Investigating Cell Mechanics with PeakForce QNM. Andrea Slade, Ph. D. Sr. Applications Scientist, Life Sciences Bruker Nano Surfaces

Transcription

1 Investigating Cell Mechanics with PeakForce QNM Andrea Slade, Ph. D. Sr. Applications Scientist, Life Sciences Bruker Nano Surfaces 1

2 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 (CO 2 ) Provides physiologically relevant environment. Allows in situ, real-time observation of cell structure and behavior. 2

3 AFM Imaging of Live Cells AFM can provide highresolution, 3D images of: Individual Cell Morphology Cell-Substrate Interactions Cell-Cell Interactions. 45 m Human endothelial cells Sample courtesy Hans Oberleithner, Muenster Univ. 3

4 AFM in Cell Mechanics Mechanobiology AFM Force Spectroscopy Approach Retract Zajac & Discher (2008) Curr Opin Cell Biol 20: z z Glycosan Biosystems & SigmaAldrich 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 4

5 Cell Membrane Elasticity Effect of Substrate Stiffness on AFM Force Curves Healthy Cells Deflection (nm) Infected Cells 20 nm Treated Cells Schematic: Modulus/stiffness derived from the contact region of the force curve. Slope of curve reflects stiffness. Stiffer surface = higher slope Softer surface = lower slope 1 Terebiznik et al. (2002) Nat. Cell Biol. 4, Scanner 06 extension 04 ( m) 02 0 Force curves on live cells (only contact region shown). Cytoskeleton disruption changes cell stiffness: Healthy > Infected > Treated 5

6 Nanomechanical Properties of Tissues Mammary Gland lymph node mammary tissue 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. 6

7 Force Volume Imaging 2D Mapping of Cell Elasticity 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. Exposure Time Radmacher et al. (2000) Biophys. J., vol 78:

8 PeakForce Tapping Technology Controls and measures force as feedback Z motion Deflection Resembles a typical force curve Z position 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). 8

9 Quantitative Nanomechanical Property Mapping Modulus Sneddon Model DMT Model Force Deformation Quantitative nanomechanical data 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. Dissipation Adhesion Imaging Separation Modified from Cox and Erler. Dis. Model. Mech. (2011) 4:

10 PeakForce QNM Mapping the Modulus of Live E. coli Cells Sneddon Modulus data painted on 3D topography Sneddon modulus Force Curves obtained from PeakForce Capture data 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) 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. 10

11 PeakForce QNM on Live Mammalian Cells Sneddon Model provides more accurate modulus for soft samples. DMT Modulus = 50kPa Sneddon Modulus = 37kPa 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) 3/12/2014 Bruker Nano Surfaces Division 11

12 PeakForce QNM on Live Mammalian Cells Combining with force volume for viscoelasticity studies PF QNM 250Hz FV 1Hz FV 5Hz 2 m 2 m 30 2 m 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. Occurrance (%) E ~20kPa Sneddon Modulus (MPa) 12

13 Mapping Cell Deformability Invasive vs. Non-invasive Glioblastoma Cells PeakForce Error Modulus Deformation (c) 20 m 20 m 20 m 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 13

14 PeakForce QNM Adhesion Measurements Molecular Recognition Mapping Adapted from Adhesion Topography Healthy Erythrocyte Infected Erythrocyte (IE) 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. Adapted from the Plasmodium Genome Resource. 14

15 PeakForce QNM Adhesion Measurements High-resolution imaging of membrane receptor distribution (CD36) Adapted from Li et al. (2011) PLoS ONE 6: Topography Adhesion AFM probes were functionalized with endothelial surface receptor CD36. Adhesion mapping found CD36 binding sites (high adhesion) to correlate to knob structures (circles). Overlay 15

16 PeakForce QNM of Adhesion Events Mapping Membrane Receptors with ~4nm resolution PF Topography PF Adhesion Bud Scars vandenla_beth/reproduction.htm. PF Modulus 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-Ni 2+ His) bond at ~306 ± 72pN. FV Modulus PF Adhesion Significantly higher spatial and temporal resolution over Force Volume imaging. Alsteens et al. (2012) Langmuir, vol 28: BioScope Catalyst operated in fluid using modified OTR4 probes. 16

