Traction force microscopy

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Traction force microscopy (TFM) is an experimental method for determining the tractions on the surface of a biological cell by obtaining measurements of the surrounding displacement field within an in vitro extracellular matrix (ECM).

Contents

Overview

The dynamic mechanical behavior of cell-ECM and cell-cell interactions is known to influence a vast range of cellular functions, including necrosis, differentiation, adhesion, migration, locomotion, and growth. TFM utilizes experimentally observed ECM displacements to calculate the traction, or stress vector, at the surface of a cell. Before TFM, efforts observed cellular tractions on silicone rubber substrata wrinkling around cells; [1] however, accurate quantification of the tractions in such a technique is difficult due to the nonlinear and unpredictable behavior of the wrinkling. Several years later, the terminology TFM was introduced to describe a more advanced computational procedure that was created to convert measurements of substrate deformation into estimated traction stresses. [2]

General Methodology

In conventional TFM, cellular cultures are seeded on, or within, an optically transparent 3D ECM embedded with fluorescent microspheres (typically latex beads with diameters ranging from 0.2-1  μm). [3] [4] [5] [6] [7] A wide range of natural and synthetic hydrogels can be used for this purpose, with the prerequisite that mechanical behavior of the material is well characterized, and the hydrogel is capable of maintaining cellular viability. The cells will exert their own forces into this substrate which will consequently displace the beads in the surrounding ECM. In some studies, a detergent, enzyme, or drug is used to disturb the cytoskeleton, thereby altering, or sometimes completely eliminating, the tractions generated by the cell.

First, a continuous displacement field is computed from a pair of images: the first image being the reference configuration of microspheres surrounding an isolated cell, and the second image being the same isolated cell surrounded by microspheres that are now displaced due to the cellular-generated tractions. Confocal fluorescence microscopy is usually employed to image the cell surface and fluorescent beads. After computing the translational displacement field between a deformed and undeformed configuration, a strain field can be computed. From the strain field, the stress field surrounding the cell can be calculated with knowledge of the stress-strain behavior, or constitutive model, of the surrounding hydrogel material. It is possible to proceed one step further, and use the stress field to compute the tractions at the surface of the cell, if the normal vectors to the cell surface can be obtained from a 3D image stack. Although this procedure is a common way to obtain the cellular tractions from microsphere displacement, some studies have successfully utilized an inverse computational algorithm to yield the traction field. [8] [9] [10]

Limitations

The spatial resolution of the traction field that can be recovered with TFM is limited by the number of displacement measurements per area. [11] The spacing of independent displacement measurements varies with experimental setups, but is usually on the order of one micrometer. The traction patterns produced by cells frequently contain local maxima and minima that are smaller. Detection of these fine variations in local cellular traction with TFM remains challenging.

Advancements

In 2D TFM, cells are cultured as a monolayer on the surface of a thin substrate with a tunable stiffness, and the microspheres near the surface of the substrate undergo deformation through cell-ECM connections. 2.5D cell cultures are similarly grown on top of a thin layer of ECM, and diluted structural ECM proteins are mixed to the medium added above the cells and substrate. Although most of the seminal work in TFM was performed in 2D, or 2.5D, many cell types require the complex biophysical and biochemical cues from a 3D ECM to behave in a truly physiologically realistic manner within an in vitro environment. [12]

When the rotation or stretch of a sub volume is large, errors can be introduced into the calculation of cell surface tractions since most TFM techniques employ a computational framework based on linear elasticity. Recent advances in TFM have shown that cells are capable of exerting deformations with strain magnitudes up to 40%, which requires usage of a finite deformation theory approach to account for large strain magnitudes. [13]

Applications

Although TFM is frequently used to observe tractions at the surface of a spatially isolated individual cell, a variation of TFM can also be used to analyze the collective behavior of multicellular systems. For example, cellular migration velocities and plithotaxis are observed alongside a computed stress variation map of a monolayer sheet of cells, in an approach termed monolayer stress microscopy. [14] The mechanical behavior of single cells versus a confluent layer of cells differ in that the monolayer experiences a "tug-of-war" state. There is also evidence of a redistribution of tractions that can take place earlier than changes in cell polarity and migration. [15]

TFM has proven particularly useful to study durotaxis as well.

