Applications of Atomic Force Microscopy in Bone Research Joseph M. Wallace IUPUI Department of Biomedical Engineering ORS Workshop March 15 th, 2014
General Seminar Outline Need for nanoscale techniques to analyze bone TEM, SEM, CryoTEM, AFM Atomic Force Microscopy (AFM): Principles and Imaging Modes AFM Applications in Bone Research: Imaging Investigating Bone Cell Activity and Structure Processing Bone for Collagen Analysis General Analysis of Collagen Structure Issues to address when considering AFM for Imaging AFM Applications in Bone Research: Mechanics Indentation: Calibration, assumptions, limitations Other cool mechanical techniques
Hierarchy Bone s Hierarchical In Collagen-Based Structure Tissues Most musculoskeletal tissues are hierarchical: defined structures can be found at multiple length scales Trabecular Bone Cortical Bone Trabecular Packet and Lamella Osteon and Lamella Mineralized Collagen Fibril Collagen / Mineral Composite Alpha Chain Crystal Lattice Kastelic et al., CTR 1978 Practical Orthopaedic Sports Medicine & Arthroscopy 2007 Tendon Fiber / Fascicle Fibril Microfibril Tropocollagen
Critical Gap in Understanding Collagen Nanoscale collagen fibril: primary building block of many collagen-based tissues Critical gap: how do nanoscale features influence more clinically-relevant tissue and structural properties? α 1 α α? 2 1 σ ult σ y Stress (σ) Modulus Elastic Region ε e Yield Area under curve: Toughness = total energy Plastic Region Strain (ε) ε t Failure
Methods For Analyzing Collagen Structure Bone researchers come from a variety of backgrounds We have borrowed analytical techniques along the way: Spectroscopic: Fourier Transform Infrared Spectroscopy (FTIR) Raman Spectroscopy Nuclear magnetic resonance (NMR) Scattering: Small Angle X Ray Scattering (SAXS) Wide Angle X Ray Diffraction (WAXD) Neutron Scattering Lack the ability to directly answer questions about nanoscale assembly and organization of collagen and mineral in bone.
Methods For Imaging Collagen Structure Quan, Methods in Enzymology, 2013 Randall: Nature and Structure of Collagen; 1953 Transmission and/or scanning electron microscopy High resolution imaging of collagen ultrastructure X Harsh sample preparation X Sectioning X Imaging conditions X Reduced ability to draw accurate conclusions
Cryo-TEM Fixation, cryosectioing and vitrification Samples maintained in a hydrated state throughout sectioning and microscopy Heavy-metal staining is not necessary Can be technically challenging to perform Quan, Methods in Enzymology, 2013
Atomic Force Microscopy (AFM) High spatial resolution Samples can remain intact Image in fluid or air Range of temperatures Minimal preparation Characteristics less likely artifacts of processing or imaging Wallace et al., Langmuir, 2010 Can be used to extract nanoscale mechanical properties
AFM Image of Pentacene (C 22 H 14 ) Gross et al., Science, 2009; 325 (5944):1110-1114
AFM Image of Hexabenzocoronene Gross et al., Science, 2012; 237:1326-1329
Basics of AFM Imaging: The Probe Nanoscale tip mounted on a microscale cantilever Kopycinska-Muller, Ultramicroscopy, 2006 Wallace, Bone, 2012 Probe X-Y-X Piezo Combination Feedback System Cantilever
Basics of AFM Imaging: System Components Piezoelectric actuators: precise movement under electric potential Raster-scanned (x-y direction) Force transducer: interaction force (typically cantilever s deflection) Cantilever deflection (z-direction) measured by photodiode Control system maintains a desired force between probe and sample and (movement in z-direction) Wallace, Bone, 2012 Probe X-Y-X Piezo Combination Feedback System Cantilever
