Raman imaging is an optical method that provides chemo-selective contrast, microscopic resolution and multiplexing capability. Medical applications, however, still suffer from weak signals and resulting long acquisition times. Plasmon resonance effects can drastically increase signals for Surface Enhanced Raman Scattering (SERS). Here, we present concepts towards an ideal, multimodal SERS agent for medical applications. Enhancement of intrinsic spectra of biological tissue is combined with contrast in Photoacoustic- and Magnetic Resonance Tomography, making this imaging agent highly promising for clinical radiology.
Distance dependant coupling of plasmon resonances between closely spaced metal nanoparticles offers an attractive
alternative for the imaging of molecular interactions. Here we analyzed interactions between molecular specific gold
nanoparticles and live cells using a combination of dark-field reflectance and hyperspectral imaging. The results of
optical imaging were correlated with transmittance electron microscopy of cell slices and theoretical simulations of
optical properties of gold aggregates. We showed that nanoparticles targeted to epidermal growth factor receptor (EGFR)
form closely spaced assemblies in the presence of the target molecule. Our experiments with living cells showed that
receptor mediated assembly and plasmon coupling of gold bioconjugates result in a spectral shift of more than 100 nm in
plasmon resonance frequency of the nanoparticles giving a very bright red signal. We demonstrated that plasmon
coupling can be used for imaging of EGFR activation and trafficking as formation of EGFR dimers and further
intracellular uptake in early and late endosomes is associated with progressive color changes from green to red,
respectively, with each stage of EGFR cycle being associated with a distinct color of EGFR bound nanoparticles. This
approach can allow imaging of molecular interactions ranging from protein pairs to multi-protein complexes with
sensitivity and SNR that cannot be currently achieved with any other method.
Photothermal therapy is a noninvasive, targeted, laser-based technique for cancer treatment. During photothermal therapy, light energy is converted to heat by tumor-specific photoabsorbers. The corresponding temperature rise causes localized cancer destruction. For effective treatment, however, the presence of photoabsorbers in the tumor must be ascertained before therapy and thermal imaging must be performed during therapy. This study investigates the feasibility of guiding photothermal therapy by using photoacoustic imaging to detect photoabsorbers and to monitor temperature elevation. Photothermal therapy is carried out by utilizing a continuous wave laser and metal nanocomposites broadly absorbing in the near-infrared optical range. A linear array-based ultrasound imaging system is interfaced with a nanosecond pulsed laser to image tissue-mimicking phantoms and ex-vivo animal tissue before and during photothermal therapy. Before commencing therapy, photoacoustic imaging identifies the presence and spatial location of nanoparticles. Thermal maps are computed by monitoring temperature-induced changes in the photoacoustic signal during the therapeutic procedure and are compared with temperature estimates obtained from ultrasound imaging. The results of our study suggest that photoacoustic imaging, augmented by ultrasound imaging, is a viable candidate to guide photoabsorber-enhanced photothermal therapy.
Gold nanoparticles functionalized with anti-EGFR antibodies undergo molecular specific aggregation on
the cellular membrane and later within the cell that leads to a red shift in the plasmon resonance frequency
of the gold nanoparticles. Capitalizing on this effect, we previously demonstrated on tissue phantoms that
highly sensitive and selective detection of cancer cells can be achieved using the combination of
photoacoustic imaging and molecular specific gold nanoparticles. To further evaluate the efficacy of
molecular specific photoacoustic imaging technique in detecting deeply situated tumors, small animal
experiments were performed. In this study, two gelatin solutions mixed with cells labeled with gold
nanoparticles and cells mixed with polyethylene glycol-thiol
(mPEG-SH) coated gold nanoparticles were
injected in a mouse abdomen ex-vivo. The photoacoustic and ultrasound images from the same crosssection
of the region before and after the injections were obtained using a 25 MHz single element
ultrasound transducer interfaced with pulsed laser system. The results of our study suggest that the
molecular specific photoacoustic imaging with plasmonic nanosensors could be used to detect deeply
embedded tumors.
