Abstract
The Single Particle Orientation and Rotational Tracking (SPORT) technique, which utilizes anisotropic plasmonic gold nanorods and differential interference contrast (DIC) microscopy, has shown potential as an effective alternative to fluorescence-based techniques to decipher rotational motions on the cellular and molecular levels. However, localizing gold nanorods from their DIC images with high accuracy and precision is more challenging than the procedures applied in fluorescence or scattering microscopy techniques due to the asymmetric DIC point spread function with bright and dark parts superimposed over a grey background. In this paper, localization accuracy and inherited uncertainties from unique DIC image patterns are elucidated with the assistance of computer simulation. These discussions provide guidance for researchers to properly evaluate their data and avoid making claims beyond the technical limits. The understanding of the intrinsic localization errors and the principle of DIC microscopy leads us to propose a new localization strategy that utilizes the experimentally-measured shear distance of the DIC microscope to improve the localization accuracy.
Highlights
Fluorescence polarization microscopy [1,2,3] and defocused fluorescence imaging techniques [4,5,6,7] have been widely used to acquire the dipole orientation of fluorescent molecules or quantum dots
Gold nanorods are imaged at their longitudinal localized surface plasmon resonance (LSPR) wavelength and exhibit most bright, most dark, or half bright – half dark image patterns depending on their orientations
To understand how a dipole’s orientation affects its localization in differential interference contrast (DIC) microscopy, we first take into account the errors that already exist in the localization based on the scattering point spread functions (PSF) from a dipole tilted relative to the horizontal plane
Summary
Fluorescence polarization microscopy [1,2,3] and defocused fluorescence imaging techniques [4,5,6,7] have been widely used to acquire the dipole orientation of fluorescent molecules or quantum dots. To overcome several critical limitations of fluorescence microscopy, such as photobleaching and photoblinking, anisotropic plasmonic nanoparticles have been chosen as rotational tracking probe in differential interference contrast (DIC) microscopy [8, 9], total internal reflection scattering (TIRS) microscopy [10,11,12], dark field polarization microscopy [13], defocused dark field microscopy [14], and planar illumination scattering microscopy [15] These techniques have enabled the studies of rotational motions in live cells. The correlation mapping algorithm does not work properly when anisotropic plasmonic nanoparticles are imaged in SPORT experiments, because the model changes constantly as a nanorod rotates to different orientations To overcome this challenge, we have developed a dual-channel imaging system to localize gold nanorods with high accuracy in the bright-field channel at the gold nanorod’s transverse localized surface plasmon resonance (LSPR) wavelength and track the rotational motions in the DIC channel at the gold nanorod’s longitudinal LSPR wavelength [26]. Based on this quantitative understanding, a new localization strategy is proposed to improve the localization accuracy of gold nanorods in SPORT experiments
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