van der Waals Potential Energy Curve Repulsive Regime Attractive Regime FORCE Net force = 0: distance between atoms is 2-3 A (~ length of a chemical bond) Net attractive force weakens as interatomic separation decreases DISTANCE Weak atomic attraction Attraction increases until electron clouds begin to electrostatically repel each other
Contact Mode Monitor Deflection Force Probe in constant contact, cantilever deflects like a spring according to Hooke s law (F = kx) Tip-Sample Separation Frictional and adhesive forces can damage sample and distort image Control system maintains user-defined deflection by vertically moving scanner using piezoelectric actuator
Tapping Mode Monitor Amplitude Force Intermittent Contact Tip-Sample Separation
Tapping Mode Monitor Amplitude Force Approach Withdrawal Tip-Sample Separation Oscillation Amplitude 1.5 1 0.5 0-0.5-1 Cantilever oscillates at userdefined amplitude near cantilever resonance frequency Setpoint Bump Bump Valley -1.5 0 5 10 15 Time
Tapping Mode Monitor Amplitude Probe in contact for a small fraction of its tapping period, lateral forces are reduced Probe must be tuned at resonance frequency can be challenging for some probes when in fluid Tuning in Air: Single resonance peak www.asylumresearch.com Tuning in Fluid: Multiple and spurious peaks
Peak Force Tapping Mode Monitor Force Peak Force Force Approach Withdrawal Tip-Sample Separation Cantilever oscillates at low frequency (1-2 khz) No need for tuning! Slow tapping: force curve is captured with each tap Control system maintains user-defined maximum force Direct control of maximum normal force Protects tip and sample wear Limits indentation depth to a few nm - increases resolution by limiting contact area Mechanical properties mapped pixel-by-pixel at same resolution as the height image (PeakForce QNM)
AFM Applications in Bone Research: Imaging
AFM Imaging of Collagen Chernoff, J of Vac Sci Tech, 1992 AFM s adoption in bone research has been slow First AFM-based study in collagen came in 1992 (a previous study in 1989 used Scanning Tunneling Microscopy) Observed fibrillar and monomeric collagen
Imaging of Osteoclast Activity Initial observations of cellular activity were qualitative (analyses of collagen morphology were not presented) Bozec, Ultramicroscopy, 2005 Sasaki, J Elec Micro, 1993 Sasaki, CTI, 1995 Hassenkam, Anat Record, 2006
Mineralized collagen deposition in vitro Collagen deposition from MLO-A5 cells Barragan-Adjemian, CTI, 2006
Imaging of Canilicular Structure Reilly, Annals of BME, 2001 Average canilicular diameter: ~400-500 nm, larger than values typically reported from TEM studies Lin, J of Microscopy, 2010
AFM Imaging in Bone In general, we are more interested in collagen in bone Unpolished 3 µm Diamond Suspension
AFM Imaging in Bone The surface is completely mineralized
Removing Mineral in Bone to Expose Collagen Dealing with mineral is a challenge to analyzing collagen some mineral must be removed 10% Citric acid (to remove mineral), 6.5 % Sodium Hypochlorite (to remove non-collagenous proteins) Habelitz, J Struct. Bio, 2002 7% or 17% Phosphoric Acid Bozek, IEEE, 2005; El Feninat, J Biomed. Mat Res, 1998 5% Formic Acid Ge, Mat. Sci. Eng, 2007 30 mm EDTA Kindt, Nanotechnology, 2007
Removing Mineral in Bone to Expose Collagen Dealing with mineral is a challenge to analyzing collagen some mineral must be removed 30 mm EDTA Kindt, Nanotechnology, 2007
Demineralization with EDTA EDTA - Ethylenediaminetetraacetic Acid Chelating agent - sequesters divalent cations (notably Ca 2+ ) Treat with 0.5M EDTA, ph 8 (15-20 minutes) Vigorously wash with ultrapure water Sonicate to release surface-bound mineral ions Repeat 3-4 times
Demineralization with EDTA
Demineralization with EDTA
