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Bimodal Dual AC™ AFM Imaging of Collagen Fiber Ultrastructure

Elena Tomas Herruzo and Ricardo Garcia
Instituto de Microelectrónica de Madrid, CSIC, Madrid, Spain
Roger B. Proksch, Irene Revenko, and Monteith G. Heaton
Asylum Research, Santa Barbara, USA

Bimodal Dual AC imaging allows high resolution, extremely low force imaging by using the first and the second cantilever resonances. The technique provides increased compositional contrast and, unlike conventional AC (tapping mode) imaging, the combination of first mode feedback and second mode amplitude with phase imaging allows high resolution at extremely gentle, pico-Newton level forces. In this example, the ultrastructure of a collagen fiber is imaged using the Asylum Research MFP-3D™ AFM.

In conventional amplitude modulation AFM (also called tapping mode or AC imaging), the phase of the cantilever oscillation is indicative of the cantilever dissipation because the feedback loop prevents contrast from conservative tip-sample interactions. On the other hand, the amplitude of the cantilever is affected by both topography and compositional forces, resulting in mixed topographic and compositional information in the height images. Recently, the combination of first and second resonance excitations (bimodal imaging) have been harnessed to separate topographic and compositional contrast.1-3 One significant benefit of bimodal imaging is that the forces between the tip and the sample are extremely small, allowing clear and non-destructive differentiation of composition, even on soft biological samples.4

Figure 1 shows the typical setup of the microscope for bimodal imaging. The cantilever is driven at the first and second resonances and the resulting response is detected with separate lock-ins, yielding the amplitude and phase at the two frequencies. Typically, the amplitude of the first mode is used for controlling the microscope’s Z feedback loop.

Type I collagen molecules form tensile-bearing structural fibers that are very common in connective tissue and the extracellular matrix. The fibril packing structure of collagen has been debated for some time. The most commonly accepted structure of the fibers corresponds to the model of Hodge and Petruska,5 where the molecules of collagen are arranged in a staggered manner, leading to a typical 68nm pattern. However this staggered arrangement has never been clearly demonstrated, and other models have been proposed. More recently AFM has been used to image collagen fibers, and newer models have been proposed.6 A few AFM studies have also followed the assembly of molecules dynamically,7 but no insight was given on the molecular level.

In Figure 2, we use bimodal imaging to probe the ultrastructure of a collagen fiber extracted from a rat tail tendon. The tail was mechanically dissected in PBS buffer. The extracted collagen fibers were torn apart and deposited on a mica surface. After rinsing with de-ionized water, the fibers were imaged. An AC240 cantilever (Olympus) was bimodally driven using the Dual AC technique on an Asylum Research MFP-3D AFM. The first resonance was at 72.1kHz and the setpoint was 5nm. The second resonance was at 437.5kHz and the amplitude was nominally ~1nm. The topographic data show the very typical 65nm banding pattern, while the second mode amplitude shows detailed features at the surface of the fibers on the nanometer scale. Note that the fundamental resonance phase signal is relatively featureless. The small elongated structures visible in the second mode amplitude channel are on a length scale consistent with individual molecules inside the fibers. The round features could correspond to the terminal parts of the molecules forming the top layer of the fiber. We anticipate that this technique will help decipher the arrangement of the molecules when applied to the visualization of real time imaging of in vivo growing fibers. Additional information on Dual AC Mode imaging can be found here.

References

  1. Rodriguez, T.R. and Garcia, R., Compositional mapping of surfaces in AFM by excitation of the second mode. Applied Physics Letters 84, 449 (2004).

  2. Proksch, R., Applied Physics Letters 89, 113121 (2006).

  3. Martinez et al., Enhanced compositional sensitivity in AFM by the excitation of the first two flexural modes. Applied Physics Letters 89, 153115 (2006).

  4. Patil, S. et al., Force microscopy imaging of individual protein molecules with sub-pico Newton force sensitivity. J. Molecular Recognition 20, 516 (2007).

  5. Hodge, A. and Petruska, J., A subunit model for the tropocollagen macromolecule. Proc. Natl. Acd. Sci. 51, 871 (1964).

  6. Bozel, L. et al., Collagen fibrils: nanoscale ropes. Biophys. J. 9, 70 (2007).

  7. Jiang, F. et al., Assembly of collagen into microribbons: effects of pH and electrolytes. J. of Structural Biol. 148, 268 (2004); Cisneros, D. et al., Observing growth steps of collagen self-assembly by time-lapse high-resolution atomic force microscopy. J. of Structural Biol. 154, 232 (2006).

 

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Figure 1: In bimodal Dual AC, the cantilever is both driven and measured at two (or more) frequencies. The sinusoidal “shake” voltage is a sum of voltages at frequencies f1 and f2. The cantilever deflection then contains information at both of those frequencies, as shown in the red curve. The amplitude and phase at the two frequencies are then separated again by the two lockins and passed on to the controller. The controller can use one or both of the resonant frequencies to operate a feedback loop. Click here to enlarge diagram.

 

Figure 2: Bimodal images of the (a) topography obtained from feeding back on the first mode amplitude, (b) first mode phase and (c) second mode amplitude of a collagen fiber (300nm scan size). Image (d) shows a zoom into a region of the second mode amplitude image and (e) shows a section taken along the red line. The first mode phase is relatively featureless. The second mode amplitude shows a fine structure with a resolution of 2-3nm. The white bar in images (a) and (d) is 50nm long.


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