolution In NSOM Scanners Probes & Feedback Allow For Singular Excellence in AFM And Glass Based Cantilevered Probes That Do Not Obscure The Optical Axis And Are Exclusively Available For Nanonics Customers Only & The Next Evolution in SPMT & Singular 3D Flat Scanners Feedback With Unprecedented Force Sensitivity Exclusive Glass Probe
  18. 18. The Next Evolution In NSOM The Feedback Advantage
  19. 19. The Next Evolution In AFM Oft Repeated Method of Beam Bounce Feedback Used In Commercial Instruments Nanonics VISTATM Method of the Ultimate in Force Sensitivity Without Any Optical Interference The Basis of VISTATM Vivid Imaging AFM The Next Evolution in SPMTM
  20. 20. The Next Evolution In AFM Besides Optical Interference No Sample Heating Beam Bounce Feedback Has Significant Problems The Next Evolution in SPMTM
  21. 21. The Next Evolution In AFM Jump To Contact & Ringing Instabilities Occur In Beam Bounce Feedback Jump to contact Uncontrolled ringing • No Jump to Contact Due To High Force Constants • No Adhesion Ringing • Sharp Frequencies or High Q (Quaiity) Factors With Associated Ultrasensitivity Beam Bounce Feedback Problems Resolving These Critical Problems Using Tuning Forks Which Have:Probe Approaches From This Point To The Right The Next Evolution in SPMTM The Technological Limitations of Soft Cantilevers
  22. 22. The Next Evolution In AFM High Q Low Q High Q factors with ultrasharp tuning fork resonances allow ultrasmall alterations in frequency to be detected Resonance Frequency Amplitude Resonance Frequency Amplitude Sharp resonances giving ultrasensitivity to monitor forces Broad resonance giving lower sensitivity to monitor forces The Next Evolution in SPMTM
  23. 23. The Next Evolution In AFM Today Exact Equations Relate Tuning Fork Frequency To Force Between The Tip & Surface The Next Evolution in SPMTM
  24. 24. The Next Evolution In AFM Amplitude Vs Distance Curve Tuning Forks Uniquely Allow Knowledge of the Point of Contact Impossible To Experimentally Know With Certainty With Beam Bounce Distance (d) Amplitude 0 ApproachRetract TR M FT FR FM IF Finteraction = 4 3 E* R(d -d0 )3/2 + Fadh DMT Equation For Interaction of Tip With Surface Using Tapping Mode Tuning Forks Give All Values of this Equation Experimentally I = Start of Approach FT = Force at Point of Touching the Surface FM = Maximum Force FR = Force at Point of Leaving The Surface F = Final Retract Point The Next Evolution in SPMTM
  25. 25. The Next Evolution In AFM Hear From Professor Kit Umbach At Cornell Of The Accuracy of the Tuning Fork Method for Measuring Forces Click On The Link Below http://www.nanonics.co.il/user-testimonials The Next Evolution in SPMTM
  26. 26. The Next Evolution In AFM Cellular Wood Cells AFM Topography and Accurate Elasticity Maps Without Need For Approximate Digitization of Approach Curves and Assumptions Height Elasticity (E*) Great details and resolution in E* map is observed due to the different elasticity of the materials More information on the Lignin Protein distribution in these maps will be seen in the Raman section of this presentation The Next Evolution in SPMTM
  27. 27. The Next Evolution In AFM True Non-Contact Demonstrated By The Ability To Switch On-line With The AFM Probe From AFM to Tunneling Feedback The Next Evolution in SPMTM
  28. 28. The Next Evolution In AFM The Feedback Allows For True Non-Contact Demonstrated By The Ability To Switch Between AFM and STM Feedback 28
  29. 29. The Next Evolution In AFM The History of Force Measurements Peak Force Force Volume<50 nN Pulsed Force Mode Dual AC Single Harmonic Tapping Mode Phase Imaging Harmonic X <20 nN <5 nN <10 nN <3 nN <5 nN <100 pN The History of Force Measurements Is Simply Better & Better Algorithms To Account For The Beam Bounce Instabilities. The Next Evolution in SPMTM
  30. 30. The Next Evolution In AFM Even The Mechanical Force Of A Photon (1.6 pN) Has Been Measured Recently With A Nanonics Tuning Fork System With Nanonics The Tuning Fork Provides The Ultimate In Force Sensitivity The Next Evolution in SPMTM
