Accurate Quantification and Imaging of Cellular Uptake Using Single-Particle Surface-Enhanced Raman Scattering.

Understanding the uptake, distribution, and stability of gold nanoparticles (NPs) in cells is of fundamental importance in nanoparticle sensors and therapeutic development. Single nanoparticle imaging with surface-enhanced Raman spectroscopy (SERS) measurements in cells is complicated by aggregation-dependent SERS signals, particle inhomogeneity, and limited single-particle brightness. In this work, we assess the single-particle SERS signals of various gold nanoparticle shapes and the role of silica encapsulation on SERS signals to develop a quantitative probe for single-particle level Raman imaging in living cells. We observe that silica-encapsulated gap-enhanced Raman tags (GERTs) provide an optimized probe that can be quantifiable per voxel in SERS maps of cells. This approach is validated by single-particle inductively coupled mass spectrometry (spICP-MS) measurements of NPs in cell lysate post-imaging. spICP-MS also provides a means of measuring the tag stability. This analytical approach can be used not only to quantitatively assess nanoparticle uptake on the cellular level (as in previous digital SERS methods) but also to reliably image the subcellular distribution and to assess the stability of NPs in cells.

A Wide-Field Imaging Approach for Simultaneous Super-Resolution Surface-Enhanced Raman Scattering Bioimaging and Spectroscopy.

High spatial resolution imaging and chemical-specific detection in living organisms is important in a wide range of fields from medicine to catalysis. In this work, we characterize a wide-field surface-enhanced Raman scattering (SERS) imaging approach capable of simultaneously capturing images and SERS spectra from nanoparticle SERS tags in cancer cells. By passing the image through a transmission diffraction grating before it reaches an array detector, we record the image and wavelength dispersed signal simultaneously on the camera sensor. Optimization of the experiment provides an approach with better spectral resolution and more rapid acquisition than liquid crystal tunable filters commonly used for wide-field SERS imaging. Intensity fluctuations inherent to SERS enabled localization algorithms to be applied to both the spatial and spectral domain, providing super-resolution SERS images that are correlated with improved peak positions identified in the spectrum of the SERS tag. The detected Raman signal is shown to be sensitive to the focal plane, providing three-dimensional (3D) sectioning abilities for the detected nanoparticles. Our work demonstrates spectrally resolved super-resolution SERS imaging that has the potential to be applied to complex physical and biological imaging applications.

Determination of lentiviral titer by surface enhanced Raman scattering.

Lentiviruses are commonly used to deliver genetic code into host cells for biomedical applications, such as gene therapy, pharmaceuticals, and vaccine development. Knowing the infectious titer of these virus particles is critical for development in these areas. Current methods of determining viral titer often require cell culture, where a cell is infected and the inserted genetic code is expressed in a known number of cells, which can require days or weeks to prepare and analyze samples. To provide a more rapid method of determining viral titer, the use of surface enhanced Raman spectroscopy (SERS) was explored. SERS provides both chemical and structural information by using plasmonic metallic nanostructures to amplify the Raman signal. Two different lentiviruses, one with a vector encoding a GFP gene and the same virus without the GFP gene included, were analyzed by SERS in viral production media at various concentrations. The SERS response was demonstrated to be sensitive to the incorporation of the GFP gene into the viral vector. Chemometric analysis using multivariate curve resolution (MCR) was able to identify a component in the observed SERS spectra that correlated with the concentration of GFP containing virus particles. Using the MCR model and the SERS response, the viral titer of lentivirus encoding for GFP was determined. The viral titer determined by SERS agreed well with expression of the GFP in infected cells. The SERS response using different metals and excitation wavelengths was also explored. Overall, this work demonstrates the utility of SERS for rapid determination of lentiviral titer.

In Vitro Imaging of Lycopene Delivery to Prostate Cancer Cells.