TFM has recently been applied to explore the mechanics of cancer cell invasion with the hypothesis that cells which generate large tractions are more invasive than cells with lower tractions. [16] It is also hoped that recent findings from TFM will contribute to the design of optimal scaffolds for tissue engineering and regeneration of the peripheral nervous system, [17] artery grafts, [18] and epithelial skin cells. [19]

Related Research Articles

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Extracellular matrix Network of proteins and molecules outside cells that provides structural support for cells

In biology, the extracellular matrix (ECM) is a three-dimensional network consisting of extracellular macromolecules and minerals, such as collagen, enzymes, glycoproteins and hydroxyapatite that provide structural and biochemical support to surrounding cells. Because multicellularity evolved independently in different multicellular lineages, the composition of ECM varies between multicellular structures; however, cell adhesion, cell-to-cell communication and differentiation are common functions of the ECM.

Hydrogel

A hydrogel is a crosslinked hydrophilic polymer that does not dissolve in water. They are highly absorbent yet maintain well defined structures. These properties underpin several applications, especially in the biomedical area. Many hydrogels are synthetic, but some are derived from nature. The term 'hydrogel' was coined in 1894.

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Cell culture Process by which cells are grown under controlled conditions

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue, they can subsequently be maintained under carefully controlled conditions. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (CO2, O2), and regulates the physio-chemical environment (pH buffer, osmotic pressure, temperature). Most cells require a surface or an artificial substrate (adherent or monolayer culture) whereas others can be grown free floating in culture medium (suspension culture). The lifespan of most cells is genetically determined, but some cell culturing cells have been “transformed” into immortal cells which will reproduce indefinitely if the optimal conditions are provided.

Total internal reflection fluorescence microscope

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed.

Focal adhesion

In cell biology, focal adhesions are large macromolecular assemblies through which mechanical force and regulatory signals are transmitted between the extracellular matrix (ECM) and an interacting cell. More precisely, focal adhesions are the sub-cellular structures that mediate the regulatory effects of a cell in response to ECM adhesion.

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Scanning ion-conductance microscopy

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A model lipid bilayer is any bilayer assembled in vitro, as opposed to the bilayer of natural cell membranes or covering various sub-cellular structures like the nucleus. They are used to study the fundamental properties of biological membranes in a simplified and well-controlled environment, and increasingly in bottom-up synthetic biology for the construction of artificial cells. A model bilayer can be made with either synthetic or natural lipids. The simplest model systems contain only a single pure synthetic lipid. More physiologically relevant model bilayers can be made with mixtures of several synthetic or natural lipids.

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Colloidal probe technique

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Role of cell adhesions in neural development

