AFM for Cell and Tissue Research

AFM is an essential tool for cell biology research. It can provide 3D topographical data of living, unfixed cells. However, AFM’s greatest strength in cell biology is its ability to provide accurate and quantitative mechanical measurements in near-physiological conditions (i.e. in culture medium and at 37°C). The elastic and viscoelastic response of a cell or substrate can be routinely measured using force maps and AFM-based microrheology techniques, respectively. The measured cell moduli can be that of unaltered cells, cells in different states of development, differentiation, or disease, or cells responding to a stimulus such as a drug or mechanical stress. Measuring the moduli of substrates and the cell microenvironment is also important due to the role the extracellular matrix (ECM) plays in such processes as cell differentiation, fate, signalling, gene transcription, cancer, cardiovascular disease and apoptosis.
 
When integrated with an inverted optical microscope (i.e. fluorescence, confocal, TIRF, etc.), data from both imaging modalities can be combined to correlate fluorescently labeled structures with AFM topography. The optics can be used to direct the AFM tip to probe a particular region of the cell, which can be crucial for hard-to-image cell types. Finally, AFM can also be used to provide a mechanical stimulus to cells and the associated response (e.g. ion handling, membrane potential changes, etc...) can be recorded optically in order to understand mechanotransduction in living cells and tissues.
 

Capabilities

  • Image live cells in culture
  • Measure the elastic or viscoelastic response of cells and substrates
  • Integrate AFM with inverted optical microscopes and fluorescent techniques
  • Use optical images to select a region of interest (ROI) for AFM images and/or force measurements
  • Overlay AFM topography or modulus maps onto optical images and 3D AFM images 
     

Common applications

  • Dynamic imaging of living cells after treatment
  • Stiffness and viscoelastic changes in cancer cells
  • Influence of cell substrates on cell differentiation
  • Response of cells to mechanical stimulation 

AFM and Combined Optical Techniques

This application note briefly describes the basics of both optical and atomic force microscopy, followed by a discussion of some of the technical challenges of integrating these two distinct imaging modalities. In certain cases, the benefits and disadvantages of different approaches to design and integration are discussed. Lastly, a few examples of successful application of these combined imaging modalities are presented.

PDF 1.61MB
Simultaneous Atomic Force and Fluorescence Microscopy Using the MFP-3D™ AFM
Using these microscopy techniques simultaneously provides the user with high resolution imaging on specific molecules and also opens the door to a wide variety of research experiments. In this application note, we will describe the instrumentation and setup, sample preparation, applications, and give experimental examples of the technique. 
PDF 1.15MB
Simultaneous AFM and Optical Phase Contrast Using the MFP-3D™ AFM

Optical phase contrast microscopy is a popular technique for imaging low contrast, transparent samples such as cells in fluid. This technique is now standard on the MFP-3D-BIO™ Atomic Force Microscope, enabling simultaneous AFM and phase contrast imaging.

PDF 588KB
Force Scanning with the MFP-3D™ AFMs: Two Capabilities In One
Force scanning is a simple to execute, broadly applicable approach to analyze compliant materials. The technique can be used for any deformable material, from agarose gels to cartilage to living cells. 
PDF 1.79MB

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Related Webinars


	"Getting Started with AFM in Biology – It's Easier Than You Think"

"Getting Started with AFM in Biology – It's Easier Than You Think"

Review of AFM measurements on single molecules and cells

Selected Publications

I. Acerbi, L. Cassereau, I. Dean, Q. Shi, A. Au, C. Park, Y. Y. Chen, J. Liphardt, E. S. Hwang, and V. M. Weaver, "Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration," Integr. Biol. 7, 1120-1134 (2015). doi:10.1039/c5ib00040h

D. B. Agus, J. F. Alexander, W. Arap, S. Ashili, J. E. Aslan, R. H. Austin, V. Backman, K. J. Bethel, R. Bonneau et al., "A physical sciences network characterization of non-tumorigenic and metastatic cells," Sci. Rep. 3, 1449 (2013). doi:10.1038/srep01449

Z. Bálint, I. A. Krizbai, I. Wilhelm, A. E. Farkas, Á. Párducz, Z. Szegletes, and G. Váró, "Changes induced by hyperosmotic mannitol in cerebral endothelial cells: an atomic force microscopic study," Eur. Biophys. J. 36, 113-120 (2006). doi:10.1007/s00249-006-0112-4

S. Bagchi, H. Tomenius, L. M. Belova, and N. Ausmees, "Intermediate filament-like proteins in bacteria and a cytoskeletal function in Streptomyces," Mol. Microbiol. 70, 1037-1050 (2008). doi:10.1111/j.1365-2958.2008.06473.x

K. B. Bernick, T. P. Prevost, S. Suresh, and S. Socrate, "Biomechanics of single cortical neurons," Acta Biomater. 7, 1210-1219 (2011). doi:10.1016/j.actbio.2010.10.018

