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1.  Fast, multi-frequency, and quantitative nanomechanical mapping of live cells using the atomic force microscope 
Scientific Reports  2015;5:11692.
A longstanding goal in cellular mechanobiology has been to link dynamic biomolecular processes underpinning disease or morphogenesis to spatio-temporal changes in nanoscale mechanical properties such as viscoelasticity, surface tension, and adhesion. This requires the development of quantitative mechanical microscopy methods with high spatio-temporal resolution within a single cell. The Atomic Force Microscope (AFM) can map the heterogeneous mechanical properties of cells with high spatial resolution, however, the image acquisition time is 1–2 orders of magnitude longer than that required to study dynamic cellular processes. We present a technique that allows commercial AFM systems to map quantitatively the dynamically changing viscoelastic properties of live eukaryotic cells at widely separated frequencies over large areas (several 10’s of microns) with spatial resolution equal to amplitude-modulation (AM-AFM) and with image acquisition times (tens of seconds) approaching those of speckle fluorescence methods. This represents a ~20 fold improvement in nanomechanical imaging throughput compared to AM-AFM and is fully compatible with emerging high speed AFM systems. This method is used to study the spatio-temporal mechanical response of MDA-MB-231 breast carcinoma cells to the inhibition of Syk protein tyrosine kinase giving insight into the signaling pathways by which Syk negatively regulates motility of highly invasive cancer cells.
PMCID: PMC4484408  PMID: 26118423
2.  Nanomechanical Property Maps of Breast Cancer Cells As Determined by Multiharmonic Atomic Force Microscopy Reveal Syk-Dependent Changes in Microtubule Stability Mediated by MAP1B 
Biochemistry  2014;54(1):60-68.
The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery.
PMCID: PMC4295795  PMID: 24914616
3.  Multiple regimes of operation in bimodal AFM: understanding the energy of cantilever eigenmodes 
One of the key goals in atomic force microscopy (AFM) imaging is to enhance material property contrast with high resolution. Bimodal AFM, where two eigenmodes are simultaneously excited, confers significant advantages over conventional single-frequency tapping mode AFM due to its ability to provide contrast between regions with different material properties under gentle imaging conditions. Bimodal AFM traditionally uses the first two eigenmodes of the AFM cantilever. In this work, the authors explore the use of higher eigenmodes in bimodal AFM (e.g., exciting the first and fourth eigenmodes). It is found that such operation leads to interesting contrast reversals compared to traditional bimodal AFM. A series of experiments and numerical simulations shows that the primary cause of the contrast reversals is not the choice of eigenmode itself (e.g., second versus fourth), but rather the relative kinetic energy between the higher eigenmode and the first eigenmode. This leads to the identification of three distinct imaging regimes in bimodal AFM. This result, which is applicable even to traditional bimodal AFM, should allow researchers to choose cantilever and operating parameters in a more rational manner in order to optimize resolution and contrast during nanoscale imaging of materials.
PMCID: PMC3701429  PMID: 23844344
atomic force microscopy; bimodal AFM; cantilever eigenmodes; polymer characterization
4.  High-resolution dynamic atomic force microscopy in liquids with different feedback architectures 
The recent achievement of atomic resolution with dynamic atomic force microscopy (dAFM) [Fukuma et al., Appl. Phys. Lett. 2005, 87, 034101], where quality factors of the oscillating probe are inherently low, challenges some accepted beliefs concerning sensitivity and resolution in dAFM imaging modes. Through analysis and experiment we study the performance metrics for high-resolution imaging with dAFM in liquid media with amplitude modulation (AM), frequency modulation (FM) and drive-amplitude modulation (DAM) imaging modes. We find that while the quality factors of dAFM probes may deviate by several orders of magnitude between vacuum and liquid media, their sensitivity to tip–sample forces can be remarkable similar. Furthermore, the reduction in noncontact forces and quality factors in liquids diminishes the role of feedback control in achieving high-resolution images. The theoretical findings are supported by atomic-resolution images of mica in water acquired with AM, FM and DAM under similar operating conditions.
PMCID: PMC3596120  PMID: 23503468
atomic force microscopy; dAFM; high-resolution; liquids
5.  Drive-amplitude-modulation atomic force microscopy: From vacuum to liquids 
We introduce drive-amplitude-modulation atomic force microscopy as a dynamic mode with outstanding performance in all environments from vacuum to liquids. As with frequency modulation, the new mode follows a feedback scheme with two nested loops: The first keeps the cantilever oscillation amplitude constant by regulating the driving force, and the second uses the driving force as the feedback variable for topography. Additionally, a phase-locked loop can be used as a parallel feedback allowing separation of the conservative and nonconservative interactions. We describe the basis of this mode and present some examples of its performance in three different environments. Drive-amplutide modulation is a very stable, intuitive and easy to use mode that is free of the feedback instability associated with the noncontact-to-contact transition that occurs in the frequency-modulation mode.
PMCID: PMC3343270  PMID: 22563531
atomic force microscopy; control systems; dissipation; frequency modulation; noncontact
6.  Resolving Structure and Mechanical Properties at the Nanoscale of Viruses with Frequency Modulation Atomic Force Microscopy 
PLoS ONE  2012;7(1):e30204.
Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25–50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called “dissipation channel” in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.
PMCID: PMC3266245  PMID: 22295076

Results 1-6 (6)