  31. 31. The Next Evolution In AFM The History of Force Measurements Peak Force Force Volume<50 nN Pulsed Force Mode Dual AC Single Harmonic Tapping Mode Phase Imaging Harmonic X <20 nN <5 nN <10 nN <3 nN <5 nN <100 pN VISTATM Vivid Imaging AFM <1.6 pN The History of Force Measurements Is Simply Better & Better Algorithms To Account For The Beam Bounce Instabilities. Tuning Forks Provide The Ultimate in Force Sensitivity at 1.6 pN Unachievable by Beam Bounce Methods The Next Evolution in SPMTM
  32. 32. The Next Evolution In AFM Proven In The Literature By Measuring Force Sensitivity on Cells Down To 5pN The Next Evolution in SPMTM
  33. 33. The Next Evolution In AFM Apetureless Force Detection Of Plasmons With Extensions To the MidIR & Thz Mechanical/Photon Induced Force (PiFM) Detection Of Plasmonic Distribution The Next Evolution in SPMTM
  34. 34. The Next Evolution In AFM Nonetheless Nanonics Systems Readily Allow For Both Tuning Fork or Beam Bounce Feedback & Any Probe Glass or Silicon Tuning Fork Feedback Advantages: • Highest force sensitivity • No feedback laser • Exact point of contact & • True non-contact Beam Bounce Feedback Advantages: • Contact mode • Mount any 3rd party probeLeading To New Directions In Research AFM Controlled Gas Delivery & Associated Kelvin Probe Alterations [Customer Publication] The Next Evolution in SPMTM
  35. 35. Therefore Nanonics Is Proven In The Literature To Image With Excellent XY Morphological Fidelity Even Compared To FESEM The Next Evolution in SPMTM The Tuning Fork Uniquely Provides A High Quality Factor, Q, For Ultra Sensitivity In AFM And In AFM Morphology. This Is Not Available With Any Beam Bounce Feedback AFM (see green highlighted customer description). Also True Non-contact Is Achieved With The Nanonics Tuning Fork Systems. Thus, Nanonics Provides The Only AFM Systems That Allow For Switching Between AFM And STM Feedback With The Same Probe. Proving Non- contact AFM Operation
  36. 36. As Shown In This Multicenter Comparison Tuning Fork Feedback Produces No Optical Artifacts Critical in Single Molecule Imaging and NSOM FCS The CONCLUSION MultiCenter Comparison Confirming Nanonics Singular Capabilities The Next Evolution in S
  37. 37. Ni Si Nanonics Results Without Beam Bounce Laser Artifacts Topographic Image Si 0V Ni Ni Ni  Nanonics MultiProbe MultiView 4000 SPM  Electrical probe using a bias of 0V  Edge of Nickel capacitor on n type silicon was scanned  During scanning feedback laser light was switched on and off.  Deminstrating that feedback Light Off Light Off Light Off Light On Light Off Light Off Light On Light On Light On Light On Tuning Fork Conductivity Image With and Without Laser Photocurren t Artifact The Next Evolution in SPMTM
  38. 38. The Next Evolution in SP Such Conductivity Images Without Interference Can Scan State of the Art Transistors At High XY Resolution
  39. 39. The Next Evolution In AFM NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Nanothermocouples Plasmonic TERS NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis Probes are also Multiprobe friendly NanoToolKitTM of Unique Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  40. 40. Glass probes also resolve critical issues in biological imaging Silicon cantilever geometry is for from ideal for biology. They:  Generally are not angled or partially angled & thus put pressure & easily penetrate cell membranes  Silicon probe tips close to straight & squeeze out water layers and protruding structures such as microvilli  Produce images with shadow  Lack high aspect ratios with long tips compromising drastically deep penetration into invaginations in cell membranes & between cells  Have flat cantilevers acting like a paddle that reduces Q (quality factor) in liquid to single digits  Use of laser beam feedback causes an ~20% error in cellular elasticity due to lack of information on point of contact Glass cantilever geometry is ideal for biology. They:  Are angled for minimal cell pressure, penetration and shadowing  Have high aspect ratios and long tips allowing even imaging of microvilli with different types of cantilevers  Allow today previously impossible functional imaging on live cells such as SECM, patch clamp, NSOM etc  Have cylindrical cantilevers with essentially no liquid damping  Accurately allow determining point of contact with single pN force sensitivity with tuning fork feedback The Next Evolution in SP
