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BME 610
Experimental & Quantitative Physiology
Prof. Shamik Sen
Contact Information:
Email: [email protected]
Office: 203, Civil Engg. Bldg.
Atomic Force Microscopy
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Extracellular Matrix (ECM)
Provides structural support to maintain tissue architecture (collagen, elastin)Provides mechanical strength to tissues (elastin)Acts as a reservoir of water molecules (proteoglycans)Supports cell adhesion (fibronectin, collagen, laminin)
Sequesters various chemical signals relevant to cell behavior
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Mechanical Properties of Cells
Stiffness /Youngs Modulus (E)
1 kPa 10 kPa 100 kPafluid
Brain MuscleBlood Collagenous Bone
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Mechanical Properties of Cells
Plasma Membrane
0.01- 0.1 mN/mfor most cellsTo
Membrane Tension (T0)
Contractility/Pre-stress (s)
Adhesion Strength/Adhesiveness
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Cell mechanical properties are altered in development & disease
Cell functioning is intimately tied to its physical properties
Cellular propertiesare dynamic in nature
Cellular propertiesevolve duringdevelopment
Alterations in cellphysical propertiesunderlie various diseases
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Biophysical techniques for probingcells & sub-cellular organelles
Apply Force to biological samples
Measure or record deformation of/in cell(s) cellular or sub-cellular level
Determine topography and/or physical propertyof cell(s) and/or sub-cellular compartments
Mechanical
Magnetic
Electrical
Optical
Force
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Atomic Force Microscope (AFM)
Invented in 1986 by Binnig & Rohrer at IBM, Zurich; commercialized in 1989
Offshoot of Scanning Tunneling Microscope (STM)
Can image non-conducting samples
Can image live samples; no requirement for fixing
Can be used for imaging as well as force measurements
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Comparison of biophysical tools
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A Typical AFM setup
Controller
Visualization Software
Inverted Microscope
AFM Head
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a sharp tip mounted on a flexiblecantilever that acts as a spring
a laser diode, whose light isfocused on the very end of thecantilever beam
a position-sensitive photodiode
that detects the laser beamreflected by the cantilever
a piezo device for positioning thesample relative to the tip
Principal Components of an AFM
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Cantilever & Tip Parameters
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Cantilever & Tip Parameters
Length (100-300 mm)
Width (10-50 mm)
Thickness (0.5 10 mm)
Shape (Rectangular, Straight, etc)
Material (Si, SiN)
Reflective coating (Al, Au)
Spring Constant (0.01-100 N/m)
Resonance Frequency (50-200 kHz)
Tip Radius (10-50 nm)
Tip Height (0-50 mm)
Tip Angle (15-infinity )
Tip Shape (3-sided, 4-sided)
Tip Material (Si, SiN, etc)
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Mode of Operation: Contact (DC) Mode
Tip & sample are in contact
Tip raster scans the surface
Sharp tip is used for contact mode
Deflection set point is specified
Used for very flat & rigid surfaces
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Mode of Operation: Tapping (AC) Mode
Tip & sample are not in perennial contact
Tip is oscillated at resonance frequency
Amplitude set-point is used Used for soft, loosely attached samples
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Mode of Operation: Force Mode
Tip & sample distance is varied
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Imaging Biological Samples: Effect of Gain
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Gain Control
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Low Gain Vs High Gain
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Setting Setpoint
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Setting Setpoint
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Setting Scan Rate
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Setting Scan Rate
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Gain, Setpoint, and Rate all greatly contributeto image quality and data fidelity.
There are no hard-and-fast rules or values for
any of these parameters.
Good image quality usually relies on aninterplay of different values for these parameters
In general, adjustments to values should startwith gain, then setpoint, and finish with rate.
Optimizing Gain, Setpoint & Rate
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Some Images with Optimized Parameters
DNA in Liquid E-Coli
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Osteoblast Cell
Endothelial Cell
Endothelial Cell(Mannitol treated)
h
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Force Measurements with AFM
What Can Force Curves Tell Us?
Adhesion: receptor-ligand bindingElasticity: cell stiffnessTethered eventProtein unfoldingBinding
Hookes Law:F = k * x = forcek= spring constant/stiffnessx= displacement of the spring
BUT First:
Calibrate the tip!!
