Dynamic Imaging of Molecular Assemblies in Live Cells Based on Nanoparticle Plasmon Resonance Coupling

Abstract

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...

Plasmonic nanoparticles and their characterization in physiological fluids

“…An example of this is shown in Fig. 5 where DF hyperspectral imaging was used to follow the journey of Au NPs upon endocytosis: small clusters of NPs that scatter green light are present in early endosomes, before they are trafficked to late endosomes that scatter red light due to the formation of larger clusters [105].…”

Illuminating Epidermal Growth Factor Receptor Densities on Filopodia through Plasmon Coupling

“…Work from different groups has demonstrated that, due to the spectral shift in the spectrum of the scattered light, the near-field interactions between NP immunolabels at high ρ can be detected using conventional far-field optics. [24][25][26][27][28] The spectral shift in the ensemble-averaged NP resonance increases as the average interparticle separation between the NPs decreases with growing ρ.…”

“…Work from different groups has demonstrated that, due to the spectral shift in the spectrum of the scattered light, the near-field interactions between NP immunolabels at high ρ can be detected using conventional far-field optics. [24][25][26][27][28] The spectral shift in the ensemble-averaged NP resonance increases as the average interparticle separation between the NPs decreases with growing ρ.In this manuscript we quantify the relationship between the peak plasmon resonance wavelength (λ res ) and ρ for 40 nm diameter Au NP immunolabels targeted at EGFR on A431 cells. Immunolabeling strategies are commonly used to investigate receptor density distributions in the electron microscope, 8,29,30 where the spatial distribution of the NP labels is used to infer information about the local concentration of the targeted receptor.…”

Plasmonic nanoparticles track dynamic behavior of molecules in live cells

“…8,9,[14][15][16][17][18][19][20] The work we describe here expands and generalizes the application of nanoparticle plasmon-resonance coupling (NPRC) to monitor the dynamic behavior of large protein complexes in living cells. 21 This has important implications, as there is currently a dearth of physical methods for monitoring the behavior of large molecular complexes with high spatial and temporal resolution in living cells. We observed the trafficking mechanisms of the epidermal growth-factor receptor (EGFR), a key receptor tyrosine kinase that controls fundamental cellular processes such as DNA replication and division.…”