Post-staining Raman analysis of histological sections following decolorization.

Hematoxylin and eosin (HE) staining of tissue sections is a powerful tool for observing changes in the tissue structure and is used as the most fundamental and vital technique in histology. However, xenobiotics such as polymers and inorganic or organic materials have low dyeability, making it difficult to observe the distribution of materials across tissues. Raman spectroscopy is an advantageous technique for identifying materials in tissues using spectroscopic fingerprints by laser irradiation without staining. In this study, we developed a combined method for morphological observation and Raman spectral acquisition on the identical tissue slide by employing a decolorization step to remove eosin-induced fluorescence in HE-stained samples. Our method eliminated the fluorescence background and allowed the identical-field pathological observation, enabling simultaneous identification of biological responses and materials in tissues.

Application of three-dimensional Raman imaging to determination of the relationship between cellular localization of diesel exhaust particles and the toxicity.

A diesel exhaust particle (DEP) is a type of particulate matter that is easily produced from combustion in a diesel power engine. It has been reported that DEPs can cause short- and long-term health problems. This is because DEPs are complex mixtures that are highly inhalable through the airways due to their small particle size. However, the relationship between intracellular localization of DEPs after their deposition in the lungs and the subsequent biological responses remains to be clarified. This is due to difficulties in distinguishing particles that are inside the cells from those that are outside. In this study, A549 human lung epithelial cells were exposed to DEPs at concentrations of 0, 25, 75, or 200 µg/mL for different periods, after that particles in the A549 cells were analyzed by three-dimensional (3D) images obtained from a Raman microscope. The cytotoxic effects of DEPs on the A549 cells were investigated by measuring cell viability, the levels of intracellular reactive oxygen species (ROS) and cell death. The Raman microscopy revealed that the particles invaded the A549 cells, and at a concentration of 200 µg/mL, they markedly decreased cell viability, increased intracellular ROS production, triggered late apoptosis/necrosis and induced nuclear damage. These results suggest that intracellular DEPs exposed at a high concentration may be highly toxic and can impair the viability of A549 cells. Furthermore, the 3D images from the Raman microscopy can be used to evaluate intracellular particle dynamics.

Role of necroptosis of alveolar macrophages in acute lung inflammation of mice exposed to titanium dioxide nanoparticles.

Titanium dioxide (TiO2) nanoparticles are indispensable for daily life but induce acute inflammation, mainly via inhalation exposure. TiO2 nanoparticles can be phagocytosed by alveolar macrophages (AMs) in vivo and cause necroptosis of exposed cells in vitro. However, the relationship between localization of TiO2 nanoparticles in the lungs after exposure and their biological responses including cell death and inflammation remains unclear. This study was conducted to investigate the intra/extracellular localization of TiO2 nanoparticles in murine lungs at 24 h after intratracheal exposure to rutile TiO2 nanoparticles and subsequent local biological reactions, specifically necroptosis of AMs and lung inflammation. We found that TiO2 exposure induced leukocyte migration into the alveolar region and increased the secretion of C-C motif ligand (CCL) 3 in the bronchoalveolar lavage (BAL) fluid. A combination of Raman spectroscopy and staining of cell and tissue samples confirmed that AMs phagocytose TiO2. AMs that phagocytosed TiO2 nanoparticles showed necroptosis, characterized by the expression of phosphorylated mixed lineage kinase domain-like protein and translocation of high mobility group box-1 from the cell nucleus to the cytoplasm. In primary cultured AMs, TiO2 also induced necroptosis and increased the secretion of CCL3. Necroptosis inhibitors suppressed the increase in CCL3 secretion in both the BAL fluid and culture supernatant of AMs and suppressed the increase in leukocytes in the BAL fluid. These data suggest that necroptosis of AMs that phagocytose TiO2 nanoparticles is involved as part of the mechanism by which TiO2 induces acute lung inflammation.

Bis(2-quinolylmethyl)ethylenediaminediacetic acids (BQENDAs), TQEN-EDTA hybrid molecules as fluorescent zinc sensors.

Molecular hybrids of TQEN (N,N,N',N'-tetrakis(2-quinolylmethyl)ethylenediamine) and EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid) were examined as fluorescent Zn(2+) sensors. Upon the addition of Zn(2+), N,N-BQENDA (N,N-bis(2-quinolylmethyl)ethylenediamine-N',N'-diacetic acid, 1a) exhibits a 30-fold emission enhancement at 456 nm (λex = 315 nm, ϕZn = 0.018) in buffer (HEPES, pH = 7.5, 100 mM KCl). The fluorescence enhancement is Zn(2+)-specific as Cd(2+) induces much smaller increases (ICd/I0 = 5 and ICd/IZn = 16%). These spectroscopic properties, as well as the excellent water-solubility, represent a significant improvement compared to the parent TQEN sensor (ϕZn = 0.007, ICd/IZn = 64%). The isoquinoline analog N,N-1-isoBQENDA (N,N-bis(1-isoquinolylmethyl)ethylenediamine-N',N'-diacetic acid, 1b) possesses a similar Zn(2+) fluorescence response to the parent 1-isoTQEN (N,N,N',N'-tetrakis(1-isoquinolylmethyl)ethylenediamine) sensor, but exhibits diminished fluorescence intensity. Apo 1a and 1b extract more than 50% of the Zn(2+) from an equimolar amount of [Zn(TPEN)](2+) (TPEN = N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine) or [Zn(EDTA)](2-), whereas TPEN and EDTA cannot effectively remove Zn(2+) from [Zn(1a)] and [Zn(1b)]. The reduction of steric crowding in [Zn(TQEN)](2+) resulting from the substitution of two quinolines with carboxylates enhances the interaction between the metal ion and the remaining quinoline nitrogen atoms. The stronger bonding interaction results in enhanced emission intensity, Zn(2+) selectivity and metal ion affinity. This is in contrast to [Zn(1-isoTQEN)](2+) where the isoquinoline-carboxylate replacement does not relieve any coordination distortion, therefore no significant changes in fluorescence or metal binding properties are observed.