National Science Foundation     |     Directorate for Engineering  (ENG)
Division of Chemical, Bioengineering, Environmental, & Transport Systems  (CBET)
CBET Award Achievements (Nuggets)
Notable Accomplishments from CBET Awards
CAREER: Side View Atomic Force Microscopy
Daniel Fletcher    University of California-Berkeley

Background:  Research over the last decade has revealed the importance of mechanics and forces in a variety of biological processes.  It has been observed that substrate rigidity can direct the differentiation of nave mesenchymal stem cells into muscle, bone, or brain cells, and it is now known that substrate stiffness is linked with the malignant phenotype of tumor cells.  A recent study found that the adhesive forces between ectoderm, mesoderm, and endoderm progenitor cells play a crucial roll in germ layer organization in zebrafish embryos.  While mechanical loads and properties play an important role in all of these processes, the cellular response to controlled mechanical inputs and the specific molecular mechanisms underlying the observed responses are not well understood, partly due to the lack of appropriate experimental tools.

Results:  With NSF support, Professor Fletcher and his team have developed a new experimental technique to help understand the role of mechanotransduction in cellular behavior.  They have demonstrated an instrument that combines atomic force microscopy (AFM) with a unique side-view fluorescent imaging path that enables direct imaging of cellular deformation and cytoskeletal rearrangements along the axis of loading.  With this instrument, the Fletcher Team from UC Berkeley was able to directly observe cell shape under load, correlate changes in shape with force ruptures, and image formation of membrane tethers during cell-cell adhesion measurements.  Additionally, the Team observed cytoskeletal reorganization and stress fiber formation while measuring the contractile force of an individual cell.  This new side-view AFM provides a useful new tool for understanding the role of mechanical properties and forces in biological processes.

Daniel Fletcher 1
Figure 1.  Side view AFM measurement of U2OS cell deformation during contraction against a load. Cell length and force of contraction are shown in the black trace (bottom) over time.  Cell width (as depicted by blue dotted arrows in the cartoon in the inset) is measured for every image taken during the experiment and shown as the blue trace (top) over time.
Daniel Fletcher 2
Figure 2.  Side view AFM measurement of U2OS cell deformation during contraction against a load.  Side-view images taken at the indicated time points of GFP actin in the cell.  The location of the surface and cantilever are represented by the yellow dotted line and blue cartoon respectively.  Formation of fibers can be seen at 15 minutes.  Scale bar is 10 m.
Credit for Images 1 & 2:  O. Chaudhuri, S. Parekh, W. Lam, and D. A. Fletcher - University of California Berkeley
This project addresses the NSF Strategic Outcome Goals, as described in the NSF Strategic Plan 2006-2011, as follows:
Primary Strategic Outcome Goal:        (1) Research Infrastructure:  Currently, the mechanical properties of individual cells and the adhesive forces between cells have been characterized using magnetic tweezers, micropipette aspiration, single cell rheometry, optical tweezers, and atomic force microscopy (AFM).  Of these, AFM has been used extensively to study mechanics on the single cell level because it enables force measurements with high resolution (~ pN) over a large dynamic range (100s of nN).  AFM is often combined with brightfield and fluorescence microscopy to image cellular shape and labeled cellular proteins while making force measurements by mounting the instrument on an epi-fluorescence imaging system that provides an image of the sample along a plane parallel to the surface, or "bottom-view".  However, the most significant cellular deformations and cytoskeletal rearrangements are expected to be aligned with the applied force in a plane perpendicular to the surface.  Imaging in this plane via a "side view" would allow one to relate specific cellular responses along the loading direction with the applied load.  Professor Fletcher and his team have contributed to research infrastructure by developing and disseminating the design of a new side view AFM for investigating cellular biomechanics.
                                                                     (1) Research Infrastructure Category:
                                                                            - Research Resources (minor facilities, infrastructure and
                                                                              instrumentation, field stations, museum collections, etc.)

Secondary Strategic Outcome Goal:    (2) Discovery:  With this new side-view AFM, Professor Fletcher and his team were able to directly observe the behavior of cells tethered between the cantilever and a surface, providing simultaneous measurements of force, displacement, and cytoskeletal organization in the direction of loading.  This discovery-oriented research with the side-view AFM demonstrates that it provides a useful new tool for understanding the role of mechanical properties and forces in biological processes.
                                                                     (2) Discovery Categories:
                                                                            - CAREER: Faculty Early Career Program
                                                                            - Engineering Research

Secondary Strategic Outcome Goal:    (3) Learning:  The side-view AFM project provided training to graduate students and postdoctoral fellows in the multi-disciplinary fields of engineering and biology.  The students learned that new experimental tools can be developed to study basic biological questions.
                                                                     (3) Learning Category:
                                                                            - Graduate Education and Graduate Student Research

In terms of Intellectual Merit, this work is notable because the development of the side view AFM provides a new tool for measurement of cellular mechanics that provides data on the connection between forces and cell shape not possible with other experimental tools.

In terms of Broader Impacts, this work is notable because the research and development of the side view AFM was conducted by graduate students and postdoctoral fellows, providing them with interdisciplinary training (engineering and biology) and teaching them that new experimental tools can be developed to address basic biological questions.

This research is Transformative.  The potentially transformative results of this research come from the new insight it will provide into the role of forces in cell behavior.  In future studies with this instrument, the research team will investigate how forces can guide cell growth for tissue engineering.

This research represents Broadening Participation.  The research in this project involves contributions from a medical doctor - - who is not normally involved in instrument development work and now better appreciates its importance.

Existing or potential Societal Benefits of this research:  Potential societal benefits will arise from a more complete understanding of how mechanical stresses control cellular behavior, particularly during tissue regeneration and wound repair.

Program Director:
Semahat Demir
CBET Program Director - Biomedical Engineering
NSF Award Number:   0348758
Award Title:
  CAREER: Biomechanics of Polymerization Motors and Cell Motility
PI Name:   Daniel Fletcher
Institution Name:   University of California-Berkeley
Program Element Code:   5345
NSF Investments:
  - Understanding Complex Biological Systems (including the
      interfaces of life, physical, and computational sciences)
- Adaptive Systems Technology
CBET Nugget:

  FY 2009

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This Nugget was Updated on 30 September 2009.