“9.61x6.69” b2815 Tissue Engineering and Nanotheranostics
Plasmonic Nanoparticles Application in Biosensor and Bioimaging 169
4.3. 3D Orientational Imaging with Dark-Field Microscopy
The orientational dynamics of nanocargos would be a key parameter
for revealing the fundamental mechanisms of vital biological pro
cesses, such as the interactions between the nanocargo and nearby
biomolecules. 3D DFM provides access to solve this problem.104,105
Recently, He and Zhu successfully recorded the 3D orientation angles
of single anisotropic nanoparticle with standard optical DFM by the
method of defocused imaging.^29 The field distribution pattern in the
defocused DFM images depends on the aspect ratio of the AuNPs,
the numerical aperture of the objective, the defocusing distance and
the polarization direction of the incident radiation, thus, by compar
ing the experiment results at fixed condition with the simulated
images3D orientations of nanoparticle was confirmed. They improved
this method subsequently by adding another focused laser to monitor
transverse oscillation modes simultaneously. The dualwavelength
DFM has been proven to obtain the localization and orientational
information at the same time by the combination of focused trans
verse illumination and defocused longitudinal mode.
Further, He et al. presented another method to directly decipher
the 3D orientation of individual AuNRs in solution or inside living
cells with planar illumination.^104 They illuminated the sample with
two orthogonal sheets of light and obtained the longitudinal and
transverse scattering spectra simultaneously. DFM was improved by
inserting a birefringent crystal in the detection path before CCD
camera. They utilized this method to directly monitor the rotational
dynamics of individual AuNRs, which modified with different surface
ligands and dispersed in glycerol/water mixtures, and with the help
of proper surface function, they track the rotational movements of
individual AuNR transported by kinesin motor protein on microtu
bules inside living cells.
Since the 3D orientation of anisotropic metallic nanoparticles was
obtained by matching the measured pattern with the simulated
images in convention 3D DFM, high degrees of uncertainty are
involved. As TIR microscopy reduces the background noise greatly,
Fang’s group developed TIR scattering microscope to follow 3D