  41. 41. Imaging Microvilli of MDCK Cells Microvilli in live MDCK cells with a cantilevered glass probe attached to tuning fork Q factor in liquid 5000 normally 4 or 5 with beam bounce methods silicon cantilevers Only in 2016 were very specialized silicon cantilevers able to image this structure and even these cantilevers could not approximate the ideal of glass probes and so were not able to measure the elasticity (see next slide) The Next Evolution in SP
  42. 42. AFM With Tuning Forks Show For The First Time MicroVilli Elasticity 11µm 0.22 Volts -0.28 Volts 11µm 546.50 nm -530.23 nm Height Experimental Amplitude Low Amplitude Soft Trampoline High Amplitude Stiff Trampoline High Amplitude MicroVilli Are Seen And Shown To Be Less Stiff Than The Cell Membrane The Tuning Fork Had A Force Constant of 5000 The Next Evolution in SP
  43. 43. Liposome Single Walled Liposome Imaging The Next Evolution in SP
  44. 44. AFM Scanning of Fibroblast Cells Cantilevered AFM Probe Fibroblast Cells 3D Topographic Images of the Fibroblast Cells Watch The scan https://www.dropbox.co m/s/76h0g3wdwhweo70 /Cellular%20Imaging% 20Scan.mov?dl=0 The Next Evolution in SPMTM
  45. 45. 6.0µm Neuroblastoma Cells in Medium Re- Trace 6.0µm • Q factor = 2600 Comparing VISTATM UltraSensitivity Above with Similar AFM Imaging With Beam Bounce Based AFM Feedback Using An Alternate AFM Trace The Next Evolution in SPMTM
  46. 46. Nikon Nanonics Multiview 2000 Probe & Sample Scanning The Next Evolution in SPMTM Readily Added To Any Beam Scanning Confocal or Non-linear Microscope
  47. 47. Two Photon Fluorescence &Topographic of CLL Cells Two Photon Image Scan Range 50 x50 µm Topographic Image Scan Range 50 x50 µm Two Photon Image of GFP Label CLL B cells Accumulate in bone marrow and blood They crowd out healthy blood cells CLL is a stage of small lymphocytic lymphoma The Next Evolution in SPMTM
  48. 48. With Tuning Forks These Geometric Restrictions of Beam Bounce Tracking Are Removed Allowing Even Water Immersion Objectives Reflection of the mount in liquid Ultra low working distances as small as 3.5 mm that provide ultra high numerical aperture objectives upto 0.8 The Next Evolution in SPMTM
  49. 49. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis Probes are also Multiprobe friendly Many Functionally Important Optically Friendly Probes The Next Evolution in SPMTM
  50. 50. Apertured Near-field Scanning Optical Microscopy Ultimately Resolves Bleaching, Correlation & Resolution NSOM illumination is localized and hence NO bleaching of fluorescent molecules occurs outside the local spot of illumination Only those molecules under the probe tip get excited Pixel by pixel AFM gives absolute correlation
  51. 51. NSOM Development Has Now Reached The Pinnacle of Live Cell Imaging With Synergism With Far-field Super-resolution Techniques The Next Evolution in SP
  52. 52. 7.0µm FLUORESCENCE GREEN EXCITATION AFM ABSORPTION BLUE EXCITATION AFM STEM Cell NSOM Imaging Correlated with Topography With diI Membrane Staining 7.0µm Excitation 514.5 nm Excitation 457 nm 7.0µm 7.0µm Absorption Fluorescence
  53. 53. Absorption NSOM Of Live MDCK Cells Stained With Di-4-AN(F)EPPTEA Shows Totally Different Contrast To Fluorescent Confocal706.66 nm 0.00 nm 7.4µm AFM NSOM Brightest point 3DAFM Confocal Fluorescence Comparison Mean AFM Normal Force  Solution: 60 µL  Q-factor in air: 2000  Q-factor in liquid: Approx The Same  Scan 40x40 micron  12 ms/pixel  488nm  Dark dots (green arrow) due to NSOM absorption of membrane emanating microvilli filled with dye  Large dark region is correlated with a large cilliary protrusion in the topography (blue arrow) The Next Evolution in SP
