Investigating Cell Mechanics with PeakForce QNMInvestigating Cell Mechanics with PeakForce QNM®...

Investigating Cell Mechanics with PeakForce QNM®

Andrea Slade, Ph. D.Sr. Applications Scientist, Life SciencesBruker Nano Surfaces

Bruker Nano Surfaces Division3/12/2014 1

AFM For Live Cell Studies

Advantages To AFM:– Single molecule/cell technique

– Non-destructive

– No staining/coating

– Operable in a fluid environment• Water, Buffer, Cell Media, etc.• Ionic strength / Salinity• pH

– Environmental Control• Temperature • Fluid perfusion / exchange• Gas (CO2)

Provides physiologically relevant environment.

Allows in situ, real-time observation of cell structure and behavior.Bruker Nano Surfaces Division3/12/2014 2

AFM Imaging of Live Cells

45 m Human endothelial cells Sample courtesy Hans Oberleithner, Muenster Univ.

• AFM can provide high-resolution, 3D images of:

• Individual Cell Morphology

• Cell-Substrate Interactions

• Cell-Cell Interactions.


Zajac & Discher (2008) Curr Opin Cell Biol 20: 609-6150

Glycosan Biosystems & SigmaAldrich

AFM in Cell MechanicsMechanobiology

AFM Force Spectroscopy

�z

ApproachRetract

• Elasticity varies between different tissues.

• Cells sense and respond to forces.• AFM force curves can measure:

• Elasticity• Adhesion• Molecular (un)folding

• Binding interactions • Response to

mechanical stimulus


∆z

Effect of Substrate Stiffness on AFM Force Curves

00 20 40 60 81

20 nm

Healthy Cells

Infected Cells

Treated Cells

Scanner extension (m) 01D

efle

ctio

n (n

m)

Force curves on live cells (only contact region shown).

Cytoskeleton disruption changes cell stiffness:

Healthy > Infected > Treated

Terebiznik et al. (2002) Nat. Cell Biol. 4, 766.Schematic: www.freesbi.ch

Cell Membrane Elasticity

Modulus/stiffness derived from the contact region of the force curve.

Slope of curve reflects stiffness.• Stiffer surface = higher slope• Softer surface = lower slope


Nanomechanical Properties of TissuesMammary Gland

• Role of forces in disease states (eg. breast cancer).

• Mammary gland becomes increasingly stiffer with tumor progression.

• Force curves show lymph node stiffness mammary tissue stiffness.

lymph node

mammary tissue


Force Volume Imaging2D Mapping of Cell Elasticity


Exposure Time

• 2-Dimensional array of force curves conducted over a defined scan area.

• Disassembly of actin filaments after treatment of living 3T3 fibroblast cells with the drug Cytochalasin B.

Radmacher et al. (2000) Biophys. J., vol 78: 520-35.

PeakForce Tapping TechnologyControls and measures force as feedback

Z motion

Deflection

Z position

Resembles a typical force

curve…

PeakForce Tapping Mode:

• Probe modulated at small amplitudes at low frequency (1-2kHz).

• Feedback signal is peak force between tip and sample.

• Direct control of imaging forces with ultra-low setpoints (<100pN).

• Images acquired at typical scan rates (1000’s force curves/sec).Bruker Nano Surfaces Division3/12/2014 8

Quantitative Nanomechanical Property Mapping

Forc

e

Separation

Sneddon ModelDMT Model

Adhesion Imaging

Dissipation

Modulus

Deformation

• Quantitative nanomechanicaldata obtained simultaneously in real-time with topography.

• Material properties measured over a wide range (kPa-GPa).

• DMT and Sneddon modulus models.

• Individual force curves analyzed offline with PeakForce Capture.

Modified from Cox and Erler. Dis. Model. Mech. (2011) 4:165-178.


Sneddon Modulus data painted on 3D topography Sneddon modulus

PeakForce QNM Mapping the Modulus of Live E. coli Cells

• Dividing cell (on the right) is significantly softer: ~2MPa vs ~15MPa.

• The substrate is stiffer than both cells (~50MPa). Note some softer components, including the bacterial flagella in the lower-right corner.