Z. Deng, T. Zink, H. yuan Chen, D. Walters, F. tong Liu, and G. yu Liu, "Impact of Actin Rearrangement and Degranulation on the Membrane Structure of Primary Mast Cells: A Combined Atomic Force and Laser Scanning Confocal Microscopy Investigation," Biophys. J. 96, 1629-1639 (2009). doi:10.1016/j.bpj.2008.11.015

P. C. D. P. Dingal, A. M. Bradshaw, S. Cho, M. Raab, A. Buxboim, J. Swift, and D. E. Discher, "Fractal heterogeneity in minimal matrix models of scars modulates stiff-niche stem-cell responses via nuclear exit of a mechanorepressor," Nat. Mater. 14, 951-960 (2015). doi:10.1038/nmat4350

A. J. Engler, "Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments," J. Cell Biol. 166, 877-887 (2004). doi:10.1083/jcb.200405004

A. J. Engler, S. Sen, H. L. Sweeney, and D. E. Discher, "Matrix Elasticity Directs Stem Cell Lineage Specification," Cell 126, 677-689 (2006). doi:10.1016/j.cell.2006.06.044

N. A. Geisse, S. P. Sheehy, and K. K. Parker, "Control of myocyte remodeling in vitro with engineered substrates," In Vitro Cellular & Developmental Biology - Animal 45, 343-350 (2009). doi:10.1007/s11626-009-9182-9

J. Gingras, R. M. Rioux, D. Cuvelier, N. A. Geisse, J. W. Lichtman, G. M. Whitesides, L. Mahadevan, and J. R. Sanes, "Controlling the Orientation and Synaptic Differentiation of Myotubes with Micropatterned Substrates," Biophys. J. 97, 2771-2779 (2009). doi:10.1016/j.bpj.2009.08.038

F. M. Hecht, J. Rheinlaender, N. Schierbaum, W. H. Goldmann, B. Fabry, and T. E. Schäffer, "Imaging viscoelastic properties of live cells by AFM: power-law rheology on the nanoscale," Soft Matter 11, 4584-4591 (2015). doi:10.1039/c4sm02718c

Z. Hong, K. J. Reeves, Z. Sun, Z. Li, N. J. Brown, and G. A. Meininger, "Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists," PLOS ONE 10, e0119533 (2015). doi:10.1371/journal.pone.0119533

Z. Hong, K. J. Reeves, Z. Sun, Z. Li, N. J. Brown, and G. A. Meininger, "Vascular Smooth Muscle Cell Stiffness and Adhesion to Collagen I Modified by Vasoactive Agonists," PLOS ONE 10, e0119533 (2015). doi:10.1371/journal.pone.0119533

I. L. Ivanovska, J.-W. Shin, J. Swift, and D. E. Discher, "Stem cell mechanobiology: diverse lessons from bone marrow," Trends Cell Biol. 25, 523-532 (2015). doi:10.1016/j.tcb.2015.04.003

J. Jaczewska, M. H. Abdulreda, C. Y. Yau, M. M. Schmitt, I. Schubert, P.-O. Berggren, C. Weber, R. R. Koenen, V. T. Moy, and E. P. Wojcikiewicz, "TNF-α and IFN-γ promote lymphocyte adhesion to endothelial junctional regions facilitating transendothelial migration," J. Leukocyte Biol. 95, 265-274 (2013). doi:10.1189/jlb.0412205

H. Jin, D. A. Heller, M. Kalbacova, J.-H. Kim, J. Zhang, A. A. Boghossian, N. Maheshri, and M. S. Strano, "Detection of single-molecule H2O2 signalling from epidermal growth factor receptor using fluorescent single-walled carbon nanotubes," Nat. Nanotechnol. 5, 302-309 (2010). doi:10.1038/nnano.2010.24

G. Kaushik, A. C. Zambon, A. Fuhrmann, S. I. Bernstein, R. Bodmer, A. J. Engler, and A. Cammarato, "Measuring passive myocardial stiffness in Drosophila melanogaster to investigate diastolic dysfunction," J. Cell. Mol. Med. 16, 1656-1662 (2012). doi:10.1111/j.1582-4934.2011.01517.x

M. S. Kellermayer, Á. Karsai, A. Kengyel, A. Nagy, P. Bianco, T. Huber, Á. Kulcsár, C. Niedetzky, R. Proksch, and L. Grama, "Spatially and Temporally Synchronized Atomic Force and Total Internal Reflection Fluorescence Microscopy for Imaging and Manipulating Cells and Biomolecules," Biophys. J. 91, 2665-2677 (2006). doi:10.1529/biophysj.106.085456

J. Liu, N. Sun, M. A. Bruce, J. C. Wu, and M. J. Butte, "Atomic Force Mechanobiology of Pluripotent Stem Cell-Derived Cardiomyocytes," PLoS ONE 7, e37559 (2012). doi:10.1371/journal.pone.0037559