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References

  1. ↑ AK Harris, P Wild, and D Stopak. Silicone rubber substrata: a new wrinkle in the study of cell locomotion Science 208(4440):177–179, 1980.
  2. ↑ Munevar, S; Wang, Y; Dembo, M (2001). "Traction force microscopy of migrating normal and h-ras transformed 3t3 fibroblasts". Biophysical Journal. 80 (4): 1744–1757. doi:10.1016/s0006-3495(01)76145-0. PMC   1301364 . PMID   11259288.
  3. ↑ Maskarinec, SA; Franck, C; Tirrell, DA; Ravichandran, G (2009). "Quantifying cellular traction forces in three dimensions". Proceedings of the National Academy of Sciences. 106 (52): 22108–22113. doi: 10.1073/pnas.0904565106 . PMC   2799761 . PMID   20018765.
  4. ↑ Franck, C; Hong, S; Maskarinec, SA; Tirrell, DA; Ravichandran, G (2007). "Three-dimensional full-field measurements of large deformations in soft materials using confocal microscopy and digital volume correlation". Experimental Mechanics. 47 (3): 427–438. doi:10.1007/s11340-007-9037-9. S2CID   137243239.
  5. ↑ TM Koch, S Munster, N Bonakdar, JP Butler, and B Fabry. 3d traction forces in cancer cell invasion PLoS ONE 7(3):e33476, 2012
  6. ↑ Legant, WR; Choi, CK; Miller, JS; Shao, L; Gao, L; Betzig, E; Chen, CS (2013). "Multidimensional traction force microscopy reveals out-of-plane rotational moments about focal adhesions". Proceedings of the National Academy of Sciences. 110 (3): 881–886. doi: 10.1073/pnas.1207997110 . PMC   3549134 . PMID   23277584.
  7. ↑ "Traction Force Microscopy". cellmechanics.de.
  8. ↑ M Dembo and Y Wang. 1999 Stresses at the cell-to-substrate interface during locomotion of fibroblasts. Biophysical journal, 76(4):2307–2316.
  9. ↑ Legant, WR; Miller, JS; Blakely, BL; Cohen, DM; Genin, GM; Chen, CS (2010). "Measurement of mechanical tractions exerted by cells in three-dimensional matrices". Nature Methods. 7 (12): 969–971. doi:10.1038/nmeth.1531. PMC   3056435 . PMID   21076420.
  10. ↑ Dong, Li; Oberai, Assad A. (2017-02-01). "Recovery of cellular traction in three-dimensional nonlinear hyperelastic matrices". Computer Methods in Applied Mechanics and Engineering. Special Issue on Biological Systems Dedicated to William S. Klug. 314: 296–313. doi: 10.1016/j.cma.2016.05.020 .
  11. ↑ Sabass, B; Gardel, ML; Waterman, CM; Schwarz, US (2008). "High resolution traction force microscopy based on experimental and computational advances". Biophysical Journal. 94 (1): 207–220. doi:10.1529/biophysj.107.113670. PMC   2134850 . PMID   17827246.
  12. ↑ LG Griffith and MA Swartz. Capturing complex 3d tissue physiology in vitro. Nature Reviews Molecular Cell Biology, 7(3):211–224, 2006
  13. ↑ Toyjanova, J; Bar-Kochba, E; Lopez-Fagundo, C; Reichner, J; Hoffman-Kim, D; Franck, C (2014). "High resolution, large deformation 3d traction force microscopy". PLOS ONE. 9 (4): e90976. doi: 10.1371/journal.pone.0090976 . PMC   3989172 . PMID   24740435.
  14. ↑ Tambe, Dhananjay T., et al. "Collective cell guidance by cooperative intercellular forces." Nature materials 10.6 (2011): 469-475.
  15. ↑ Munevar, Steven, Yu-li Wang, and Micah Dembo. "Traction force microscopy of migrating normal and H-ras transformed 3T3 fibroblasts." Biophysical journal 80.4 (2001): 1744-1757.
  16. ↑ Koch, Thorsten M.; et al. (2012). "3D Traction forces in cancer cell invasion". PLOS ONE. 7 (3): e33476. doi: 10.1371/journal.pone.0033476 . PMC   3316584 . PMID   22479403.
  17. ↑ López-Fagundo, Cristina; et al. (2014). "Three-dimensional traction forces of Schwann cells on compliant substrates". Journal of the Royal Society Interface. 11 (97): 20140247. doi: 10.1098/rsif.2014.0247 . PMC   4208357 . PMID   24872498.
  18. ↑ George, J. C., et al. "The contractile strength of vascular smooth muscle myocytes is shape dependent." Integrative Biology 6.2 (2014): 152-163.
  19. ↑ Vedula, Sri Ram Krishna, et al. "Epithelial bridges maintain tissue integrity during collective cell migration." Nature materials (2013).
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