PeakForce QNM image obtained using a standard DNP-A probe (k~0.65N/m) in fluid at 250 Hz. The Sneddon model was used to calculate modulus (Scan size 5µm)

Force Curves obtained from PeakForce Capture data


DMT Modulus = 50kPa

Sneddon Modulus = 37kPa

PeakForce QNM on Live Mammalian Cells Sneddon Model provides more accurate modulus for soft samples.

3D image of the lamellipodium of a live B16 mouse cell. Cells were imaged in PeakForce

Tapping QNM mode at 250Hz using a MLCT-D probe (k=0.048N/m)


PeakForce QNM on Live Mammalian Cells Combining with force volume for viscoelasticity studies

The Sneddon modulus obtained on B16 cell was ~20kPa independent of ramp frequency (PF QNM or FV). Higher resolution PeakForce QNM images (above) showed actin fibers that appear stiffer than rest of membrane surface.


PF QNM250Hz

2m

FV1Hz

2m

FV5Hz

2m

Occ

urra

nce

(%)

Sneddon Modulus (MPa)

00.0 0.1 0.2

10

20

30

E ~20kPa

Mapping Cell Deformability Invasive vs. Non-invasive Glioblastoma Cells

(c)

20m 20m 20m

• Link between cancer and changes in cell mechanical properties.

• Both PeakForce QNM and force volume found non-invasive cells significantly stiffer and less deformable than cancerous cells.

• PeakForce QNM allows for better statistical measurements


PeakForce Error Modulus Deformation

Adhesion Topography

Healthy Erythrocyte Infected Erythrocyte (IE)

Adapted from the Plasmodium Genome Resource.

Adapted from http://cmr.asm.org/cgi/content-nw/full/13/3/439/F1.

• Malaria-infected erythrocytes (IE’s) are misshapen with knob-like structures on surface.

• IE’s exhibit cytoadherence.

• Prevents IE elimination by the spleen and causes vascular blockages.

PeakForce QNM Adhesion Measurements Molecular Recognition Mapping


• AFM probes were functionalized with endothelial surface receptor CD36.

• Adhesion mapping found CD36 binding sites (high adhesion) to correlate to knob structures (circles).

Topography Adhesion

Overlay

(CD36)

Adapted from Li et al. (2011) PLoS ONE 6: 1-10.

PeakForce QNM Adhesion Measurements High-resolution imaging of membrane receptor distribution


PeakForce QNM of Adhesion EventsMapping Membrane Receptors with ~4nm resolution

Alsteens et al. (2012) Langmuir, vol 28: 16738-44.

Bud Scars

BioScope Catalyst operated in fluid using modified OTR4 probes.

http://bioweb.uwlax.edu/bio203/s2012/vandenla_beth/reproduction.htm.

PF Topography

PF Modulus

FV Modulus

PF Adhesion

PF Adhesion

PF Adhesion

• PF-QNM enabled researchers to map the localization and mechanics of individual proteins of the surface of living yeast cells.• Position of individual proteins mapped at

unprecedented spatial resolution of ~4nm.

• Quantitative measurement of single NTA-Ni2+–His) bond at ~306 ± 72pN.

• Significantly higher spatial and temporal resolution over Force Volume imaging.


20 m

BioScopeTM Catalyst: MIRO SoftwareMicroscopy Image Registration & Overlay

Combined AFM / CLSMFibroblast Cells labeled with Alexa Fluo 546 Phalloidin (red) & DAPI (blue). Confocal fluorescence images obtained using a 40x oil immersion objective.

AFM images obtained in contact mode in buffer solution at 37°C.


Integrated PF-QNM & Light MicroscopyCell Wall Mechanics in Plant Morphogenesis

BioScope Catalyst integrated with confocal microscope operated in fluid using ScanAsyst-Fluid probes.

Milani et al. (2011) Plant Journal, vol 67: 1116-23.

Meristem Cells

http://www.nature.com/scitable/content/stem-cells-located-in-the-shoot-apical-14263982

• PF-QNM integrated with confocal fluorescence microscopy allowed researchers to clearly identify individual cells within the plant meristem and directly correlate their structure to localized modulus measurements.


Differences in the Mechanical Properties of Live & Dead Bacteria:Integrated AFM & Fluorescence Imaging

• The BioScopeTM Catalyst and MIROTM Software allowed correlation of live/dead fluorescence images with PeakForceTM QNM images of individual E. coli cells.

