“9.61x6.69” b2815 Tissue Engineering and Nanotheranostics
Plasmonic Nanoparticles Application in Biosensor and Bioimaging 167
that it provides high contrast images and makes it possible to collect
the scattering light of plasmon nanoparticles at the singleparticle
level. To date, DFM is widely utilized to monitor biological events,
such as protein association and overexpression. Many modifications to
the conventional darkfield system like employing different illumina
tion methods, light sources and various CCDs, have been developed
to improve the signal and time resolution of DFM to identify single
molecular binding events.
4.1. Conventional Dark-Field Microscopy
In a typical DFM system, the white light of a Halogen lamp pass
through a condenser to tilt the incident illumination and are focused
at the sample to excite the plasmons. The condenser is equipped with
a circular block at the lens to permit light with high angular transmit.
Most of the light is directly transmitted through the sample and is not
collected by the objective below the stage because the numerical aper
ture of objective is smaller than the condenser, causing the dark back
ground. Only the scattered light from the sample could be collected
to image sample in truecolor or to analyze single particle spectra.
4.2. Improvements in Sensitivity and the Speed of
Dark-Field Microscopy
The main limitation of conventional DFM is the low signal intensity
for nanoparticles smaller than 50 nm. To obtain sufficient SNR of
single nanoparticle, an intense light source and long recording time
(up to seconds) are both required, which makes DFM in suitable for
fast dynamic observation. To improve SNR and the temporal resolu
tion, several optical modifications have been developed. Noji et al.
developed the objectivetype evanescent illumination DFM by simply
replacing the dichroic mirror with a perforated mirror in a
conventional microscope.^13 In this system with total internal reflec
tion (TIR)illumination, the incident laser is reflected at the water–
glass interface and forms the evanescent field on the coverslip surface.
The evanescent field induces the oscillating dipoles within the
