Nerve-sparing surgery is essential to avoid functional deficits of the limbs and organs. Raman scattering, a label-free, minimally invasive, and accurate modality, is one of the best candidate technologies to detect nerves for nerve-sparing surgery. However, Raman scattering imaging is too time-consuming to be employed in surgery. Here we present a rapid and accurate nerve visualization method using a multipoint Raman imaging technique that has enabled simultaneous spectra measurement from different locations (n=32) of a sample. Five sec is sufficient for measuring n=32 spectra with good S/N from a given tissue. Principal component regression discriminant analysis discriminated spectra obtained from peripheral nerves (n=863 from n=161 myelinated nerves) and connective tissue (n=828 from n=121 tendons) with sensitivity and specificity of 88.3% and 94.8%, respectively. To compensate the spatial information of a multipoint-Raman-derived tissue discrimination image that is too sparse to visualize nerve arrangement, we used morphological information obtained from a bright-field image. When merged with the sparse tissue discrimination image, a morphological image of a sample shows what portion of Raman measurement points in arbitrary structure is determined as nerve. Setting a nerve detection criterion on the portion of “nerve” points in the structure as 40% or more, myelinated nerves (n=161) and tendons (n=121) were discriminated with sensitivity and specificity of 97.5%. The presented technique utilizing a sparse multipoint Raman image and a bright-field image has enabled rapid, safe, and accurate detection of peripheral nerves.
Raman microscopy enables a sensitive, label-free molecular imaging of cells. Employing deep-UV (DUV) light for Raman excitation allows selective measurement of nucleotide bases and aromatic amino acids in a cell, without spectral overlapping of components with a large quantity (i.e. lipid, peptide), because their Raman scattering are specifically enhanced due to the resonance effect. To implement DUV resonance Raman imaging of cells, I previously established a home-built Raman microscope equipped with a DUV laser (λ = 257.2 nm). Raman image representing the distribution of cellular nucleic acid can be reconstructed with the intensity of a Raman band selectively assigned to adenine and guanine. Unfortunately, DUV resonance Raman imaging of cells is severely hindered by molecular photodegradation that occurs after a molecule absorbs DUV light during Raman measurement, precluding a high signal-to-noise ratio and repetitive measurement. To address this issue, I developed a technique for molecular protection under DUV exposure; the trivalent ions of lanthanide group including terbium, europium, and thulium could significantly suppress the molecular photodegradation by relaxing the DUV-excited molecules. The buffer solution containing any of these lanthanide ions with the concentration of 100 µM or higher could provide less destruction of the cellular structures, including nucleotide bases, than the one without the ions, under DUV exposure. Utilizing such protective effects of the lanthanide ions, I successfully achieved a twice higher signal-to-noise ratio and repetitive DUV Raman imaging of cells.
Deep-UV (DUV) plasmonics can expand the possibilities of DUV-based techniques (i.e. UV lithography, UV spectroscopy, UV imaging, UV disinfection). Here we present that indium is useful for research of DUV plasmonics. According to dielectric function, indium and aluminum are low-loss, DUV plasmonic metals, of which the imaginary parts are far smaller than those of other metals (i.e. rhodium, platinum) in the DUV range. Additionally, the real parts in the whole DUV range are close to but smaller than -2, allowing efficient generation of surface plasmon polaritons on an indium or aluminum nanosphere. In comparison to aluminum, indium provides a distinctive feature for fabricating DUV-resonant substrates. It is highly apt to form a grainy deposition film on a standard, optically transparent substrate (i.e. fused silica). The surface plasmon resonance wavelength becomes promptly tailored by simply varying the deposition thickness of the films, resulting in different grain sizes. Thus, we fabricated indium-coated substrates having different plasmon resonance wavelengths by varying the deposition thicknesses from 10 to 50 nm. DUV resonance Raman scattering of adenine molecules was best enhanced using the 25 nm deposition thickness substrates by the factor of 2. Furthermore, the FDTD calculation simulated the electromagnetic field enhancement over a grainy, indium-coated fused silica substrate. Both results indicate how indium plays an indispensable role in study of DUV plasmonics.
We report the first demonstration of deep ultraviolet (DUV) Raman imaging of a cell. Nucleotide distributions in a HeLa cell were observed without any labeling at 257 nm excitation with resonant bands attributable to guanine and adenine. Obtained images represent DNA localization at nucleoli in the nucleus and RNA distribution in the cytoplasm. The presented technique extends the potential of Raman microscopy as a tool to selectively probe nucleic acids in a cell with high sensitivity due to resonance.