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Surface-enhanced Raman spectroscopy (SERS), as induced by noble metal nanoparticles, is used for medical applications. In this study, gold nanoparticle parameters such as size, shape, separation, substrate etc. are varied systematically using electron beam lithographic fabrication methods. The resultant Raman spectra intensities are correlated with the nanoparticle surface plasmon energies as determined by electron energy loss spectroscopy (EELS) in a scanning transmission electron microscope (STEM) at nanometer-scale resolution. It is found that the largest enhancement is achieved when the illuminating laser energy closely matches, or is slightly higher than, the surface plasmon energies. In general, nanoparticle size is a strong determinant factor, and higher intensities are generated by sharply defined features. This approach allows a procedure to identify the optimum combination of nanoparticle parameters and laser energy to generate the largest Raman signals, thus enabling detection, for instance, of smaller, early-stage cancer tumors.

Background: Recent evidence suggests that the accumulation of iron, specifically ferrous Fe2+, may play a role in the development and progression of neurodegeneration in Alzheimer’s disease (AD) through the production of oxidative stress. Objective: To localize and characterize iron deposition and oxidation state in AD, we analyzed human hippocampal autopsy samples from four subjects with advanced AD that have been previously characterized with correlative MRI-histology. Methods: We perform scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and electron energy loss spectroscopy (EELS) in the higher resolution transmission electron microscope on the surface and cross-sections of specific iron-rich regions of interest. Results: Specific previously analyzed regions were visualized using SEM and confirmed to be iron-rich deposits using EDS. Subsequent analysis using focused ion beam cross-sectioning and SEM characterized the iron deposition throughout the 3-D volumes, confirming the presence of iron throughout the deposits, and in two out of four specimens demonstrating colocalization with zinc. Analysis of traditional histology slides showed the analyzed deposits overlapped both with amyloid and tau deposition. Following higher resolution analysis of a single iron deposit using scanning transmission electron microscope (STEM), we demonstrated the potential of monochromated STEM-EELS to discern the relative oxidation state of iron within a deposit. Conclusion: These findings suggest that iron is present in the AD hippocampus and can be visualized and characterized using combined MRI and EM techniques. An altered relative oxidation state may suggest a direct link between iron and oxidative stress in AD. These methods thus could potentially measure an altered relative oxidation state that could suggest a direct link between iron and oxidative stress in AD. Furthermore, we have demonstrated the ability to analyze metal deposition alongside commonly used histological markers of AD pathology, paving the way for future insights into the molecular interactions between Aβ, tau, iron, and other putative metals, such as zinc.

Current clinical brain tumour therapy practices are based on tumour resection and post-operative chemotherapy or X-ray radiation. Resection requires technically challenging open-skull surgeries that can lead to major neurological deficits and, in some cases, death. Treatments with X-ray and chemotherapy, on the other hand, cause major side-effects such as damage to surrounding normal brain tissues and other organs. Here we report the development of an integrated nanomedicine-bioelectronics brain-machine interface that enables continuous and on-demand treatment of brain tumours, without open-skull surgery and toxicological side-effects on other organs. Near-infrared surface plasmon characteristics of our gold nanostars enabled the precise treatment of deep brain tumours in freely behaving mice. Moreover, the nanostars' surface coating enabled their selective diffusion in tumour tissues after intratumoral administration, leading to the exclusive heating of tumours for treatment. This versatile remotely controlled and wireless method allows the adjustment of nanoparticles' photothermal strength, as well as power and wavelength of the therapeutic light, to target tumours in different anatomical locations within the brain.

The ability to tune the localized surface plasmon resonance (LSPR) of nanostructures is desirable for surface enhanced Raman spectroscopy (SERS), plasmon-assisted chemistry and other nanophotonic applications. Although historically the LSPR is mainly studied by optical techniques, with the recent advancement in electron monochromators and correctors, it has attracted considerable attention in transmission electron microscopy (TEM). Here, we use electron energy loss spectroscopy (EELS) in a scanning TEM to study individual gold nanodiscs and bowties in lithographic arrays with variable LSPRs by adjusting the size, interspacing, shape and dielectric environment during the nanofabrication process. We observe the strongest Raman signal enhancement when the LSPR frequency is close to the incident laser frequency in Raman spectroscopy. A simplified harmonic oscillator model is used to estimate SERS enhancement factor (EF) from EELS, bridging the connection between electron and photon excitation of plasmonic arrays. This work demonstrates that STEM-EELS shows promise for revealing the contributions of specific LSPR modes to SERS EF. Our results provide guidelines to fine-tune nanoparticle parameters to deliver the maximum signal enhancement in biosensing applications, such as early cancer detection.

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging.

Abnormal accumulation of inorganic trace elements in a human brain, such as iron, zinc and aluminium, oftentimes manifested as deposits and accompanied by a chemical valence change, is pathologically relevant to various neurodegenerative diseases. In particular, Fe(2+) has been hypothesized to produce free radicals that induce oxidative damage and eventually cause Alzheimer’s disease (AD). However, traditional biomedical techniques, e.g. histology staining, are limited in studying the chemical composition and valence states of these inorganic deposits. We apply commonly used physical (phys-) science methods such as X-ray energy dispersive spectroscopy (EDS), focused-ion beam (FIB) and electron energy loss spectroscopy (EELS) in transmission electron microscopy in conjunction with magnetic resonance imaging (MRI), histology and optical microscopy (OM) to study the valence states of iron deposits in AD patients. Ferrous ions are found in all deposits in brain tissues from three AD patients, constituting 0.22–0.50 of the whole iron content in each specimen. Such phystechniques are rarely used in medical science and have great potential to provide unique insight into biomedical problems.

