Although biological cells are mostly transparent, they are phase objects that differ in shape and refractive index. Any image that is projected through layers of randomly oriented cells will normally be distorted by refraction, reflection, and scattering. Counterintuitively, the retina of the vertebrate eye is inverted with respect to its optical function and light must pass through several tissue layers before reaching the light-detecting photoreceptor cells. Here we report on the specific optical properties of glial cells present in the retina, which might contribute to optimize this apparently unfavorable situation. We investigated intact
Metal nanoparticles with surface plasmon resonance (SPR) in the near-infrared region (NIR) are of great interest for imaging and therapy. Presently, gold nanoparticles with NIR absorbance are typically larger than 50nm, above the threshold size of ~5 nm required for efficient renal clearance. As these nanoparticles are not biodegradable, concerns about long-term toxicity have restricted their translation into the clinic. Here, we address this problem by developing a flexible platform for the kinetically-controlled assembly of sub-5 nm ligand-coated gold particles to produce metal/polymer biodegradable nanoclusters smaller than 100 nm with strong NIR absorbance for multimodal application. A key novel feature of the proposed synthesis is the use of weakly adsorbing biodegradable polymers that allows tight control of nanocluster size and, in addition, results in nanoclusters with unprecedented metal loadings, and thus optical functionality. Over time, the biodegradable polymer stabilizer degrades under physiological conditions that leads to disassembly of the nanoclusters into sub-5nm primary gold particles which are favorable for efficient body clearance. This synthesis of polymer/inorganic nanoclusters combines the imaging contrast and therapeutic capabilities afforded by the NIR-active nanoparticle assembly with the biodegradability of a polymer stabilizer.
We used molecular-specific gold nanoparticles to monitor epidermal growth factor receptors (EGFR) in live A431 cells over time. Dark-field hyperspectral imaging, electron microscopy, and electrodynamic modeling were used to correlate optical properties of EGFR-bound plasmonic nanoparticles with receptor regulation state. We showed that receptor trafficking resulted in a progressive red-shift of greater than 100nm in the nanoparticle plasmon resonance wavelength over a time period of 60 minutes. Furthermore, we demonstrated that changes in peak scattering wavelengths of gold nanoparticles from 546±15 nm to 574±20 nm and, to 597±44 nm are associated with EGFR trafficking from the cell membrane, to early endosomes and to late endosomes/multivesicular bodies, respectively. Finally, we used the changes in scattering spectra of EGFR-bound nanoparticles and a straightforward statistical analysis of RGB-channel color images of labeled cells to create near real-time maps of EGFR regulatory states in living cells.Keywords plasmonic nanoparticles; molecular imaging; functional imaging; growth factor receptors; molecular trafficking Detecting and monitoring the vast number of bio-molecular interactions in the cell is a central effort in biology, as these interactions largely govern the behavior of nearly all cell types. Imaging methods are an indispensable approach for measuring the spatio-temporal characteristics of protein assemblies in intact cells. In this work, we expand the application of nanoparticle plasmon resonance coupling (NPRC) and demonstrate a novel, generalized method for imaging and characterizing molecular assemblies at the nanometer length-scale in living cells.Over the past decades, fluorescence resonance energy transfer (FRET) has allowed many investigators to elucidate important functional associations between pairs of proteins at submicroscopic resolution, all without destroying the cell 1 . More recently, techniques such as image correlation microscopy (ICM) and its variants have been widely used to characterize larger protein assemblies and clusters, including EGFR, in-situ2 , 3. Although all of these techniques are eminently useful, FRET is typically limited to detecting two very closely separated (< 5nm) molecules of different types1 , 4. While the various ICM methods can be used to evaluate associations and clustering between many molecules on the submicron scale, this additional information is an ensemble average and it does not directly reveal the distribution of cluster sizes or any additional information related to nanometer-scale organization of biomolecules forming the clusters2 , 3. Furthermore, ICM methods are in general highly sensitive to background interference, and the resolution of the imaging system is often not fully utilized. In addition, both FRET and ICM can be critically limited by photobleaching. These limitations are avoided in electron microscopy (EM), and in particular, the immuno-gold method to image assemblies of biomolecules at the nanometer length scale 5 . Howe...
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.
The dual-beam laser trap is a versatile tool with many possible applications. In order to characterize its thermal properties in a microfluidic trap geometry we have developed a non-intrusive fluorescence ratio technique using the temperature sensitive dye Rhodamine B and the temperature independent reference dye Rhodamine 110. We measured temperature distribution profiles in the trap with submicron spatial resolution on a confocal laser-scanning microscope. The maximum heating in the center of the trap amounts to (13 +/- 2) degrees C/W for a wavelength of lambda = 1064 nm and scales linearly with the applied power. The measurements correspond well with simulated temperature distributions.
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