Issues to Consider: Effects of Acid Immature collagen crosslinks are very sensitive to acid Using acids to demineralize bone may inadvertently destroy crosslinks, distort structure Fixation of tissue may be necessary prior to demineralization Fixation (glutaraldehyde or paraformadehyde) may preserve structural integrity Fixation effects on collagen structure are not fully known
100 µm x 100 µm 50 µm x 50 µm 25 µm x 25 µm 10 µm x 10 µm 3.5 µm x 3.5 µm
3.5 µm x 3.5 µm
Analysis of Collagen Structure in Bone Spatial organization of newly formed bone following a surgically-induced injury (rat tibial hole) statistical diameter of the fibers is 159 nm in good agreement with the literature values for type I collagen fibers. Baranauskas, J. Vac. Sci. Tech., 2001 Baranauskas, App. Surface Sci., 2005
Analysis of Collagen in Bone: Fibril Diameter Fibril Diameter: easiest to measure property, first attempts were a bit crude, better with increased resolution Thalhammer, J. Arch. Sci, 2001 Sasaki, J. Mat. Sci. Tech., 2002 Bozec, Ultramicroscopy, 2005
Collagen in Bone: Fibril Diameter Wallace et al., Langmuir, 2010 Caution must be taken when reporting and interpreting diameters measured from fibrils in bone: Fibril packing - width may not be fully exposed
Collagen in Bone: Fibril Diameter Rigozzi J Struct Bio - 2011 Caution must be taken when reporting and interpreting diameters measured from fibrils in bone: Fibril packing - width may not be fully exposed
Collagen in Bone: Fibril D-Spacing HO CH 2 CH 2 HO CH 2 H O HC CH 2 H O H 2 C CH 2 O H O HC CH 2 H H H H N C H C N C C N C H C N C C N C H C N C C N C H C N C C N C H C H O H 2 C CH 2 O H O HC CH 2 H O H 2 C CH 2 O CH 2 glycine X - proline Y - hydroxyproline 1.5 nm HO CH 2 300 nm CH 2 Collagen Fibril Axial D-Periodicity Hodge and Petruska, 1963
http://remf.dartmouth.edu/imagesindex.html TEM Height (nm) 20 15 10 5 0 Randall: Nature and Structure of Collagen; 1953 D-Spacing = 67 nm? AFM SEM 0 0.2 0.4 0.6 0.8 1 Length (microns)
Collagen in Bone: Fibril D-Spacing Many studies have shown observable D-spacing, content that near theoretical value: Sasaki, 2002: 67 ± 2 nm Bozek, 2005: 66.5 ± 1.4 nm Ge, 2007: 66.6 ± 3.8 nm A 2002 study in dentin got me interested Habelitz, J Structural Bio, 2002 n = 322 wet n = 363 dry
Collagen in Bone: Fibril D-Spacing D-periodic spacing: important structural feature that captures aspects related to: Mouse Femur, Mid-diaphysis Internal structure Wallace et al., Bone, 2010; 46: 1349-1354 Wallace et al., J Stuct Bio, 2011; 173: 146-152 Kemp et al., J Stuct Bio, 2012; 180: 428-438 Warden et al., Endocrinology 2013; 154(9): 3178-3187 Gallant et al., Bone 2014; 61; 191-200 Bart et al., Conn. Tiss Res, 2014: In Press Enzymatic and nonenzymatic cross-linking Hammond et al., Bone, 2014; 60: 26-32 Diaz Gonzalez et al., J Biomech 2014; 47(3): 681-686 Voytik-Harbin et al., In Preparation, 2014
2D Fast Fourier Transform Analysis Raw AFM Image Erickson B, Wallace JM et al., Biotechnology Journal, 2013 2D FFT Power Spectrum Harmonics Fibril D-Spacing
2D Fast Fourier Transform Analysis Amplitude (µv 2 ) 120000 100000 80000 60000 40000 20000 Raw AFM Image 0 2D FFT Power Spectrum Harmonics Fibril D-Spacing 0 0.01 0.02 0.03 0.04 0.05 0.06 Position (1/nm) D-periodic spacing 0.015 (1/nm) ~ 67 nm
D-Spacing Distribution Group Samples (%) 100 90 80 70 60 50 40 30 20 10 0 Overall Mean: 67.0 ± 0.0 nm 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm)
D-Spacing Distribution Group Samples (%) 100 90 80 70 60 50 40 30 20 10 0 4 Samples Overall Mean: 67.0 ± 0.0 nm Total of n = 255 fibrils Overall Mean: 68.2 ± 1.2 nm 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm)
Cumulative Distribution Function Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 0 This population: 255 fibrils - Each fibril: 1/255 = 0.392% - Data range: 62.8-71.6 nm 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm)
