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Atomic force microscopy of red-light photoreceptors using peakforce quantitative nanomechanical property mapping

Kroeger, Marie E ; Sorenson, Blaire A ; Thomas, J Santoro ; Stojković, Emina A ; Tsonchev, Stefan ; Nicholson, Kenneth T

Journal of visualized experiments, 2014-10 (92), p.e52164-e52164 [Periódico revisado por pares]

United States: MyJove Corporation

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  • Título:
    Atomic force microscopy of red-light photoreceptors using peakforce quantitative nanomechanical property mapping
  • Autor: Kroeger, Marie E ; Sorenson, Blaire A ; Thomas, J Santoro ; Stojković, Emina A ; Tsonchev, Stefan ; Nicholson, Kenneth T
  • Assuntos: Aluminum Silicates - chemistry ; Mechanical Phenomena ; Microscopy, Atomic Force - instrumentation ; Microscopy, Atomic Force - methods ; Nanotechnology ; Photoreceptor Cells - chemistry ; Physics ; Phytochrome - chemistry
  • É parte de: Journal of visualized experiments, 2014-10 (92), p.e52164-e52164
  • Notas: ObjectType-Article-2
    SourceType-Scholarly Journals-1
    ObjectType-Undefined-1
    ObjectType-Feature-3
    content type line 23
    Correspondence to: Kenneth T. Nicholson at k-nicholson@neiu.edu
  • Descrição: Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or "tapping mode" AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials. The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials.
  • Editor: United States: MyJove Corporation
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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