b2815 Tissue Engineering and Nanotheranostics “9.61x6.69”
168 Tissue Engineering and Nanotheranostics
particle, leading to conversion of the nonpropagating nearfield
light into the conventional propagating scattered light. The scattered
light is detected by camera while the returning laser is stopped,
inducing darkfield effects. Taking advantage of the focus on a thin
layer by TIR illumination, background noise was decreased and
images with high SNR were acquired. In a study of the rotary mecha
nism of F 1 –ATPase 1–2 nm, spatial precision and 9.1 μs temporal
resolution were demonstrated by the successful application of this
homebuilt TIRDFM system. Later, to achieve higher sensitivity,
white light laser with average power output of 2.8 W was reported to
supply a strong light source to illuminate the particles in TIR geom
etry. With the help of the improved system, the dynamic evolution of
a single protein binding to a single AuNP was observed with milli
second time resolution.^102
Although TIR illumination can decrease the background noise,
this technique is limited to a thin region adjacent to the interface.
Baumberg et al. used a supercontinuum laser to perform DF spec
troscopy in a focused mode, they equipped a circular block in the
illumination beam path to focus the ringshaped laser on the sam
ple, and the illumination intensities yielded were up to 10 MW/
cm^2.^103 Under intense illumination, the growth of a single gold
nanorod was observed in real time with 8 ms time resolution. More
importantly, this method can be used to probe deeply into a
sample.
To reduce the toxicity of the illumination light to living cells, He
et al. developed quiet a different method. They utilized a new tech
nique that named dualwavelength difference (DWD) imaging to
remove noise in the living cell environment.^27 Briefly, the scattering
intensity of nanoparticle probe at resonance frequency is much higher
than noise signal from living cells, while at nonresonance frequency
scattering intensity of nanoparticle is similar with noise signals. Then,
noise free images could be obtained by subtracting the signal of refer
ence beam, of which wavelength is far away from the resonance fre
quency, from probe poem beam at the resonance. With this novel
strategy, the diffusion dynamics of nucleic acids functionalized AuNPs
on the cell membranes were explored.