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C d f i h dh i
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Force Curve: Hard surface with adhesion
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Cantilever Stiffness & Resonance Frequency
1/2
2
2
~ ( / )
0.5
0.5
/
c eff
B
c
c B
k m
E k T
E k d
k k T d
Non-destructiveEasy to do
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F C S ft f ith dh i
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Force Curve: Soft surface with adhesion
Di t d F C Fib bl t
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Directed Force Curves: Fibroblasts
E t ti Stiff V l
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Extracting Stiffness Values
2
2
2
tan 1
cellEF
3/ 2 1/ 2
2
4
3 1
cellEF R
Sneddons Model Hertz Model
Q tit ti El ti it M t
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Patterned cardiac myocyte on PDMS/glassCourtesy: Prof. K. Parker & Dr. N. Geisse,Harvard University
Quantitative Elasticity Measurements
Q tit ti El ti it M t
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Quantitative Elasticity Measurements
Topography/stiffness map of a lamellipodia of a spreading fibroblast(Solon et al., Biophys 07)
D t V A tif t i F M t
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Data Vs Artifact in Force Measurements
Do these varying values reflect heterogeneity of thecell or are these artifacts??
Data Vs Artifact in Force Measurements:
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Data Vs Artifact in Force Measurements:What are you probing?
Membrane Vs Cytoskeleton
A
B
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Choice of tip geometry in probing cells
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Choice of tip geometry in probing cells
Sharp tips may rupture cell membrane
Sharp tips may pass through pores in the cell membrane
Sharp tip provides very localized information of stiffness
Spherical tip more gentle on cells
Spherical tip provides averaged stiffness information
Effect of location of probing more important for sharp tips compared tospherical tips
St d i Adh i ith AFM
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Studying Adhesion with AFM
Receptor-ligand interactionsCell-cell interactions
Q tif i R d Bl d C ll M h
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Phagocyte
Signaling Self
Clustering
flow
RBC
Binding
CD47
SIRPa
Quantifying Red Blood Cell-MacrophageInteractions
Model System: Adhesively spread red cell
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Spread erythrocyte
glass (-)
poly-lysine layer (+++)
Model System: Adhesively spread red cell
negatively charged membrane constant cell volume constant membrane area
2.5 m
Imaged in AFM contact mode after glutaraldehyde fixation.
Strong Adhesion Limit Tensed Spherical Caps
AFM probing of unfixed cell membrane
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AFM probing of unfixed cell membrane
Indentation
z(nm)
Force(pN)
Indentation
Retraction
Adhesive detachment
0 200 400 600 800 1000
0
4000
8000
12000
z
Adhesion ?
Retraction with detachment
Probing CD47 SIRP interactions
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80
60
40
20
0
0 1 2 3 4
frequency
SIRP (human)
1
2
34
Human RBC
Rat RBC
# of peaks
Probing CD47- SIRP interactions
CD47 SIRP interaction is species restricted
Probing human CD47- human SIRP interactions
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0 100 200 300 400 500
0
500
1000
1500
2000
2500
Extension (nm)
Force(pN)
0 100 200 300 400 500
0
10
20
30
Peak to peak length (nm)
frequency
0 50 100 150 2000
10
20
30
Force (pN)
frequency
0 5 00 1 00 0 15 00 20 00 2 50 0 30 00
0
10
20
30
Force (pN)
frequency 0.5 mg/ml 0.05 mg/ml
2nd sawtooth1st sawtooth
SIRP SIRP
g
1500 pN first peak forces; 600 pN second peak forces at highconcentrations
At low concentration, peaks ~ 100 pN
215 nm mean extension
P k #1 Cl t #1
CD47-SIRP adhesion: Rate-dependence, & Valency of clusters
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Peak #2 = Cluster #2
Peak #1 = Cluster #1
~ 7 bonds break in parallel
< 4 bonds in parallel
Single bond strength:
70 pN @ 5 mm/sec
Second cluster shows:
fewer bonds
longer time needed
velocity ( m/s)
Indentation time (msec)
100
1000
0 5 10 15 20 25
e -time/500 msec
firstpkfo
rces(pN)
500 20 500 20
0 5 10 15 20 25
100
e -time/1200 msec
velocity ( m/s)
Indentation time (msec)
second
pkfo
rces(pN)
3 bonds break in parallel
~1 bond
CD47-SIRP Rate-dependence of Clustering -
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Phagocyte
Signaling Self
Clustering
flow
RBC
Binding
CD47
SIRPa
CD47-SIRP Rate-dependence of Clustering -implications for Signaling
tx ~ crossover from one cluster to two clusters
%
peaks
1 10
10
20
30
40
50
60
70
80
90
500 20Indentation time (msec)
Studying Force Spectroscopy with AFM
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Studying Force Spectroscopy with AFM
Protein Unfolding of Titin
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Protein Unfolding of Titin
Combined AFM & Microscopy
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Combined AFM & Microscopy
AFM: Provides high resolution images, and can characterize
biological forces and mechanics
Optical Microscopy: provides chemical/functional sensitivity; canlook below the surface of a specimen
Both: Can correlate fluorescently labeled structures withtopography
Mechanical stimulation with AFM
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Mechanical stimulation with AFM
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