Optimizing force spectroscopy by modifying commercial cantilevers: Improved stability, precision, and temporal resolution.
J Struct Biol
; 197(1): 13-25, 2017 01.
Article
em En
| MEDLINE
| ID: mdl-26804584
ABSTRACT
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) enables a wide array of studies, from measuring the strength of a ligand-receptor bond to elucidating the complex folding pathway of individual membrane proteins. Such SMFS studies and, more generally, the diverse applications of AFM across biophysics and nanotechnology are improved by enhancing data quality via improved force stability, force precision, and temporal resolution. For an advanced, small-format commercial AFM, we illustrate how these three metrics are limited by the cantilever itself rather than the larger microscope structure, and then describe three increasingly sophisticated cantilever modifications that yield enhanced data quality. First, sub-pN force precision and stability over a broad bandwidth (Δf=0.01-20Hz) is routinely achieved by removing a long (L=100µm) cantilever's gold coating. Next, this sub-pN bandwidth is extended by a factor of â¼50 to span five decades of bandwidth (Δf=0.01-1000Hz) by using a focused ion beam (FIB) to modify a shorter (L=40µm) cantilever. Finally, FIB-modifying an ultrashort (L=9µm) cantilever improves its force stability and precision while maintaining 1-µs temporal resolution. These modified ultrashort cantilevers have a reduced quality factor (Q≈0.5) and therefore do not apply a substantial (30-90pN), high-frequency force modulation to the molecule, a phenomenon that is unaccounted for in traditional SMFS analysis. Currently, there is no perfect cantilever for all applications. Optimizing AFM-based SMFS requires understanding the tradeoffs inherent to using a specific cantilever and choosing the one best suited to a particular application.
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MEDLINE
Assunto principal:
Proteínas
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Microscopia de Força Atômica
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Imagem Individual de Molécula
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article