  54. 54. All Modes Possible 8.0µm NSOM Absorption The Next Evolution in SPMTM
  55. 55. Dye Staining Was With Voltage Sensitive Dye That Undergoes A Stark Effect With Such A Dye We Can Prove Live Cell Glass Probe NSOM Imaging The Membrane Binding Dye Is Di-4- AN(F)EPPTEA Cell Membrane With Membrane Voltage With KCL Membrane Voltage Reduction Depolarization l - + + - Absorption Fluorescence 514nm
  56. 56. Imaging Membrane Potential With NSOM Proving Live Cell Imaging Proving The Imaging Was Of Live MDCK Cells By Imaging Depolarization With Addition of 5mM KCl NSOM Fluorescence Imaging of Membrane Potential With Di-4- AN(F)EPPTEA Stained Membranes Using a Large Probe [Live cell near- field optical imaging and voltage sensing with ultrasensitive force control, OPTICS EXPRESS Vol. 25, 29 May 2017 https://doi.org/10.1364/OE.25.0121 31] The Next Evolution in SP
  57. 57. 10µm 10µm 13.08 KHz -0.00 KHz 37.18 KHz -0.00 KHz NSOM Before added KCl NSOM After added KCl Before KCl After KCl Near-field Fluorescence Robust Change With Membrane Voltage  Robust change indicative of NSOM measurement  NSOM depth of focus very low  NSOM is membrane centric with little out-of-focus noise  Thus, the brightest pixel in the image before and after KCl shows a considerable reduction
  58. 58. NSOM Illumination without Background Is Also Very Important for FCS Attoliter Illumination Volumes Alloswing 3 Orders of Magnitude Increased Sensitivity 1E-3 0.01 0.1 1 10 100 1.00000 1.00002 1.00004 1.00006 G(t)Amplitude 10nM 100nM 1m correlation time (s) The Next Evolution in SPMTM
  59. 59. Super-resolution Fluorescence Single Molecule Biology Of Amyloid Fibrils With Nanonics The Next Evolution in SPMTM
  60. 60. The Next Evolution in SPMTM Synergistically Confirmed 3 years Later Using Far-field Single Molcule Super-resolution Fluorescence by Moerner the 2014 Nobel Prize winner in Chemistry
  61. 61. AFM image Lifetime image with decay analysis Lifetime image without analysis Integrated NSOM Super-resolution Fluorescence Lifetime Imaging of Liposomes Stained with Di-4-AN(F)EPPTEA The Next Evolution in SPMTM
  62. 62. And Many Other Functional Imaging Tasks Capable of Being Accomplished Only With Nanonics Probes
  63. 63. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Thermal Conductivity or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis And We Have NanoToolKitTM of Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  64. 64. From: D. Ossola, M.-Y. Amarouch, P. Behr, J. Vö rö s, H. Abriel, and T. Zambelli, “Force-controlled patch clamp of beating cardiac cells,” Nano Lett. 15(3), 1743–1750 (2015). Pioneers In Force Sensing Nanopipettes
  65. 65. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Thermal Conductivity or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis And We Have NanoToolKitTM of Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  66. 66. Fountain Pen Nanochemistry To Write and NanoDispense as NanoDrops a Variety of Molecules Including Proteins, DNA etc From Solution or Dispersion The Nanopipette Aligns As It Draws The Next Evolution in SPMTM Watch A Movie Of Drawing With An AFM Fountain Pen https://www.dropbox.com/s/puk5hzjp8ge nhm9/Drawing%20SWCNTs%20on%20Si O2.mp4?dl=0
  67. 67. The crucial factor is that the 'fountain pen' can have different inks channeled into it automatically, simply by connecting it up to standard high- performance liquid chromatography instrumentation. This should make writing a multi-protein nanoarray much easier than by using DPN, and without the need for any complex pre-treatment of the substrate. NanoFountain Pen Protein Printing
  68. 68. 32.521.510.50 24 22 20 18 16 14 12 10 8 X[µm] Z[nm] Deposition of a Copper Line of ~15nm Between Two Electrodes The Next Evolution in SPMTM The Ultimate In Resolution