CDF vs. Histogram Group Samples (%) 50 45 40 35 30 25 20 15 10 5 0 62 63 64 65 66 67 68 69 70 71 72 73 74 Histo CDF 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm) 100 90 80 70 60 50 40 30 20 10 0
Changes in D-Spacing with Disease Group Samples (%) 20 15 10 5 0 Control: n=6, 182 Fibrils Estrogen Depleted: n=5, 168 fibrils KS Test p<0.001 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Wallace et al., Bone, 2010; 46: 1349-1354 D-Periodic Spacing (nm) Control 68.0 ± 2.6 nm Estrogen Dep 65.9 ± 3.1 nm t-test on mean p=0.048
Changes in D-Spacing with Disease Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 0 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Wallace et al., Bone, 2010; 46: 1349-1354 Control: n=6, 182 Fibrils Estrogen Depleted: n=5, 168 fibrils D-Periodic Spacing (nm) KS Test p<0.001
Question Model and Tissue Morphology Publication Tissue Specificity in Mice Gender Effects Estrogen Depletion Estrogen Depletion Osteoporosis and Mechanical Loading Osteogenesis Imperfecta Osteogenesis Imperfecta Osteogenesis Imperfecta and Tissue Hydration Type 2 Diabetes Type 2 Diabetes Raloxifene (in vivo and in vitro) Male mouse, 8 weeks Femur, dentin, tail tendon Male and female mice, 16 weeks Femur OVX in sheep, 5 years + 2 Radius OVX in female, 5 years + 2 Skin (dermis) OVX in ulnar loading in rats Ulna Brtl/+ model, male mice, 2 months Femur Oim/oim model, male mice, 12 weeks Femur Brtl/+ model, male mice, 6 months Tail Tendon Male ZDSD rats, 30 weeks Tibia Female ZDSD rats, 32 weeks Tail Tendon Female dog, treated with Raloxifene Femur No differences in bone vs. dentin, both vs. tendon No differences Mean and population shift Significant population shift No loading effect, population shift with OVX No mean, widening of distribution Mean and population shift Significant population shift Significant population shift Significant population shift Significant population shift Wallace et al., Langmuir, 2011; 26(10): 7349-7354 N/A Wallace et al., Bone, 2010; 46: 1349-1354 Erickson et al., Biotech. J., 2013; 8(1): 1174-126 Warden et al., Endocrinology 2013; 154(9): 3178-3187 Wallace et al., J Stuct Bio, 2011; 173: 146-152 Bart et al., Conn. Tiss Res, 2014: In Press Kemp et al., J Stuct Bio, 2012; 180: 428-438 Hammond et al., Bone, 2014; 60: 26-32 Diaz Gonzalez et al., J Biomech 2014; 47(3): 681-686 Gallant et al., Bone 2014; 61; 191-200
Issues to Consider: Site Selection and Sampling 15 mm = 15000 µm 100 µm x 100 µm 3.5 µm x 3.5 µm Wallace et al., Langmuir, 2011; 26(10): 7349-7354 KS Test p<0.001 0.023% of bone s length
Issues to Consider: Site Selection and Sampling 1 2 3 4 5 6 7 8 9 D-Periodic Gap/Overlap Spacing (nm) 74 72 70 68 66 64 62 60 (# of Fibrils) 1 (35) 2 (33) 3 (40) 4 (39) 5 (35) 6 (29) 7 (26) 8 (23) 9 (28) Axial Location (Proximal End = 1, Distal End = 9) Wallace et al., Langmuir, 2011; 26(10): 7349-7354 288 fibrils for population, n=1 for mean comparisons! WT (288)
Issues to Consider: Effects of Hydration Collagen and bone exist in a hydrated environment in vivo Hydration state can impact morphology and mechanics Wet Tendon Kemp et al., J Struct.Bio, 2012; 180 (3): 428-438. Dry Tendon
Issues to Consider: Effects of Hydration Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 Group Samples (%) 50 40 30 20 10 0 62 64 66 68 70 72 74 D-Spacing (nm) 0 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm) WT Wet (n=255) 68.2 ± 1.2 nm WT Dry (n=257) 68.6 ± 0.8 nm WT Dry WT Wet Nonsignificant mean increase with drying (p=0.200) Loss of fibrils < 66 nm, population more uniform (p<0.001) Kemp et al., J Struct.Bio, 2012; 180 (3): 428-438.
