Multispectral Raman Differentiation of Malignant Skin Neoplasms In Vitro: Search for Specific Biomarkers and Optimal Wavelengths.

Confocal scanning Raman and photoluminescence (PL) microspectroscopy is a structure-sensitive optical method that allows the non-invasive analysis of biomarkers in the skin tissue. We used it to perform in vitro diagnostics of different malignant skin neoplasms at several excitation wavelengths (532, 785 and 1064 nm). Distinct spectral differences were noticed in the Raman spectra of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), compared with healthy skin. Our analysis of Raman/PL spectra at the different excitation wavelengths enabled us to propose two novel wavelength-independent spectral criteria (intensity ratios for 1302 cm-1 and 1445 cm-1 bands, 1745 cm-1 and 1445 cm-1 bands), related to the different vibrational "fingerprints" of cell membrane lipids as biomarkers, which was confirmed by the multivariate curve resolution (MCR) technique. These criteria allowed us to differentiate healthy skin from BCC and SCC with sensitivity and specificity higher than 95%, demonstrating high clinical importance in the differential diagnostics of skin tumors.

FT-IR Analysis of P. aeruginosa Bacteria Inactivation by Femtosecond IR Laser Radiation.

We report the successful inactivation of P. aeruginosa strain by femtosecond infrared (IR) laser radiation at the resonant wavelengths of 3.15 µm and 6.04 µm, chosen due to the presence of characteristic molecular vibrations in the main structural elements of the bacterial cells in these spectral ranges: vibrations of amide groups in proteins (1500-1700 cm-1), and C-H vibrations in membrane proteins and lipids (2800-3000 cm-1). The underlying bactericidal structural molecular changes were revealed by the stationary Fourier-transform IR spectroscopy, with the spectral peaks parameters being obtained by Lorentzian fitting with the hidden peaks revealed by the second derivative calculations, while no visible damage to the cell membranes was identified by scanning and transmission electron microscopy.

Direct Bactericidal Comparison of Metal Nanoparticles and Their Salts against S. aureus Culture by TEM and FT-IR Spectroscopy.

We report the bactericidal effect of Ag and Cu NPs with different concentrations on methicillin-resistant S. aureus strain in comparison to the effect of AgNO3 and CuCl2 solutions, characterized by microbiological tests, TEM and Fourier-transform infrared spectroscopy. NPs were produced by nanosecond laser ablation in distilled water and characterized by scanning electron microscopy, UV-vis, energy dispersive X-ray, FT-IR spectroscopy, as well as X-ray diffraction, dynamic light scattering size and zeta-potential measurements. Microbiological tests showed antibacterial activity of NPs and metal ion-containing salts. Comparative FT-IR spectroscopy of bacteria, treated with metal NPs and salts, showed the broadening of amide I and II bands, a CH2-related peak and its frequency decrease, indicating the increase of membrane fluidity. The main mechanisms of the antibacterial effect were proposed: Ag and Cu NPs release ions and ROS, which result in lipid peroxidation; AgNO3 forms precipitates on the cell surface, which lead to the mechanical rupture of the membrane and subsequent possible penetration of the precipitates in the emerged damaged spots, complete destruction of the membrane and bacterial death; Cu ions from the CuCl2 solution cause damage to phosphorus- and sulfur-containing biomolecules, which leads to disruption of intracellular biochemical processes. The theories were confirmed by FT-IR spectroscopy and TEM.

Spectrally-selective mid-IR laser-induced inactivation of pathogenic bacteria.

Micrometer-thick layers of Pseudomonas aeruginosa bacteria were prepared on fluorite substrates and scanned by focused mid-IR femtosecond laser radiation that was spectrally tuned to achieve the selective excitation of either the stretching C-H vibrations (3 µm), or stretching C = O, C-N vibrations (6 µm) of the amide groups in the bacteria. The enhanced biocidal efficiency of the latter selective excitation, compared to the more uniform 3-µm laser excitation, was demonstrated by performing viability assays of laser-treated bacterial layers. The bacterial inactivation by the 6-µm ultrashort laser pulses is attributed to dissociative denaturation of lipids and proteins in the cell membranes and intra-cell nucleic acids.