  69. 69. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Thermal Conductivity or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis And We Have NanoToolKitTM of Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  70. 70. Nonetheless Nanonics Systems Readily Allow For Both Tuning Fork or Beam Bounce Feedback & Any Probe Glass or SiliconTuning Fork Feedback Advantages: • Highest force sensitivity • No feedback laser • Exact point of contact & • True non-contact Beam Bounce Feedback Advantages: • Contact mode • Mount any 3rd party probeLeading To New Directions In Research AFM Controlled Gas Delivery & Associated Kelvin Probe Alterations [Customer Publication] The Next Evolution in SPMTM
  71. 71. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Thermal Conductivity or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis And We Have NanoToolKitTM of Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  72. 72. Scanning Ion Conductance Microscopy Advantages • SICM cantilevered nanopipette probes monitors the ion conductance & topography in separate channels • High resolution SICM with 10-30 nm apertures • AFM Feedback holds the tip in ultrasensitive feedback with the sample and monitors the topography as a separate channel • AFM of the cells can be obtained with the highest sensitivity • Largest Z range available: <85µ
  73. 73. AFM Controlled Conductance Microscopy Porous Sample or Biological Membrane with Pores or Channels Cantilevered NanoPipette Electrode in NanoPipette with counter electrode in Solution in which Porous Sample is Sitting Measuring the Porosity of MicroPores Non-Destructively with AFM Controlled Conductance Imaging
  74. 74. Scanning Ion Conductance Microscopy With A Cantilevered NanoPipette With Normal Force Topography Correlated With SICM Signal Of Nucleopore Filters 2.0µm2.0µm Height SICM
  75. 75. Similar Imaging Correlation between (a) Topographic and (b) Current image.
  76. 76. Line Scan
  77. 77. Direct Correlation of Peak Contact With Current
  78. 78. Nanonics Atomic Force For Real Samples Opens New Horizons: C. elegans The Next Evolution in SPMTM
  79. 79. AFM C. elegans Only Achievable With Nanonics
  80. 80. All modes of optical microscopy on-line including fluorescence & DIC (differential interference contrast) To Make Things Easier to View The Next Evolution in SPMTM
  81. 81. Differential Interference Contrast (DIC) & Phase Imaging Glass AFM probe in feedback on a cell surface in physiological media as a function of the focus of a 50 X objective in a DIC microscope. Notice that when the cell is in focus (lower right) the probe is completely transparent & invisible.
  82. 82. Thus Controlled Nanopipette Penetration & Injection of C. elegans With Nanonics MultiView System Is Made Very Easy Nanopipette Controlled By Atomic Force Microscopy Injecting a Fluorescent Dye Around A Specific Neuron With On-line Fluorescence Microscopy Nanonics Patented Cantilevered Nanopipette Controlled Point of Penetration with Nanonics Atomic Force Microscope Stained Neuron The Next Evolution in SPMTM
  83. 83. Readily Moved Onto The Stage Of Any Renishaw Microscope Atomic Force Microscopy Raman MicroSpectroscopy AFM Raman The Next Evolution in SPMT
  84. 84. AFM Sample Z Feedback Allows For Accurate Raman Spectral Intensity Comparisons At Different Sample Positions With AFM Autofocus & Nanonics AFM Large Z Range Is Required For The Large C. elegans Dimensions
  85. 85. On-line Raman Chemical Indentification Obtained on C. Elegans AFM Controlled Nanopipette Lipid Injection Laser 785 nm Power 2mW Exposure time 600s CCD image of the point of AFM injection of lipid & Raman investigation in C. elegans with on-line chemical identification Raman Spectrum of C-H vibration of lipid injected into C. elegans collected at region indicated in the image to the left The Next Evolution in SPMTM