17 BioScope TM Catalyst: MIRO Software Microscopy Image Registration & Overlay 20 m Combined AFM / CLSM Fibroblast 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. 17

18 Integrated PF-QNM & Light Microscopy Cell Wall Mechanics in Plant Morphogenesis Meristem Cells BioScope Catalyst integrated with confocal microscope operated in fluid using ScanAsyst-Fluid probes. 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. Milani et al. (2011) Plant Journal, vol 67:

19 Differences in the Mechanical Properties of Live & Dead Bacteria: Integrated AFM & Fluorescence Imaging The BioScope TM Catalyst and MIRO TM Software allowed correlation of live/dead fluorescence images with PeakForce TM QNM images of individual E. coli cells. Definitive identification of live & dead cells. Peak Force Error Image Live/Dead Fluoresecence Image 3 m 3 m Images were obtained on a BioScope TM Catalyst integrated with a Zeiss Axiovert 200 IOM and operated in PeakForce TM 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. 19

20 Differences in the Mechanical Properties of Live & Dead Bacteria: PeakForce TM QNM Mechanical Property Mapping 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. 3 m 3 m Images were obtained on a BioScope TM Catalyst integrated with a Zeiss Axiovert 200 IOM and operated in PeakForce TM Tapping Mode in fluid. Images courtesy J. Shaw, Bruker-Nano Inc. 20

21 Optically Guided Force Spectroscopy Eliminates need to acquire an overview image of sample: time to data. 10 m Courtesy C. Callies, H. Oberleithner. Institute for Physiology II, University of Muenster, Germany. Non-disruptive to sensitive samples (live cells). Preserves functionalized probes. 21

22 Differences in the Mechanical Properties of Live & Dead Bacteria: Optically-Guided Point & Shoot Force Spectroscopy 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). Live/Dead Fluorescence Image Substrate > Live Cells > Dead Cells m Force (nn) 1nN Black: Live Blue: Dead Pink: Substrate Separation (nm) Images were obtained on a BioScope TM Catalyst integrated with a Zeiss Axiovert 200 IOM and operated in Force Spectroscopy Mode in fluid. Images courtesy J. Shaw, Bruker-Nano Inc. 22

23 Mechanobiology and Neuronal Signaling AFM measurements of Mechanotransduction DIC image showing the neurite ending connected to the cell of interest. 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. The AFM probe is modified with a 10µm bead. Cells are labeled with the calcium indicator dye Fluo8/acetoxymethyl ester (AM). 23

24 Mechanobiology and Neuronal Signaling AFM measurements of Mechanotransduction cluster Cells are instantly activated upon contact. Primary and secondary stimulations are visible, far away from the contact point in both clusters and isolated cells. Isolated cells Demonstrate potential to detect signal propagation in living neurons to obtain better insight into the mechanotransduction. 3/12/2014 Bruker Nano Surfaces Division 24

25 FastScan Bio AFM of Cell Dynamics Mechanics of Cell Migration Process of cell migration m Mouse embryonic stem cells. Obtained over 12hrs with a 40x phase contrast objective. The Exploratorium, AFM images obtained over ~1hr at 40sec/frame at 512 x 128 pixel resolution. Phase images shown. (~10 m scan size) 25

26 FastScan Bio NanoTrack Imaging DNA during Heating HEAT ON 0 8 min Compensation ON Compensation OFF 50nm 50nm Plasmid DNA during heating from room temperature to 37 C in HEPES buffer. Images captured at 5 sec/frame at 256x256 pixels. (Video playback at 6fps) 26

27 Summary AFM offers various modes for studying the nanomechanical properties 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 2 m PF QNM 250Hz 27

28 contact me at: Andrea Slade, PhD More Info: nanoscaleworld.bruker-axs.com Thank-you for your attention! 28