In photothermal therapy, a localized temperature increase is achieved by using a continuous wave laser and optically
tuned metal nanoparticles. However, the successful outcome of therapy depends on identifying the presence of
nanoparticles in the tumor before therapy and monitoring temperature rise during the photothermal procedure. In this
paper, we investigate the utility of photoacoustic and ultrasound imaging to guide photothermal therapy. Differences in
the optical properties of tissue, enhanced by the presence of nanoparticles, provide a contrast for photoacoustic imaging.
Thus, an uptake of nanoparticles in the tumor can be detected by monitoring a photoacoustic image over time. A
temperature rise causes the photoacoustic signal amplitude to increase. In addition, a temperature change also leads to
time shifts in an ultrasound signal, primarily due to the change in speed of sound. Therefore, by measuring the change in
the photoacoustic signal, and differential motion of ultrasound speckle, the temperature rise during photothermal
therapy can be computed. Combined imaging was performed with a tunable pulsed laser and an array-based ultrasound
transducer. Experiments were carried out on ex-vivo animal tissue injected with composite and broadly absorbing gold
nanoparticles. The photoacoustic imaging identified the presence of nanoparticles in tissue. In addition, a localized
temperature increase, obtained during therapy, was monitored using photoacoustic and ultrasound imaging. The
temperature profiles, obtained by both imaging techniques, were spatially and temporally co-registered. Therefore, the
experimental results suggest that photoacoustic and ultrasound imaging can be used to guide and monitor photothermal
therapy.
We demonstrate the use of gold nanorods as molecularly targeted contrast agents for two-photon luminescence (TPL)
imaging of cancerous cells 150 μm deep inside a tissue phantom. We synthesized gold nanorods of 50 nm x 15 nm size
with a longitudinal surface plasmon resonance of 760 nm. Gold nanorods were conjugated to antibodies against
epidermal growth factor receptor (EGFR) and labeled to A431 human epithelial skin cancer cells in a collagen matrix
tissue phantom. Using a 1.4 NA oil immersion objective lens, we found that excitation power needed for similar
emission intensity in TPL imaging of labeled cells was up to 64 times less than that needed for two-photon
autofluorescence (TPAF) imaging of unlabeled cells, which would correspond to a more than 4,000 times increase in
emission intensity under equal excitation energy. However, the aberrations due to refractive index mismatch of the
immersion oil and the sample limit imaging depth to 75 μm. Using a 0.95 NA water immersion objective lens, we
observe robust two-photon emission signal from gold nanorods in the tissue phantoms from at depths of up to 150 μm.
Furthermore, the increase in excitation energy required to maintain a constant emission signal intensity as imaging depth
was increased was the same in both labeled and unlabeled phantom, suggesting that at the concentrations used, the
addition of gold nanorods did not appreciably increase the bulk scattering coefficient of the sample. The remarkable TPL
brightness of gold nanorods in comparison to TPAF signal makes them an attractive contrast agent for early detection of
cutaneous melanoma.
We demonstrate the use of gold nanorods as molecularly targeted contrast agents for two-photon luminescence (TPL)
imaging of cancerous cells 150 µm deep inside a tissue phantom. We synthesized gold nanorods of 50 nm x 15 nm size
with a longitudinal surface plasmon resonance of 760 nm. Gold nanorods were conjugated to antibodies against
epidermal growth factor receptor (EGFR) and labeled to A431 human epithelial skin cancer cells in a collagen matrix
tissue phantom. Using a 1.4 NA oil immersion objective lens, we found that excitation power needed for similar
emission intensity in TPL imaging of labeled cells was up to 64 times less than that needed for two-photon
autofluorescence (TPAF) imaging of unlabeled cells, which would correspond to a more than 4,000 times increase in
emission intensity under equal excitation energy. However, the aberrations due to refractive index mismatch of the
immersion oil and the sample limit imaging depth to 75 µm. Using a 0.95 NA water immersion objective lens, we
observe robust two-photon emission signal from gold nanorods in the tissue phantoms from at depths of up to 150 µm.
Furthermore, the increase in excitation energy required to maintain a constant emission signal intensity as imaging depth
was increased was the same in both labeled and unlabeled phantom, suggesting that at the concentrations used, the
addition of gold nanorods did not appreciably increase the bulk scattering coefficient of the sample. The remarkable TPL
brightness of gold nanorods in comparison to TPAF signal makes them an attractive contrast agent for early detection of
cutaneous melanoma.
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