Norikazu Sugiyama

Label-free characterization of living human induced pluripotent stem cells by subcellular topographic imaging technique using full-field quantitative phase microscopy coupled with interference reflection microscopy

There is a need for a noninvasive technique to monitor living pluripotent stem cell condition without any labeling. We present an optical imaging technique that is able to capture information about optical path difference through the cell and cell adhesion properties simultaneously using a combination of quantitative phase microscopy (QPM) and interference reflection microscopy (IRM) techniques. As a novel application of QPM and IRM, this multimodal imaging technique demonstrated its ability to distinguish the undifferentiated status of human induced pluripotent stem (hiPS) cells quantitatively based on the variation of optical path difference between the nucleus and cytoplasm as well as hiPS cell-specific cell adhesion properties.

Milligauss magnetic field applied pure water exert firefly luciferin-luciferase luminescence and induce intracellular calcium elevation of CHO cells without ATP

In the previous paper, we demonstrated that milligauss ultra-low-frequency alternative current (mG ULF-AC) magnetic field applied phosphate buffered saline solution (PBS) per se stimulated elevation of intracellular Ca/sup 2+/ level and enhanced phagocytic activity of human poly-morphonuclear (PMN) leukocyte. During the course of the study to explore the mechanism of the biological effect of such mG magnetic field applied PBS, we found extraordinary phenomena which challenges our understanding of physical chemistry of water and bioenergetics. PBS and highly purified water were exposed to mG ULF-AC which was generated by a Helmholtz coil under visible light (mG/light). When mG/light-applied pure water or PBS was added to the medium, CHO cells which respond to exogenous adenosine triphosphate (ATP) resulting in increase of intracellular Ca/sup 2+/ level, were found to elevate intracellular Ca/sup 2+/ level without exogenous ATP. This activity of mG ULF-AC applied water or PBS was equivalent to 100 nM ATP. Furthermore, when magnetic field was applied on highly purified water under visible light, for more than 12 h at 40/spl deg/C, the pure water became to exert luminescence of luciferin in the presence of luciferase. This reaction did not require ATP nor Mg/sup 2+/ in the reaction mixture at all. These facts indicate that mG/light-applied PBS or water mimics ATP activity.

A photon counting TV camera equipped with an image guide for rapid detection and counting of single bacteria in a wide field

Abstract— We developed a photon counting TV camera system, which uses an image guide consisting of tapered optical fibers, for the rapid detection and counting of microscopic sized luminous particles in a wide field. Using a luminous bacterium as a standard, we compared this image guide‐coupled TV camera to a lens‐coupled TV camera by determining their light collecting powers and detection abilities for single bacteria on a membrane filter. The image guide shortened the detection time by an order of magnitude, making the photon counting TV camera a practical system for rapid counting of bacteria.

Three-dimensional morphological imaging of human induced pluripotent stem cells by using low-coherence quantitative phase microscopy

There is an increasing need for non-invasive imaging techniques in the field of stem cell research. Label-free techniques are the best choice for assessment of stem cells because the cells remain intact after imaging and can be used for further studies such as differentiation induction. To develop a high-resolution label-free imaging system, we have been working on a low-coherence quantitative phase microscope (LC-QPM). LC-QPM is a Linnik-type interference microscope equipped with nanometer-resolution optical-path-length control and capable of obtaining three-dimensional volumetric images. The lateral and vertical resolutions of our system are respectively 0.5 and 0.93 μm and this performance allows capturing sub-cellular morphological features of live cells without labeling. Utilizing LC-QPM, we reported on three-dimensional imaging of membrane fluctuations, dynamics of filopodia, and motions of intracellular organelles. In this presentation, we report three-dimensional morphological imaging of human induced pluripotent stem cells (hiPS cells). Two groups of monolayer hiPS cell cultures were prepared so that one group was cultured in a suitable culture medium that kept the cells undifferentiated, and the other group was cultured in a medium supplemented with retinoic acid, which forces the stem cells to differentiate. The volumetric images of the 2 groups show distinctive differences, especially in surface roughness. We believe that our LC-QPM system will prove useful in assessing many other stem cell conditions.

Firefly luciferin-luciferase luminescence by milligauss ultra-low frequency pulsed magnetic field applied pure water without ATP

This paper reports on the biological activities of milligauss magnetic field (mG) applied PBS and also of mG-applied highly purified water on biological systems including CHO cells and luciferin-luciferase system. PBS and highly purified water were exposed to ultra low frequency magnetic field (mg-ULF) which was generated by an Helmholtz coil under visible light . When mG/light-applied pure water or PBS was added to the medium, CHO cells which have UTP receptor on the cell surface and which responds to exogenous ATP which in turn causes an increase of intracellular Ca/sup 2+/ level were found to elevate intracellular Ca/sup 2+/ level and suramin, an antagonist for the receptor, blocked the mG/light-applied PBS or water activity on the cells. When magnetic field was applied on pure water under visible light, for more than 12 hours at 40/spl deg/C, the pure water caused the luminescence of luciferin in the presence of luciferase. This reaction did not require ATP nor Mg/sup 2+/ in the reaction mixture at all. However, without luciferase in the reaction mixture no luminescence was observed. This mG/light-applied water did not scavenge luciferin-luciferase luminescence by the water. This is the first report that luciferin-luciferase luminescence without ATP and Mg/sup 2+/ and this raises the question of what kind of molecular species play stable energy donor in place of ATP. One hypothesis is that the magnetic energy applied onto pure water was stored in certain water (cluster) molecule: H/sub 3/O/sup +/(H2O)/sub n/, as a form with stable hydrogen bond resonance, and transferred to the enzyme protein molecule resulting to form oxy-luciferin with luminescence.