The ability to monitor the uptake and distribution of food nutrients in in vitro cell culture models is key to understanding the efficacy of these nutraceuticals to treat and prevent disease. Lycopene is a carotenoid found in chloroplasts and chromoplasts of tomatoes, providing the familiar red color, and a bioactive that inhibits prostate carcinogenesis. We employed live-cell Raman microscopy to visualize lycopene delivery from tween 80 micelles into PC-3 prostate cancer cells. The tween 80 micelle provides a mimic of natural lipoprotein complexes that deliver lycopene in vivo, overcomes the low aqueous solubility of lycopene and challenges replicating physiological uptake to cells, and provides a stable signal to assess cellular uptake of the nutraceutical formulation. The Raman images indicate subcellular localization of the lycopene within the cells. The lycopene Raman signal is resonantly enhanced at an excitation wavelength of 532 nm, providing a convenient, sensitive, and label-free technique to detect and quantify lycopene uptake in living cells. Analysis of the acquired Raman spectra in the maps determines the concentration of lycopene at each point in the cell. In addition to the expected lycopene Raman signal, Raman scattering from the tween 80 vehicle is also mapped in the cells. The Raman data correlates with scattering features observed in darkfield microscopy images of the cells, which display the cell membrane and other features for reference. Overall, the Raman maps indicate lycopene likely accumulates in lipid membranes of cytoplasmic organelles.

Spectrally Resolved Surface-Enhanced Raman Scattering Imaging Reveals Plasmon-Mediated Chemical Transformations.

Challenges investigating molecules on plasmonic nanostructures have limited understanding of these interactions. However, the chemically specific information in the surface-enhanced Raman scattering (SERS) spectrum can identify perturbations in the adsorbed molecules to provide insight relevant to applications in sensing, catalysis, and energy conversion. Here, we demonstrate spectrally resolved SERS imaging, to simultaneously image and collect the SERS spectra from molecules adsorbed on individual nanoparticles. We observe intensity and frequency fluctuations in the SERS signal on the time scale of tens of milliseconds from n-mercaptobenzoic acid (MBA) adsorbed to gold nanoparticles. The SERS signal fluctuations correlate with density functional theory calculations of radicals generated by the interaction between MBA and plasmon-generated hot electrons. Applying localization microscopy to the data provides a super-resolution spectrally resolved map that indicates the plasmonic-induced molecular charging occurs on the extremities of the nanoparticles, where the localized electromagnetic field is reported to be most intense.

Comparison of 4-Mercaptobenzoic Acid Surface-Enhanced Raman Spectroscopy-Based Methods for pH Determination in Cells.

Measurements of cellular pH are used to infer information such as stage of cell cycle, presence of cancer and other diseases, as well as delivery or effect of a therapeutic drug. Surface-enhanced Raman spectroscopy (SERS) of nanoparticle-based pH probes have been used to interrogate intracellular pH, with the significant advantage of avoiding photobleaching compared to fluorescent indicators. 4-Mercaptobenzoic acid (MBA) is a commonly used pH-sensitive reporter molecule. Intracellular pH sensing by SERS requires analysis of the observed MBA spectrum and spectral interference can affect the pH determination. Background from common cell containers, imaging too few particles, signal-to-noise ratios, and degradation of reporter molecules are among the factors that may alter appropriate SERS-based pH determination in cells. Here, we have compared common methods of spectral analysis to see how different factors alter the calculated pH in Raman maps of MBA functionalized Au nanostars in SW620 cancer cells. The methods included in our comparison use the relative intensity of the ν(COO-) stretch, chemometric analysis of the ν8a mode, and analyzing the frequency shift of the ν8a mode. These methods show different sensitivity to some of these sources of error in live cell experiments. pH determination based on Raman frequency shift appears to give a more reliable pH determination, though in high signal-to-noise environments, intensity ratios may provide better sensitivity to small changes in pH for cellular imaging.

Protein corona-resistant SERS tags for live cell detection of integrin receptors.

Chemical signals are conveyed to cells through ligand-receptor binding, triggering cascades of biochemical reactions and resulting in pivotal cellular functions. These binding events are important in understanding membrane signaling and drug interactions. To probe ligand-receptor binding, surface enhanced Raman scattering (SERS) tags are a promising tool. SERS tags are plasmonic nanostructures functionalized with a protective coating, a Raman reporter molecule, and a biorecognition element. In biological fluids, native proteins have affinity for bare nanoparticles and form a protein corona. SERS tags have a protective shell which eliminates this complication. It is important to analyze ligand-receptor binding with SERS tags in live cells since cell fixatives alter protein structure, leading to spectral changes and data misinterpretation. In this study, we synthesized a novel SERS tag by creating a mixed monolayer of the small cyclic arginine-glycine-aspartic acid-phenylalanine-cysteine (RGDFC) peptide and 4-mercaptobenzonitrile (MBN) on the surface of spherical gold nanoparticles (Au NP). Au-RGDFC-MBN NP showed resistance to PC formation and were successfully detected in both fixed and living human metastatic colon cancer cells.