Parkinson's disease (PD) is a severe neurological disorder that affects more than 1 million people in the U.S. alone. A hallmark of PD is the formation of intracellular α-synuclein (α-Syn) protein aggregates called Lewy bodies (LBs). Although this protein does not have a particular localization in the central neural system, α-Syn aggregates are primarily found in certain areas of the midbrain, hypothalamus, and thalamus. Microscopic analysis of LBs reveals fragments of lipidrich membranes, organelles, and vesicles. These and other pieces of experimental evidence suggest that α-Syn aggregation can be triggered by lipids. In this study, we used atomic force microscope infrared spectroscopy (AFM-IR) to investigate the structural organization of individual α-Syn oligomers grown in the presence of two different phospholipids vesicles. AFM-IR is a modern optical nanoscopy technique that has single-molecule sensitivity and subdiffraction spatial resolution. Our results show that α-Syn oligomers grown in the presence of phosphatidylcholine have a distinctly different structure than oligomers grown in the presence of phosphatidylserine. We infer that this occurs because of specific charges adopted by lipids, which in turn governs protein aggregation. We also found that the protein to phospholipid ratio has a substantial impact on the structure of α-Syn oligomers. These findings demonstrate that α-Syn is far more complex than expected from the perspective of the structural organization of oligomeric species.

Viruses are infections species that infect a large spectrum of living systems. Although displaying a wide variety of shapes and sizes, they are all composed of nucleic acid encapsulated into a protein capsid. After virions enter the host cell, they replicate to produce multiple copies of themselves. They then lyse the host, releasing virions to infect new cells. The high proliferation rate of viruses is the underlying cause of their fast transmission among living species. Although many viruses are harmless, some of them are responsible for severe diseases such as AIDS, viral hepatitis, and flu. Traditionally, electron microscopy is used to identify and characterize viruses. This approach is time- and labor-consuming, which is problematic upon pandemic proliferation of previously unknown viruses, such as H1N1 and COVID-19. Herein, we demonstrate a novel diagnosis approach for label-free identification and structural characterization of individual viruses that is based on a combination of nanoscale Raman and infrared spectroscopy. Using atomic force microscopy–infrared (AFM-IR) spectroscopy, we were able to probe structural organization of the virions of Herpes Simplex Type 1 viruses and bacteriophage MS2. We also showed that tip-enhanced Raman spectroscopy (TERS) could be used to reveal protein secondary structure and amino acid composition of the virus surface. Our results show that AFM-IR and TERS provide different but complementary information about the structure of complex biological specimens. This structural information can be used for fast and reliable identification of viruses. This nanoscale bimodal imaging approach can be also used to investigate the origin of viral polymorphism and study mechanisms of virion assembly.

Abrupt aggregation of α-synuclein (α-Syn) leads to a formation of highly toxic protein oligomers. These aggregates are the underlying molecular cause of an onset of the irreversible degeneration of dopaminergic neurons in midbrain, hypothalamus, and thalamus, a pathology known as Parkinson's disease. The transient nature of oligomers, as well as their structural and morphological heterogeneity, limits the use of cryo-electron microscopy and solid-state NMR, classical tools of structural biology, for elucidation of their secondary structure. Despite this limitation, numerous pieces of experimental evidence suggest that phospholipids can uniquely alter the structure and toxicity of oligomers. In this study, we utilize an innovative nano-infrared imaging technique, also known as atomic force microscopy infrared (AFM-IR) spectroscopy, to examine the structure of individual α-Syn oligomers grown in the presence of phosphatidylcholine (α-Syn:PC) and phosphatidylserine (α-Syn:PS). We determined the amount of the parallel and the antiparallel β-sheets, as well as the amount the αhelix and the unordered protein, in the secondary structure of α-Syn:PC and α-Syn:PS formed at day 2 (D2), 8 (D8), and 15 (D15) after initiation of protein aggregation. We found a gradual decrease in the amount of the parallel β-sheet in both α-Syn:PC and α-Syn:PS from D2 to D15 together with an increase in the α-helix and the unordered protein secondary structure. We infer that this is due to the presence of lipids in the structure of oligomers that prevent an expansion of the parallel β-sheet upon interaction of the oligomers with monomeric α-Syn.

Atomic force microscopy infrared (AFM-IR) spectroscopy is an emerging analytical technique that can be used to probe the structural organization of specimens with nanometer spatial resolution. A growing body of evidence suggests that nanoscale structural analysis of very small (<10 nm) biological objects, such as viruses and amyloid aggregates, requires substrates that must fit strict criteria of low surface roughness and low IR background, simultaneously. In this study, we examine the suitability of a broad range of substrates commonly used in AFM and IR fields, and we determined that silicon, zinc sulfide, and calcium fluoride are the most ideal substrates for nanoscale imaging of amyloid oligomers, protein aggregates that are directly linked to the onset and progression of neurodegenerative diseases. Our data show that these substrates provide the lowest roughness and the lowest background in the 800− 1800 cm −1 spectral window from all examined AFM and IR substrates. We also investigate a contribution of surface enhancement in AFM-IR by the direct comparison of signal intensities from oligomers located on silicon and gold-coated silicon surfaces. We found that metallization of such substrates provides a factor of ∼7 enhancements to the IR signal and induces an equivalent enhancement of the sample background in the 950−1250 cm −1 spectral region.

Biotic and abiotic stresses cause substantial changes in plant biochemistry. These changes are typically revealed by high-performance liquid chromatography (HPLC) and mass spectroscopy-coupled HPLC (HPLC-MS). This information can be used to determine underlying molecular mechanisms of biotic and abiotic stresses in plants. A growing body of evidence suggests that changes in plant biochemistry can be probed by Raman spectroscopy, an emerging analytical technique that is based on inelastic light scattering. Non-invasive and non-destructive detection and identification of these changes allow for the use of Raman spectroscopy for confirmatory diagnostics of plant biotic and abiotic stresses. In this study, we couple HPLC and HPLC-MS findings on biochemical changes caused by Candidatus Liberibacter spp. (Ca. L. asiaticus) in citrus trees to the spectroscopic signatures of plant leaves derived by Raman spectroscopy. Our results show that Ca. L. asiaticus cause an increase in hydroxycinnamates, the precursors of lignins, and flavones, as well as a decrease in the concentration of lutein that are detected by Raman spectroscopy. These findings suggest that Ca. L. asiaticus induce a strong plant defense response that aims to exterminate bacteria present in the plant phloem. This work also suggests that Raman spectroscopy can be used to resolve stress-induced changes in plant biochemistry on the molecular level.