Few Percent Efficient Polarization-Sensitive Conversion in Nonlinear Plasmonic Interactions Inside Oligomeric Gold Structures.

The backscattering spectra of a 500 nm thick gold film, which was excited near the 525 nm transverse localized plasmon resonance of its constituent, self-organized, vertically-aligned nanorods by normally incident 515 nm, 300 fs laser pulses with linear, radial, azimuthal and circular polarizations, revealed a few-percent conversion into Stokes and anti-Stokes side-band peaks. The investigation of these spectral features based on the nanoscale characterization of the oligomeric structure and numerical simulations of its backscattering response indicated nonlinear Fano-like plasmonic interactions, particularly the partially degenerate four-wave mixing comprised by the visible-range transverse plasmon resonance of the individual nanorods and an IR-range collective mode of the oligomeric structure. Such oligomeric structures in plasmonic films may greatly enhance inner nonlinear electromagnetic interactions and inner near-IR hotspots, paving the way for their engineered IR tunability for broad applications in chemosensing and biosensing.

Polarization-Sensitive Surface-Enhanced In Situ Photoluminescence Spectroscopy of S. aureus Bacteria on Gold Nanospikes.

We report the possibility of a time-resolved bacterial live/dead dynamics observation with the use of plasmonic nanospikes. Sharp nanospikes, fabricated on a 500-nm thick gold film by laser ablation with the use of 1030-nm femtosecond pulses, were tested as potential elements for antibacterial surfaces and plasmonic luminescence sensors. Staphylococcus aureus bacteria were stained by a live/dead viability kit, with the dead microorganisms acquiring the red colour, caused by the penetration of the luminescent dye propidium iodide through the damaged cell membrane. Photoluminescence was pumped by 515-nm femtosecond laser pulses with linear (Gaussian beam), circular, azimuthal and radial (Laguerre-Gaussian beam) polarizations, exciting the transverse plasmon resonance of the nanospikes and their apex lightning-rod near-field. According to the numerical electrodynamic modeling, the observed strong increase in the photoluminescence yield for radial polarization, while slightly lower for circular and azimuthal polarizations, compared with the low luminescence intensities for the linear laser polarization, was related to their different laser-nanospike coupling efficiencies.

Surface-Enhanced IR-Absorption Microscopy of Staphylococcus aureus Bacteria on Bactericidal Nanostructured Si Surfaces.

Surface-enhanced IR absorption (SEIRA) microscopy was used to reveal main chemical and physical interactions between Staphylococcus aureus bacteria and different laser-nanostructured bactericidal Si surfaces via simultaneous chemical enhancement of the corresponding IR-absorption in the intact functional chemical groups. A cleaner, less passivated surface of Si nanoripples, laser-patterned in water, exhibits much stronger enhancement of SEIRA signals compared to the bare Si wafer, the surface coating of oxidized Si nanoparticles and oxidized/carbonized Si (nano) ripples, laser-patterned in air and water. Additional very strong bands emerge in the SEIRA spectra on the clean Si nanoripples, indicating the potential chemical modifications in the bacterial membrane and nucleic acids during the bactericidal effect.

Nanosecond-Laser Generation of Nanoparticles in Liquids: From Ablation through Bubble Dynamics to Nanoparticle Yield.

A comprehensive picture of the nanosecond-laser generation of colloidal nanoparticles in liquids is nowadays the demand of their high-throughput industrial fabrication for diverse perspective biomedical, material science, and optoelectronic applications. In this study, using silicon as an example, we present a self-consistent experimental visualization and theoretical description of key transient stages during nanosecond-laser generation of colloidal nanoparticles in liquids: plasma-mediated injection of ablated mass into the liquid and driving the vapor bubble, finalized by the colloid appearance in the liquid. The explored fundamental transient stages envision the basic temporal and spatial scales, as well as laser parameter windows, for the demanded high-throughput nanosecond-laser generation of colloidal nanoparticles in liquids.