Fluorescence fluctuation analysis of dilute biomolecules can provide a powerful method for fast
and accurate determination of diffusion dynamics, local concentrations, and aggregation states in
complex environments. However, spectral overlap among multiple exogenous and endogenous
fluorescent species, photobleaching, and background inhomogeneities can compromise
quantitative accuracy and constrain useful biological implementation of this analytical strategy in
real systems. In order to better understand these limitations and expand the utility of fluctuation
correlation methods, spatiotemporal fluorescence correlation analysis was performed on
spectrally resolved line scanned images of modeled and real data from mixed fluorescent
nanospheres in a synthetic gel matrix. It was found that collecting images at a pixel sampling
regime optimal for spectral imaging provides a method for calibration and subsequent temporal
correlation analysis which is insensitive to spectral mixing, spatial inhomogeneity, and
photobleaching. In these analyses, preprocessing with multivariate curve resolution (MCR)
provided the local concentrations of each spectral component in the images, thus facilitating
correlation analysis of each component individually. This approach allowed quantitative
removal of background signals and showed dramatically improved quantitative results compared
to a hypothetical system employing idealized filters and multi-parameter fitting routines.
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.
Near-field coupling between plasmonic resonant nanoparticles and the associated shifts in scattering spectra
enables the accomplishment of unprecedented observation of the co-localization dynamics of in-situ biomolecules
on nanometer length-scales. We have recently shown that resonant nanoparticles conjugated to antibodies for
cell-surface receptors provide a sensitive probe allowing the unambiguous resolution of not only the time sequence,
but also the details of the intracellular pathway, for receptor-mediated endocytosis in live cells.
In terms of general principles, the classical electrodynamics determining the scattering cross-section for
nanoparticle aggregates is straightforward. However, the specifics of the angular dependence of the differential
cross-section at a single wavelength, the wavelength dependence of this cross-section, and the correct implementation
and interpretation of statistical averages of cross-section properties over an ensemble of aggregate
morphologies are generally quite complicated, and in fact are often misinterpreted in the literature.
Despite this complexity, we have constructed a set of few-parameter formulae describing optical scattering
from nanoparticle aggregates by judicious combination of experimental results with extensive, near-exact simulation
using the T-matrix technique. These phenomenological results facilitate the practical use of nanoparticle
aggregates for biological measurement and clinical therapeutic applications.
An effective cancer control strategy requires improved early detection methods, patient-specific drug selection, and the ability to assess response to targeted therapeutics. Recently, plasmon resonance coupling between closely spaced metal nanoparticles has been used to develop ultrasensitive bioanalytical assays in vitro. We demonstrate the first in vivo application of plasmon coupling for molecular imaging of carcinogenesis. We describe molecular-specific gold bioconjugates to image epidermal growth factor receptor (EGFR); these conjugates can be delivered topically and imaged noninvasively in real time. We show that labeling with gold bioconjugates gives information on the overexpression and nanoscale spatial relationship of EGF receptors in cell membranes, both of which are altered in neoplasia. EGFR-mediated aggregation of gold nanoparticles in neoplastic cells results in more than a 100-nm color shift and a contrast ratio of more than tenfold in images of normal and precancerous epithelium in vivo, dramatically increasing contrast beyond values reported previously for antibody-targeted fluorescent dyes.
We are developing a multi-modal miniature microscope (4M device) for imaging morphology and cytochemistry in vivo and providing better delineation of tumors. The 4M device is designed to be a complete microscope on a chip, including optical, micro-mechanical, and electronic components. It has advantages such as compact size and capability for microscopic-scale imaging. This paper presents the recent imaging experiment of 4M device including trans-illumination imaging, TIR illumination imaging and fluorescent imsging. We built a multi-modal imaging test-bed to demonstrate multi-modality of 4M device. In this paper, we present imaging experiment results by implementing various imaging modality with cervical cancer cells. In order to enhance image contrast, some imaging modality uses cells attached with contrast agency such as silver nano-particles. Imaging results indicate that the 4M prototype can resolve cellular detail necessary for detection of precancer.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.