Sota Takanezawa

Two-Dimensional Raman Correlation Analysis of Diseased Esophagus in a Rat

Generalized two-dimensional (2D) Raman correlation analysis effectively distinguished a benign tumor from normal tissue. Line profiling Raman spectra of a rat esophagus, including a benign tumor, were measured and the generalized 2D synchronous and asynchronous spectra were calculated. In the autocorrelation area of the amide I band of proteins in the asynchronous map, a cross-like pattern was observed. A simulation study indicated that the pattern was caused by a sharp band component in the amide I band region. We considered that the benign tumor corresponded to the sharp component.

Verifying of endocrine disruptor chemical affect to the mouse testes: can raman spectroscopy support histology study?

One of suspect environmental endocrine disruptors that affect mouse male reproduction by altering the morphology of Sertoli cells and spermatogenic cells is phthalate. The effects of mono(2-ethylhexyl)phthalate (MEHP), one of metabolites of di(2-ethylhexyl)phthalate , on immature mouse testes in vivo were examined. We have recently shown that MEHP induced Sertoli cells necrosis and spermatogenic cells apoptosis in mice by TUNEL method, F-actin staining, and ultrastructural study, but there is no data for biochemical changing of testes due to those methods could not explore. To verify in detail of it, we conducted Raman spectroscopy study with 785 nm wavelength laser line, 50mW of laser power and 3 minutes of exposure time to analysis the MEHP-treated testicular tissue, which has been fixatived by 4% paraformaldehyde (PFA). Five weeks old (5 w.o) male mice were used in this experiment. As the results, the alterations were observed by Raman spectroscopy that there are significantly differences of DNA, actin filament, type IV collagen and amide I between control group (0 μM MEHP) and treatment group (100 μM MEHP). These results significantly support histology staining observation (such as the apoptotic spermatogenic cells which is associated with DNA fragmentation and F-actin disruption) and ultrastructural observation (such as mitochondria rupture and disintegration of nucleus membrane). Raman spectroscopy can be used for 4% PFA-fixatived tissue observation. However, we recommend that Raman spectroscopy may be able to be expanded as an armamentarium not just for the clarification of histology staining and ultrastructural study, but furthermore, it may be as a non-invasion assessment for screening animal tissue toxicity of chemical in future.

Detection of changes in bone quality of osteoporotic model induced by sciatic nerve resection by using Raman spectroscopy

To detect the bone quality loss in osteoporosis, we performed Raman spectroscopic analysis of sciatic nerve resection (NX) mice. Eight months after surgery, lower limbs were collected from the mice and fixed with 70% ethanol. Raman spectra of anterior cortical surface of the proximal tibia at 5 points in each bone were measured by RENISHAW inVia Raman Microscope. Excitation wave length was 785 nm. We also performed DXA and micro CT measurement to confirm the bone mineral density and bone microstructure in the osteoporotic model induced by sciatic nerve resection. In the result of Raman spectroscopy, we detected changes of Raman peak intensity ratio in carbonate/phosphate, mineral/combined proline and hydroxyproline and mineral/phenylalanine. In addition, in the result of micro CT, we found significant changes in VOX BV/TV, Trabecular number, thickness, cancellous bone mineral density, cortical thickness and cortical bone mineral density. The results suggest that not only the bone mineral density but also bone quality reduced in the NX mice. We conclude that Raman spectroscopy is a useful for bone quality assessment as a complementary technique for conventional diagnostics.

2P199 Raman Micro-spectroscopy of the Dynamics of Cellular Chemical State upon Stimulation with Growth Factors(12. Cell biology,Poster)

Cell fate changes stimulated by external factors, often show large cell-tocell variation in a clonal population. Such variation should reflect differences in the internal chemical states of cells. By defining and tracking the chemical state of individual cells, it would be possible to find a pathway of cell fate determination. Raman microspectroscopy is a powerful method to obtain information about the comprehensive cellular states. In this study, we tracked the cellular chemical states in the principal components plane defined from the time-course Raman spectra of the single living cells upon stimulation with growth factors. Results of the analysis indicated that differentiation process is affected by the initial chemical states. While proliferation looked stochastic.

Raman Spectroscopic Analysis of H2O2-Stimulated Three-Dimensional Human Skin Models Containing Asian, Black, and Caucasian Melanocytes

Reactive oxygen species generated in dermal cells of human skin is related to skin disorders or diseases. In this study, Raman analysis effectively clarified the identities of three types of human skin models after the models were stimulated with hydrogen peroxide. With the Caucasian skin model, the major Raman bands underwent large intensity changes within 4-5 days of stimulation. With the Black skin model, the Raman bands remained almost unchanged. The changes in the Asian skin model were unique compared to those in the above two. Eumelanin and pheomelanin are probably the main compounds that differentiate dermal cells in terms of their sensitivity to hydrogen peroxide.