Issues to Consider: Effects of Hydration Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 Group Samples (%) 50 40 30 20 10 0 62 64 66 68 70 72 74 D-Spacing (nm) 0 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm) WT Wet 68.2 ± 1.2 nm WT Dry 68.6 ± 0.8 nm WT Dry WT Wet Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 Group Samples (%) 50 40 30 20 10 0 62 64 66 68 70 72 74 D-Spacing (nm) 0 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm) KS Test: p<0.001 WT Case: 0.4 nm mean increase with drying (p=0.200) Brtl/+ Wet 67.5 ± 1.4 nm Brtl/+ Dry 68.9 ± 1.0 nm Brtl Dry Brtl Wet Loss of fibrils < 66 nm, population more uniform (p<0.001) Brtl/+ Case: 1.4 nm increase with drying (p<0.001) Entire population shifted upward (p<0.001) Kemp et al., J Struct.Bio, 2012; 180 (3): 428-438.
Issues to Consider: Effects of Hydration Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 WT Wet Brtl Wet 0 62 63 64 65 66 67 68 69 70 71 72 73 74 D-Periodic Spacing (nm) KS Test: p<0.001 0 62 63 64 65 66 67 68 69 70 71 72 73 74 Brtl/+ distribution shifted downward vs. WT when wet Cumulative Group Total (%) 100 90 80 70 60 50 40 30 20 10 WT Dry Brtl Dry D-Periodic Spacing (nm) Brtl/+ distribution shifted upward vs. WT when dry Kemp et al., J Struct.Bio, 2012; 180 (3): 428-438. KS Test: p<0.001
Issues to Consider: AFM calibration Absolute distance measurements require accurate piezo calibration AFM systems have large scan ranges (150 µm to < 1 µm) Scanner non-linearities can introduce substantial error over that range Erickson B, Wallace JM et al., Biotechnology Journal, 2013
Issues to Consider: AFM calibration Manufacturers suggest a 10 µm pitch standard at full range 150 times larger than D-spacing, image at 2.3% of max range Standard Scan size (µm) # of pitches Fast Scan Error (%) Slow Scan Error (%) 10 µm 70 6 1.8 2.73 Erickson B, Wallace JM et al., Biotechnology Journal, 2013
Issues to Consider: AFM calibration Manufacturers suggest a 10 µm pitch standard at full range 150 times larger than D-spacing, image at 2.3% of max range Standard Scan size (µm) # of pitches Fast Scan Error (%) Slow Scan Error (%) 10 µm 70 6 1.8 2.73 10 µm 50 4 3.01 5.39 10 µm 40 3 2.90 4.60 1 µm 35 28 5.32 7.96 1 µm 20 16 6.46 11.32 1 µm 10 8 7.96 14.59 100 nm 10 80 6.02 11.98 100 nm 7 60 8.27 14.39 100 nm 5 40 8.14 14.27 Erickson B, Wallace JM et al., Biotechnology Journal, 2013 100 nm 3.5 20 9.17 17.24 100 nm 2 10 9.37 17.74 Erickson B, Wallace JM et al., Biotechnology Journal, 2013
AFM calibration Take Away Message Calibration addresses absolute accuracy, not precision (differential sensitivity between measurements) Does not limit ability to differentiate between groups using the same AFM with the same calibration parameters. Calibration using a scan size and feature size comparable to features of interest is highly recommended
AFM Applications in Bone Research: Indentation Mechanics
AFM-Based Indentation Force (nn) In addition to high resolution imaging, AFM can be used to extract nanoscale mechanical data Approach Withdrawal Tip-Sample Separation (nm)
AFM-Based Indentation: Cantilever Calibration Cantilever Deflection (Volts) 1. Ramp into a rigid sample measure the slope Deflection sensitivity, x nnnn VV 2. Calculate cantilever spring constant Spring constant, k NN mm Z-Piezo Height (nm) 3. Hooke s law: takes deflection (volts) and converts to units of force: F = kx
AFM-Based Indentation: Cantilever Calibration Force (nn) Distance traveled in z-direction measured by z-piezo height Sample deformation: must also consider cantilever deflection: Separation = z height - deflection Z-Piezo Height (nm)
AFM-Based Indentation: Cantilever Calibration Force (nn) Distance traveled in z-direction measured by z-piezo height Sample deformation: must also consider cantilever deflection: Separation = z height - deflection Tip-Sample Separation (nm)
AFM-Based Indentation: Parameters Peak Force Force (nn) Adhesion Force Indentation Depth (δ) Approach Withdrawal Contact Mechanics Fit for Elastic Modulus (E) Tip-Sample Separation (nm) Area Between Curves Energy Dissipation