J. I. Lopez, I. Kang, W.-K. You, D. M. McDonald, and V. M. Weaver, "In situ force mapping of mammary gland transformation," Integr. Biol. 3, 910 (2011). doi:10.1039/c1ib00043h

J. L. Maciaszek, and G. Lykotrafitis, "Sickle cell trait human erythrocytes are significantly stiffer than normal," J. Biomech. 44, 657-661 (2011). doi:10.1016/j.jbiomech.2010.11.008

J. M. Maloney, D. Nikova, F. Lautenschläger, E. Clarke, R. Langer, J. Guck, and K. J. V. Vliet, "Mesenchymal Stem Cell Mechanics from the Attached to the Suspended State," Biophys. J. 99, 2479-2487 (2010). doi:10.1016/j.bpj.2010.08.052

J. K. Mouw, Y. Yui, L. Damiano, R. O. Bainer, J. N. Lakins, I. Acerbi, G. Ou, A. C. Wijekoon, K. R. Levental, P. M. Gilbert, E. S. Hwang, Y.-Y. Chen, and V. M. Weaver, "Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression," Nat. Med. 20, 360-367 (2014). doi:10.1038/nm.3497

M. F. Murphy, M. J. Lalor, F. C. Manning, F. Lilley, S. R. Crosby, C. Randall, and D. R. Burton, "Comparative study of the conditions required to image live human epithelial and fibroblast cells using atomic force microscopy," Microsc. Res. Tech. 69, 757-765 (2006). doi:10.1002/jemt.20339

M. Prabhune, G. Belge, A. Dotzauer, J. Bullerdiek, and M. Radmacher, "Comparison of mechanical properties of normal and malignant thyroid cells," Micron 43, 1267-1272 (2012). doi:10.1016/j.micron.2012.03.023

M. Prass, "Direct measurement of the lamellipodial protrusive force in a migrating cell," J. Cell Biol. 174, 767-772 (2006). doi:10.1083/jcb.200601159

A. Raman, S. Trigueros, A. Cartagena, A. P. Z. Stevenson, M. Susilo, E. Nauman, and S. A. Contera, "Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy," Nat. Nanotechnol. 6, 809-814 (2011). doi:10.1038/nnano.2011.186

F. Rehfeldt, A. E. X. Brown, M. Raab, S. Cai, A. L. Zajac, A. Zemel, and D. E. Discher, "Hyaluronic acid matrices show matrix stiffness in 2D and 3D dictates cytoskeletal order and myosin-II phosphorylation within stem cells," Integr. Biol. 4, 422 (2012). doi:10.1039/c2ib00150k

J. Rother, H. Noding, I. Mey, and A. Janshoff, "Atomic force microscopy-based microrheology reveals significant differences in the viscoelastic response between malign and benign cell lines," Open Biology 4, 140046-140046 (2014). doi:10.1098/rsob.140046

S. Sen, S. Subramanian, and D. E. Discher, "Indentation and Adhesive Probing of a Cell Membrane with AFM: Theoretical Model and Experiments," Biophys. J. 89, 3203-3213 (2005). doi:10.1529/biophysj.105.063826

E. Spedden, and C. Staii, "Neuron Biomechanics Probed by Atomic Force Microscopy," Int. J. Mol. Sci. 14, 16124-16140 (2013). doi:10.3390/ijms140816124

E. Spedden, J. D. White, E. N. Naumova, D. L. Kaplan, and C. Staii, "Elasticity Maps of Living Neurons Measured by Combined Fluorescence and Atomic Force Microscopy," Biophys. J. 103, 868-877 (2012). doi:10.1016/j.bpj.2012.08.005

J. R. Tse, and A. J. Engler, "Stiffness Gradients Mimicking In Vivo Tissue Variation Regulate Mesenchymal Stem Cell Fate," PLoS ONE 6, e15978 (2011). doi:10.1371/journal.pone.0015978

K. R. Wilhelm, E. Roan, M. C. Ghosh, K. Parthasarathi, and C. M. Waters, "Hyperoxia increases the elastic modulus of alveolar epithelial cells through Rho kinase," FEBS Journal 281, 957-969 (2013). doi:10.1111/febs.12661

Y. Xiong, A. C. Lee, D. M. Suter, and G. U. Lee, "Topography and Nanomechanics of Live Neuronal Growth Cones Analyzed by Atomic Force Microscopy," Biophys. J. 96, 5060-5072 (2009). doi:10.1016/j.bpj.2009.03.032

W. Xu, R. Mezencev, B. Kim, L. Wang, J. McDonald, and T. Sulchek, "Cell Stiffness Is a Biomarker of the Metastatic Potential of Ovarian Cancer Cells," PLoS ONE 7, e46609 (2012). doi:10.1371/journal.pone.0046609

E. K. Yim, E. M. Darling, K. Kulangara, F. Guilak, and K. W. Leong, "Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells," Biomaterials 31, 1299-1306 (2010). doi:10.1016/j.biomaterials.2009.10.037

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