Abstract
Precise nanometre-scale imaging of soft structures at room temperature poses a major challenge to any type of microscopy because fast thermal fluctuations lead to significant motion blur if the position of the structure is measured with insufficient bandwidth. Moreover, precise localization is also affected by optical heterogeneities, which lead to deformations in the imaged local geometry, the severity depending on the sample and its thickness. Here we introduce quantitative thermal noise imaging, a three-dimensional scanning probe technique, as a method for imaging soft, optically heterogeneous and porous matter with submicroscopic spatial resolution in aqueous solution. By imaging both individual microtubules and collagen fibrils in a network, we demonstrate that structures can be localized with a precision of ∼10 nm and that their local dynamics can be quantified with 50 kHz bandwidth and subnanometre amplitudes. Furthermore, we show how image distortions caused by optically dense structures can be corrected for.
Highlights
Background correction for probe position signalIn absence of optically heterogeneous material, the position of the probe is determined by reading out the interference of light forward scattered by the probe with light of the trapping beam on the photonic force microscope (PFM)’s quadrant photodiode
Thermal noise imaging was introduced more than a decade ago[10], but it has until now remained challenging to turn it into a quantitative method for three-dimensional imaging of soft nanostructures
Thermal noise imaging exploits the thermal motion of a nanoparticle as a natural scanner, similar to a protein searching for a binding site
Summary
Background correction for probe position signalIn absence of optically heterogeneous material, the position of the probe is determined by reading out the interference of light forward scattered by the probe with light of the trapping beam on the PFM’s quadrant photodiode. If the probe as well as the collagen fibril are close to the focus, both light scattered by the probe and by the fibril will strike the detector. The output signal no longer correctly reflects the probe’s position and must be corrected for the fibril contributions. It can be shown[30,31] that to first order approximation the total detector signals along the x, y and z directions. Sa (bp, bf), are the sum of the signals on the detector caused by the fibril in absence of the probe Sa, fibril (bf), and the signal caused by the particle in absence of the fibril Sa,probe (bp) (a 1⁄4 x, y, z and bp and bf are the position vectors of the particle and fibril with respect to the focus, Supplementary Fig. 3) ÀÁ. If the offsets are known for each grid position, the particle’s position can be calculated from the measured signals as
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