  86. 86. Multiprobe AFM Force Excitation & Detection One Probe Periodically Excites While A Second Probe Monitors C. elegans Oscillation 0 2 4 6 8 10 12 14 16 18 20 0.1 0 2 4 6 8 10 12 14 16 18 20 0.2 0.4 0.6 0.8 Time, msec Voltage,V 0 2 4 6 8 10 12 14 16 18 20 0 200 400 600 800 1000 1200 Time, msec Voltage,mV 0.2 0.3 0.4 Time, msec Voltage,V 0 2 4 6 8 10 12 14 16 18 20 0 200 400 600 800 1000 1200 Time, msec Voltage,mV 0 2 4 6 8 10 12 14 16 18 20 0.1 0.2 0.3 0.4 Time, msec Voltage,V 0 2 4 6 8 10 12 14 16 18 20 0 200 400 600 800 1000 1200 Time, msec Voltage,mV Set Point = 15 nN, Tip size = 0.2 um First 1 sec Bottom Excitation Probe & Top Detection Probe Set Point = 15 nN, Tip size = 0.2 um After 10 secs Bottom Excitation Probe & Top Detection Probe Both After 10 and 15 secs (below) of AFM Probe Periodic Excitation Adaptation Is Seen Of The Mechanical Response Detected With A Second AFM Probe On-line dissipates Touch Receptors with GFP mec-2 Chimeras in Mechanosensory ALM Touch Receptors Next Evolution in SPMTM
  87. 87. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Thermal Conductivity or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis And We Have NanoToolKitTM of Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  88. 88. Cantilevered SECM probes for simultaneous normal force sensing with full SECM functionality A continuous nanowire of platinum is embedded in glass and as can be seen in the electron micrograph below the wire is flush with the nanometric glass end SEM of the nanowire at the nanometric tip 70 nm Diagrammatic representation of the SECM probe Platinum nanowire Glass The Next Evolution in SPMTM
  89. 89. Nanonics Founded By Aaron Lewis With Contributions to Both Optics and Electrochemistry
  90. 90. Exemplary approach curve of a Nanonics Nanoelectrode SECM Probe To A Non-conducting Surface 0.00 0.20 0.40 0.60 0.80 1.00 1.20 0 2 4 6 8 10 12 14 16 18 20 NormalizedCurrent L= d/r Nanoelectrode_1.3 Neg_4 Neg_3 Theor_Neg_RG5 Theor_Neg_RG10 Theor_Neg_RG20 Neg_4 Neg_3 Theor_Neg_RG5 Theor_Neg_RG10 Theor_Neg_RG20 Negative feedback approach curve of a Nanonics Nanoelectrode SECM Probe The experimental approach curve (smooth lines) was fitted with theoretical approach curve (points) A determination of the effective radius is 180-200nm Approach Speed: 0.05µm/ 0.0167sec The Next Evolution in SPMTM
  91. 91. 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 0 2 4 6 8 10 12 14 16 18 20 NormalizedCurrent L= d/r Nanoelectrode_1.3 Theor_Post Post_2ndtime Positive feedback approach curve of previous 180-200nm electrode Exemplary Approach Curve of a Nanonics Nanoelectrode SECM Probe Relative To A Conducting Surface Without Sample Voltage The Next Evolution in SPMTM
  92. 92. -9.00E-12 -8.00E-12 -7.00E-12 -6.00E-12 -5.00E-12 -4.00E-12 -3.00E-12 -2.00E-12 -1.00E-12 0.00E+00 1.00E-12 2.00E-12 -400-350-300-250-200-150-100-500 Current,Amp Potential, mV (vsHg/Hg2SO4) 10mM Ferrocyanide & 0.1M Na2SO4 CVFC# 4 Radius= 4nm Cyclic voltammogram of 4 nm radius Nanonics Nanoelectrode SECM probe in presence of 10mM ferrocyanide and 0.1M Na2SO4 Exemplary approach curve of a Nanonics Nanoelectrode SECM Probe This is the ultimate and should not be taken as a standard The Next Evolution in SPMTM
  93. 93. ΔX=250 nm Line Scan of the SECM Image An Order of Magnitude Better Resolution Than All Other SECMs Along With Topography Due To On Line AFM The Next Evolution in SPMTM
  94. 94. Unique Designs of Electrochemical Cells With Environmental Control & Optical Integration Reference Electrode
  95. 95. Details of Electrochemical Cell Counter Electrode Completes Circuit Allowing Current To Flow From Probe and/or Sample or Vice Verse Back Electrical Contact For Allowing Voltage To The Sample Relative To The Reference Electrode SECM Probe or Working Electrode With Voltage Relative To Reference Electrode Sample Voltage Between SECM Probe & Reference Electrode Reference Electrode Voltage Between Sample & Reference Electrode Counter Electrode To Probe & Sample
  96. 96. An Electrochemical Liquid Cell Designed To Protect From Spillage
  97. 97. The Next Evolution In AFM Top Left: Exposed SECM Cell. Sample is placed in ring in the middle. Changeable wire electrodes on the sides. Top Right: SECM Cell that is partially covered to minimize evaporation. Left: SECM Cell from backside. Contains back contact for applying voltage to the sample. SECM Cell