Gold plasmonic nanostructures with several different adhesion layers have been studied with monochromated electron energy loss spectroscopy in the scanning transmission electron microscope (STEM-EELS) and with surface enhanced Raman spectroscopy (SERS). Compared to samples with no adhesion layer, those with 2nm of Cr or Ti show broadened, lower intensity plasmon peaks as measured with EELS. This broadening is observed in both optically active (“bright”) and inactive (“dark”) plasmon modes. When the former are probed with SERS, the signal enhancement factor is lower for samples with Cr or Ti, another indication of reduced plasmon resonance. This work illustrates the capability of STEM-EELS to provide direct near-field measurement of changes in plasmon excitation probability with nano-scale spatial resolution. Additionally, it demonstrates that applications which require high SERS enhancement, such as biomarker detection and cancer diagnostics, can be improved by avoiding the use of a metallic adhesion layer.

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 μL) of blood.

High resolution electron microscopy has been applied to study the structure of metallic nanoparticles. These have sparked considerable interest as contrast agents in the field of biological imaging, including in magnetic resonance imaging (MRI) and computed tomography (CT). Here, we describe a method of synthesizing sub-10nm superparamagnetic metal and alloy nanoparticles by reduction of metallic salts. Annealing at 900°C in a methane/hydrogen environment forms a thin graphitic-carbon shell which is expected to improve stability, biocompatibility, and functionalization. Subsequent high resolution electron microscopy verifies graphitization and allows for crystallographic analysis. Most particles consist of single crystals in the phase predicted for the bulk material at the annealing temperature. Electron energy loss spectroscopy, energy dispersive X-ray spectroscopy and lattice constant measurements show large variation in composition for alloy nanoparticles from a single synthesis. The magnetization relaxation time (T2) measurements demonstrate that Fe and AuFe nanoparticles compete with commercially available iron oxide MRI contrast agents. X-ray attenuation measurements of an AuFe alloy nanoparticle solution gave a relative radiodensity of 280 Hounsfield Units, demonstrating promise as a dual-purpose contrast agent in CT and MRI. Long term stability in an atmospheric environment was also tested, with no signs of corrosion or oxidation after several years of storage.

Surface Enhanced Raman Spectroscopy (SERS) is a powerful technique for detection of small concentrations of molecules which makes use of inelastic scattering of light near nanostructured metal surfaces. It has been widely proposed that the scattering efficiency is improved by a local enhancement in the electric field from surface plasmons in the noble metal particles [1]. In spite of this, there has been no definitive correlation between localized surface plasmon resonances, SERS enhancement and morphology. [2] The approach herein outlines a procedure to produce repeatable and adjustable arrays of metallic nanoparticles via electron beam lithography for analysis by optical and electron techniques. The procedure allows for controlled adjustment of particular morphological feature while holding all others constant. By modifying a single feature at a time, the effect on both plasmon frequency and SERS intensity can be established. Synthesis is performed using a JEOL 6300 electron beam (e-beam) lithography tool. This procedure uses an electron beam to trace out a programmed pattern on a layer of poly(methyl methacrylate) [an electron beam sensitive positive resist] with 8nm resolution, which is then developed into a mask for nanoparticle deposition. The latter are generated by evaporating a 2nm Cr layer followed by 20 to 30nm of Au onto the substrate and lifting off the mask by dissolving the remaining PMMA in acetone. This process gives control over not only nanoparticle size and shape, but also orientation of the nanoparticles and periodicity of the array. Both SEM and AFM imaging were carried out. Characterization is important to assess the quality of the lithography and the repeatability of the synthesized structures. SEM imaging was performed using an FEI Magellan 400 XHR. Images were taken at 3 to 25kV and 25 to 100pA current. Figure 1 shows a series of SEM images of disk shaped nanoparticles fabricated with this technique. The grain structure is visible in the nanoparticles, demonstrating that they are polycrystalline. Figure 1 Low and high magnification SEM images of an array of Au disks fabricated by e-beam lithography. Two hundred disk shaped nanoparticles were measured from SEM images to determine the control and reproducibility of patterning. The radius averaged 73 nm with a standard deviation of 3 nm, compared to an aim of 74 nm. In another study using equilateral triangular nanoparticles (Figure 2) of side length 72±4 nm (75nm aim), corner radii were measured at 6±2 nm. Nanoparticle gap sizes below 20nm were consistently achievable. Figure 2 Array of “bow-tie” nanoparticles showing small gaps between them. AFM was used to characterize the height of the nanoparticles. While convolution of the tip and the nanoparticle reduces in-plane resolution, AFM is a widely used tool for height mapping. The AFM results (Figure 3) show an average height difference from peak to trough of 33 nm, which is very close to the aim of 2nm Cr plus 30nm Au. Figure 3 AFM image used to measure particle height. Greyscale colormap shows relative height in nm of a 1µm × 1µm section of particles. This study demonstrates that electron beam lithography, as expected, is a powerful technique for creating arrays of tunable nanoparticles, with controlled pitch and orientation relative to one another, in order to gain a better understanding of the relationship between nanoparticle structure and SERS properties. Subsequent work includes deposition of nanoparticles directly onto TEM grids for analysis of the localized surface plasmons by electron energy loss spectroscopy [3]. This work sets the foundation for this type of fabrication and characterization.