• Definitive identification of live & dead cells.

3 m 3 m

Peak Force Error Image Live/Dead Fluoresecence Image

Images were obtained on a BioScopeTM Catalyst integrated with a Zeiss Axiovert 200 IOM and operated in PeakForceTM Tapping Mode in fluid. E. coli cells were labeled with live /dead assay (green/red, respectively) and

immobilized on a gelatin-coated glass bottom Petri dish. Images courtesy J. Shaw, Bruker-Nano Inc. Bruker Nano Surfaces Division3/12/2014 19

• Live E. coli cells were fairly uniform in deformation (elasticity) while dead cells were more hetergeneous with areas of increased deformation (softer – less elastic).

• Live and dead cells showed no differences in adhesion.

Images were obtained on a BioScopeTM Catalyst integrated with a Zeiss Axiovert 200 IOM and operated in PeakForceTM Tapping Mode in fluid. Images courtesy J. Shaw, Bruker-Nano Inc.

Differences in the Mechanical Properties of Live & Dead Bacteria:PeakForceTM QNM Mechanical Property Mapping

3 m 3 m


• Eliminates need to acquire an overview image of sample:

• time to data.

• Non-disruptive to sensitive samples (live cells).

• Preserves functionalized probes.

10 m

Optically Guided Force Spectroscopy

Courtesy C. Callies, H. Oberleithner. Institute for Physiology II, University of Muenster, Germany.


• Fluorescence images can be used to navigate the AFM probe to specific points on a surface to conduct localized nanomechanical property measurements (eg. Membrane Elasticity).

Substrate > Live Cells > Dead Cells

Images were obtained on a BioScopeTM Catalyst integrated with a Zeiss Axiovert 200 IOM and operated in Force Spectroscopy Mode in fluid. Images courtesy J. Shaw, Bruker-Nano Inc.

Differences in the Mechanical Properties of Live & Dead Bacteria:Optically-Guided Point & Shoot Force Spectroscopy

Separation (nm)

Forc

e (n

N)

Black: Live Blue: DeadPink: Substrate

1nN

0 50 1003 m

Live/Dead Fluorescence Image

+

+

+++

+++

+++



Mechanobiology and Neuronal SignalingAFM measurements of Mechanotransduction

• A colloidal probe used to stimulate the neurites of living dorsal root ganglion cells.

• Change in fluorescence intensity away from probe indicates the cell is mechanically activated and signal is propagated along axon to cell body.

DIC image showing the neurite ending connected to the cell of interest.

The AFM probe is modified with a 10µm bead. Cells are labeled with the calcium indicator dye Fluo8/acetoxymethyl ester (AM).

• Cells are instantly activated upon contact.

• Primary and secondary stimulations are visible, far away from the contact point in both clusters and isolated cells.

• Demonstrate potential to detect signal propagation in living neurons to obtain better insight into the mechanotransduction.

clus

ter

Isol

ated

cel

lsMechanobiology and Neuronal SignalingAFM measurements of Mechanotransduction


Mouse embryonic stem cells. Obtained over 12hrs with a 40x phase contrast objective.

© The Exploratorium, www.exploratorium.edu

50 m

AFM images obtained over ~1hr at 40sec/frame at 512 x 128 pixel resolution. Phase images shown. (~10 m scan size)

FastScan Bio AFM of Cell DynamicsMechanics of Cell Migration


Process of cell migration. www.cellmigration.org

Plasmid DNA during heating from room temperature to 37C in HEPES buffer. Images captured at 5 sec/frame at 256x256 pixels. (Video playback at 6fps)

Compensation ‘ON’ Compensation ‘OFF’

0 8min

50nm 50nm

HEAT ON

FastScan Bio NanoTrack™ Imaging DNA during Heating


Summary

• AFM offers various modes for studying the nanomechanicalproperties of living cells.

• PeakForce QNM • Force volume• Force spectroscopy

• Advances in Bruker’s AFM Technology are providing new methods of experimentation in mechanobiology research.

• PeakForce QNM • BioScope Catalyst & MIRO• Dimension FastScan Bio


PF QNM250Hz

2m

contact me at:

Andrea Slade, [email protected] More Info: nanoscaleworld.bruker-axs.com

Thank-you for your attention!

www.bruker.com/webinars_afm

[email protected]


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