  98. 98. Thus All Nanonics Systems Permit On-line Raman Chemical Characterization Atomic Force Microscopy Raman MicroSpectroscopy AFM Raman The Next Evolution in SPMTM
  99. 99. The Nanonics MultiView 2000 System Are Flexible Under The Lens of A Bruker microRaman MV2000
  100. 100. MultiView 2000 with the Renishaw InVia System Combinations with Raman & Fluorescence Spectroscopy Especially Effective with Water Immersion Objectives from Above on Opaque Materials • Chemically image the electrochemical process MV2000
  101. 101. STFMTM Probes Allow For Water Immersion Objectives From Above to Be Used Reflection of the mount in liquid Ultra low working distances as small as 3.5 mm that provide ultra high numerical aperture objectives upto 0.8 Ultra high Raman signals The Next Evolution in SPMTM
  102. 102. Substrate: Thin layer of Cu on Si Working Electrode and AFM probe: When voltage is applied on it, it creates oxidants which oxidize the Cu. When Cu is etched, the Si is exposed. A spectro- chemical signal of Si is detected. SECM-Raman SECM Applications: Ionic Dissolution + Electron-Kinetic Transfer
  103. 103. Results SECM- Raman Optical image Raman signal BEFORE SECM Applications: Ionic Dissolution + Electron- Kinetic Transfer
  104. 104. IN REAL TIME Raman signal in real time SECM Applications: Ionic Dissolution + Electron-Kinetic Transfer Results SECM- Raman
  105. 105. NanoOptical Light Source Nanopipettes for Ionic Conductance NanoFountain Pens for Liquid & Gas Delivery NanoVacuum NanoHeaters or Thermal Conductivity or Nanothermocouples Plasmonic NanoProbes with Single Gold NanoParticles Glass Insulated Coaxial NanoElectrical & Cantilevered NanoElectrochemical Probes All Probes Are Electron & Ion Optically Friendly With Non-Obscuring Cantilevers & With Probe Tips Exposed To The Optical Axis And We Have NanoToolKitTM of Optically, Electron Optically & Multiprobe Friendly Probes The Next Evolution in SPMTM
  106. 106. Thermal conductivity imaging of Sematech produced voids 150100500 75 70 65 60 55 50 45 40 35 X[nm] Z[mV] ΔX=30 nm The line-scan across the thermal conductivity image demonstrates lateral resolution 30 nm on a test sample of voids in Silicon Provided By Sematech The Next Evolution in SPMTM AFM / Thermocouple or Thermoresistive Probe
  107. 107. Thermal Coonductivity & Topography Imaging of Nanotubes & Quantum Dot Decorated DNA Imaging Semiconductor Quantum Dots With Thermal Conductivity Image (Above) SiGe Structures on Silicon Imaged With Topography, Phase and Thermal Conductivity (Aux Channel) The Next Evolution in SPMTM
  108. 108. NanoThermal Imaging of Polymer Blend & Thermal Analysis Select a Point for Thermal Analysis & Choose The Experimental Parameters
  109. 109. ThermoMechanical and Differential Scanning Calorimtery
  110. 110. ThermoMechanical Analysis of Nylon
  111. 111. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation Other AFM Imaging Tasks The Next Evolution in SPMTM
  112. 112. Quantum Dot Decorated DNA With Tuning Fork Quality Factors of Thousands Resolution X Y: Uncompromised NanoImaging The Next Evolution in SPMTM
  113. 113. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  114. 114. Resolution Z: UltraLow Noise Image Atomic Steps Strontium Titanate (001) 7006005004003002001000 10 9 8 7 6 5 4 3 X[nm]Z[Å] ΔZ=0.28 nm Tuning-fork based feedback with Nanonics glass cantilevered AFM probe Noise in this image is 0.01 nm peak to peak with the RMS generally three times lower The Next Evolution in SPMTM Noise Is At The 0.01nm Level
  115. 115. Low Noise & Ultrahigh Resolution Comparative Examples Comparing tuning Fork UltraSensitivtty with Similar HOPG Beam Bounce Image Omnicrom Single atomic steps on Highly Oriented Pyrolytic Graphite HOPG is a standard of choice for AFM resolution in Z The Next Evolution in SPMTM