Hertz Contact Equation The Hertz equation models contact of a spherical indenter with an elastic half space www.wikipedia.com
Hertz Contact Equation The Hertz equation models contact of a spherical indenter with an elastic half space www.wikipedia.com
Hertz Contact Equation z δ www.wikipedia.com r R FF = 4 3 EE ss 1 νν ss 2 RR δδ3 2 E s : Sample modulus ν s : Poisson s ratio R: tip radius of curvature δ: load point displacement Relevant Hertz Assumptions: Strains are small and within the elastic limit Indenter is infinitely stiff (only sample deforms) Indentation depth << radius of curvature of indenter Surfaces are continuous, non-conforming (must be flat) Load is normal to surface
Proper Indentation Model is Essential δ FF = 4 3 r EE ss 1 νν ss 2 rr δδ3 2 Hertz Model: Spherical Indenter depth << radius of curvature E s : Sample modulus ν s : Poisson s ratio R: tip radius α: opening angle FF = 2 ππ EE ss δ 1 νν ss 2 α tan αα δδ2 Sneddon Model: Conical Indenter depth radius of curvature
Indentation in OI Tendon Study y Brtl/+ and WT mice, 6 month old males z 4 tails per group 2-3 fascicles per tail 25-30 fibrils per tail 4-5 locations per fibril Indent force: 20 nn wet (Sneddon) 50 nn dry (Hertz) x
OI Tendon: Differential Effects of Dehydration Wet: 30.5 ± 24.3 MPa Dry: 1268 ± 718 MPa Kemp et al., J Struct.Bio, 2012; 180 (3): 428-438. Wet: 30.1 ± 20.2 MPa Dry: 1737 ± 973 MPa Significant stiffening of fibrils with dehydration More pronounced modulus modulus as a function of disease
OI Tendon: Differential Effects of Dehydration Group Samples (%) 60 50 40 30 20 10 0 KS Test: p=0.129 t Test: p=0.464 0 20 40 60 80 100 120 140 160 180 200 Indent Modulus (MPa) Diseased phenotype not present when wet, phenotype observed when dried Kemp et al., J Struct.Bio, 2012; 180 (3): 428-438. WT Wet Brtl Wet 555 WT Indents: 30.5 ± 24.3 MPa 614 Brtl/+ Indents: 30.1 ± 20.2 MPa Group Samples (%) 20 18 16 14 12 10 8 6 4 2 0 KS Test: p<0.001 T Test: p<0.001 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 2750 3000 3250 3500 3750 4000 4250 4500 4750 5000 Indent Modulus (MPa) WT Dry Brtl Dry 464 WT Indents: 1268 ± 718 MPa 479 Brtl/+ Indents: 1737 ± 973 MPa
Indentation in Diabetic Tendon (Poster #0495) y Control and Diabetic Rat Tail Tendon 4-5 rats per group 2-3 fascicles per rat z ~70 fibrils/tail 4-5 locations/fibril 20 nn (Sneddon) x
Percentage of Group Samples Multiscale Diabetes Study (Poster #0495) 30 25 20 15 10 5 0 p<0.001 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 >25 Elastic Modulus (MPa) CD (1530 indents) ZDSD (913 indents) CD (n=5) 3.81 ± 1.49 MPa ZDSD (n=3) 7.06 ± 2.63 MPa Increased indentation modulus in diabetic tendon Microscale Modulus (MPa) 900 800 700 600 500 400 300 200 100 0 r 2 =0.531 p=0.040 0 2 4 6 8 10 Nanoscale Modulus (MPa) Strong relationship between nanoscale indent modulus and microscale tensile modulus
Indentation: Limitations and Weaknesses Proper probe selection for intended application Cantilever must be stiff relative to sample Sharp probes may damage sample, easily dull R or α must be determined for each probe Properties provided by manufacturer are unreliable Vary from probe to probe, can dull during indent process Spring constant must be determined for each probe Value provided by manufacturer is inaccurate Thermal tuning: OK for soft probes, poor performance for stiffer probes Sader method (geometry), added mass, reference cantilevers
Indentation: Limitations and Weaknesses Veeco Application Technical note: Ben Ohler, Practical Advice on the Determination of Cantilever Spring Constants
Indentation: Limitations and Weaknesses Sample processing Sample hydration Proper force range Substrate effects for thin samples Proper modulus model and range of data, goodness of fit Bearing in mind these assumptions and limitations, AFM can provide true nanoscale mechanical properties both in terms of forces and deformations!
