Combined Optical-AFM Microscope for Life Sciences

Caldarola, Martín; Bilderling, Catalina von; Masip, Martín E.; Pietrasanta, Lı́a I.; Bragas, Andrea V.

doi:10.1364/fio.2012.ftu1c.6

Cited by 1 publication

(2 citation statements)

References 6 publications

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“…AFM force spectroscopic capabilities in terms of force detection sensitivity were measured to reach single ligand-receptor interaction forces in a model system as biotin-streptavidin. 21 The AFM head works on top of an inverted multipurpose optical microscope, which was operated as a wide field fluorescence microscope, with a blue LED (Tolket, SRL) as the light source, conventional GFP filter set (Chroma), and a CCD camera (Apogee Imaging Systems, Alta U2000). Two notch filters block the red AFM laser from the optical microscope light path.…”

Section: B Combined Afm and Fluorescence Microscopymentioning

confidence: 99%

“…However, these studies generally analyze global cell responses such as cell shape or total protein area, and very few of them quantify the temporal evolution of these responses with spatial resolution. In this work, we describe the integration of an AFM with an epifluorescence microscope 21 to investigate the dynamics of adhesion proteins within single FA in response to a controlled local mechanical stimulus. Indeed, AFM combines the possibility of applying forces from pN to nN, localized in a contact area that can be down to a few nm for typical AFM-probes.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Monitoring in real-time focal adhesion protein dynamics in response to a discrete mechanical stimulus

Bilderling

Caldarola

Masip

et al. 2017

Review of Scientific Instruments

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The adhesion of cells to the extracellular matrix is a hierarchical, force-dependent, multistage process that evolves at several temporal scales. An understanding of this complex process requires a precise measurement of forces and its correlation with protein responses in living cells. We present a method to quantitatively assess live cell responses to a local and specific mechanical stimulus. Our approach combines atomic force microscopy with fluorescence imaging. Using this approach, we evaluated the recruitment of adhesion proteins such as vinculin, focal adhesion kinase, paxillin, and zyxin triggered by applying forces in the nN regime to live cells. We observed in real time the development of nascent adhesion sites, evident from the accumulation of early adhesion proteins at the position where the force was applied. We show that the method can be used to quantify the recruitment characteristic times for adhesion proteins in the formation of focal complexes. We also found a spatial remodeling of the mature focal adhesion protein zyxin as a function of the applied force. Our approach allows the study of a variety of complex biological processes involved in cellular mechanotransduction. Published by AIP Publishing. [http://dx

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Section: B Combined Afm and Fluorescence Microscopymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%