  116. 116. Single atomic steps on Highly Oriented Pyrolytic Graphite HOPG is a standard of choice for AFM resolution in Z Low Noise & Ultrahigh Resolution Comparative Examples Comparing tuning Fork UltraSensitivtty with Similar HOPG Beam Bounce Image The Next Evolution in SPMTM
  117. 117. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  118. 118. UltraLow Drift Two of the Same Area Taken 20mins Apart Only Single Nanometers Per Hour The Next Evolution in SPMTM
  119. 119. Resolution Test Fischer Structure 0.05 mm Heightnm 1 2 3 4 5 4nm 0.1
  120. 120. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  121. 121. Monolayer Polymer Film Decorated With Gold Nanoparticles Showing Packed Polymer Strands 600nm The Next Evolution in SPMTM
  122. 122. Soft Sample Imaging: Molecular Pentacene Dendrimers The Next Evolution in SPMTM
  123. 123. Soft Sample Imaging: Monolayer of Associated Insulin Associated Insulin Is Used As Exemplary of Other Associated Proteins Such As Amyloid Proteins The Next Evolution in SPMTM Y: 1 mm X: 1 mm
  124. 124. Soft Sample Imaging: BioMolecular Imaging of Collagen Protein Fibers Comparing tuning Fork UltraSensitivtty with Similar Collagen Beam Bounce Images A Tuning Fork Image of Collagen As Compared With Similar Images From Other Vendors With Beam Bounce Feedback The Next Evolution in SPM
  125. 125. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  126. 126. Phase Image Overlaid on Topography Phase Imaging NanoParticle Impact-Modified Heterogeneous PMMA Thin Film Coating of A Thermoplastic and Transparent Coating The Next Evolution in SPMTM
  127. 127. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  128. 128. Excellent 3D and Deep Trench Capabilities With High Aspect Ratio Glass Probes Can Be Imaged Only By Nanonics Due To Availability of:: • Large Z Scanning Range 85m • The Long Tip Length of 100m or greater • Very High 10:1 Aspect Ratio Glass Probes • VISTATM Soft Touch AC Mode
  129. 129. Glass Probes Combined with Unprecedent Z Range 20 Micron PMMA Polymer Microspheres Large 100 micron z scanning range of the Nanonics 3D Flat Scanning System and the ability to use glass cantilevered AFM probes with 100 micron or more tip length allow such large topographic alterations to be readily imaged. AFM Imaging Only Nanonics Can Provide +
  130. 130. The large Z range of the Nanonics 3D Flat Scanner allows us to readily measure the height of these microspheres. 20 Micron PMMA Polymer Microspheres
  131. 131. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Magnetic Force Imaging • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  132. 132. Even MFM Has A Unique Nanonics Contribution Single Co Particle Probes In Glass Nanopipettes Like Our Plasmonic Probes Only At The Tip Of The Probe Without Magnetic Interference From Coated MFM Probes The Next Evolution in SPMTM
  133. 133. Magnetic Force Microscopy (MFM) AFM Topography A close up AFM Height image of Co nanoparticles (bar is 200nm) A close up MFM image of Co nanoparticles presented at left (bar is 200nm) The Next Evolution in SPMTM MFM
  134. 134. Magnetic Force Microscopy (MFM) Videotape The Next Evolution in SPMTM AFM Topography MFM AFM Topography MFM
  135. 135. • Resolution In X Y & Z Mapping • UltraLow Noise • Low Drift & High Resolution • Soft Sample Imaging • Phase Imaging Heterogeneous Thin Film • Deep Trench Imaging of Real Samples • Fine Nanomanipulation The Next Evolution in SPMTM Other AFM Imaging Tasks
  136. 136. Fine Nanomanipulation and Imaging of Chromosomes with Fine Glass ProbesGlass probes with their slender profiled tip are ideal for imaging and nanomanipulation of soft structures such as this human chromosome Silicon AFM Probes Cannot Compete With Glass AFM Sensors Both In Imaging & Nanomanipulation
  137. 137. Placing A Single Gold Nanoparticle Onto a Single Single Walled Carbon Nanotube AFM (Left) and SEM (Right) Image93.71 nm 580nm The Next Evolution in SPMTM AFM Image SEM Image
  138. 138. 138 Placement of a Nanoparticle On a Plasmonic Structure 200n m 200n m 100n m The Next Evolution in SPMTM

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