A Note on Peak Force QNM for Bruker AFMs Mechanical properties mapped pixel-by-pixel at same resolution as the height image (PeakForce QNM) Height Modulus
A Note on Peak Force QNM Correct probe stiffness must be used based on sample Pittenger Veeco Product Bulletin
A Note on Peak Force QNM Extremely sensitive to calibration (details rarely given) Absolute: radius and spring constant known especially important from probe to probe radius with indent depth must determine R at depth you expect to image Depth may vary as a function of sample (or at different regions within the sample). Relative: reference material of known modulus used to determine a ratio of kk/ RR known modulus may come from tension, compression and not indentation R is related to depth, indentation depth on sample must be same as on reference
A Note on Peak Force QNM Modulus fit on the fly no ability to monitor fit (or range) Local sample topography and sample tilt play a role in measured parameters Assuming all modulus values in an image are correct, how to quantify? 512 x 512 pixels = 262144 measurements n=1? n=262144? n from selected ROIs?
Lamprou et al., PLoS ONE, 2013 Imaging in air on mica substrate effect? Same imaging force or indent depth for all? For each area, were all modulus values pooled?
AFM Applications in Bone Research: Other Cool Mechanical Techniques
Sacrificial Bonds and Molecular Glue Bonds within or between collagen fibrils in bone that rupture to dissipate energy as a toughening mechanism. Thompson et al., Nature, 2001 Hansma et al., JMNI, 2005 Fantner et al., Nature Mat, 2005 Fantner et al., Nano Letters, 2007
Other Mechanical Techniques Jimenez-Palomar, J Mech Beh Bio Mat, 2012 van der Rijt, Macro Bio, 2004 Yang, J Biomed Mater Res, 2007 Yang, Biophysical Journal, 2008
Other Cool Techniques Piezoresponse Force Microscopy (PFM) Electrochemical AFM (EC-AFM) Force Modulation Microscopy (FMM) Lateral Force Microscopy (LFM) Magnetic Force Microscopy (MFM) Conductive AFM (C-AFM) Force Pulling Cantilever Bending Tensile testing of individual fibrils 3 point bending of fibrils
Summary: Things to remember Imaging mode, imaging medium and probe selection Morphology Measurements Sample preparation and hydration state Measuring fibril diameter Site selection and proper sampling Appropriate system calibration Mechanical Measurements Probe selection (stiffness and tip radius) Probe calibration (radius, angle, spring constant) Indentation force range and mechanical model Hydration in biological samples PeakForce QNM
Acknowledgements BBML Max Hammond Armando Diaz Gonzalez Zachary Bart Silvia Canelon Creasy Clauser IU School of Medicine Dr. David Burr Dr. Matt Allen Dr. Stuart Warden Dr. Lilian Plotkin NIH, NICHD Dr. Joan Marini Funding Sources NIH/NIDCR 1F32DE018840-01 A1 IUPUI BME Departmental Startup Funds IUPUI Research Support Funds Grant IUPUI Biomechanics and Biomaterials Research Center
Bone Biology and Mechanics Lab (BBML) www.iupui.edu/~bbml Facebook: BoneBiologyMechanicsLab