Browsing by Author "Huang, Chuan-Keng"

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Disentangling Dynamic Changes of Multiple Cellular Components during the Yeast Cell Cycle by in Vivo Multivariate Raman Imaging
(2012-07-03)
Huang, Chuan-Keng
;
Ando, Masahiro
;
Harnaguchi, Hiro-o
;
Shigeto, Shinsuke
;
應用化學系
Cellular processes are intrinsically complex and dynamic, in which a myriad of cellular components including nucleic acids, proteins, membranes, and organelles are involved and undergo spatiotemporal changes. Label-free Raman imaging has proven powerful for studying such dynamic behaviors in vivo and at the molecular level. To construct Raman images, univariate data analysis has been commonly employed, but it cannot be free from uncertainties due to severely overlapped spectral information. Here, we demonstrate multivariate curve resolution analysis for time-lapse Raman imaging of a single dividing yeast cell. A four-dimensional (spectral variable, spatial positions in the two-dimensional image plane, and time sequence) Raman data "hypercube" is unfolded. to a two-way array and then analyzed globally using multivariate curve resolution. The multivariate Raman imaging thus accomplished successfully disentangles dynamic changes of both concentrations and distributions of major cellular components (lipids, proteins, and polysaccharides) during the cell cycle of the yeast cell. The results show a drastic decrease in the amount of lipids by similar to 50% after cell division and uncover a protein-associated component that has not been detected with previous univariate approaches.
Dissociation of energy-selected CH3CN+ in a region 15.1-16.5 eV: Vibrationally enhanced dissociation and mechanisms
(2007-05-25)
Huang, Chuan-Keng
;
Lin, I.-Feng
;
Chiang, Su-Yu
;
應用化學系
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Department of Applied Chemistry
Threshold photoelectron-photoion coincidence spectra of CH3CN were measured in a region 15.1-16.5 eV. We observed a small vibrational enhancement on dissociation of CH3CN+ and obtained kinetic energy releases for channels C2HN+ + H-2 and CH2+ + HCN. The kinetic energies released in channel C2HN+ + H-2 are substantial; a linearly extrapolated dissociation threshold of 14.68 +/- 0.01 eV agrees with a Gaussian-3 B3LYP prediction 14.70 eV for formation of HCCN+ + H-2. In contrast, the small releases for channel CH2+ + HCN agree with a statistical calculation. We describe a plausible dissociation mechanism with the Gaussian-3 B3LYP method to rationalize experimental results. (c) 2007 Elsevier B.V. All rights reserved.
In vivo multimode Raman imaging reveals concerted molecular composition and distribution changes during yeast cell cycle
(2011)
Huang, Chuan-Keng
;
Hamaguchi, Hiro-o
;
Shigeto, Shinsuke
;
應用化學系
;
應用化學系分子科學碩博班
In vivo time-lapse Raman imaging reveals highly dynamic and concerted changes in concentration and distribution of phospholipids and proteins during and after cell division of a single living Schizosaccharomyces pombe cell.
In Vivo Probing of the Temperature Responses of Intracellular Biomolecules in Yeast Cells by Label-Free Raman Microspectroscopy
(2013-05-27)
Chiu, Yu-Fang
;
Huang, Chuan-Keng
;
Shigeto, Shinsuke
;
應用化學系
;
應用化學系分子科學碩博班
Environmental temperature is an essential physical quantity that substantially influences cell physiology by changing the equilibria and kinetics of biochemical reactions occurring in cells. Although it has been extensively used as a readily controllable parameter in genetic and biochemical research, much remains to be explored about the temperature responses of intracellular biomolecules in vivo and at the molecular level. Here we report in vivo probing, achieved with label-free Raman microspectroscopy, of the temperature responses of major intracellular components such as lipids and proteins in living fission yeast cells. The characteristic Raman band at 1602 cm1, which has been attributed mainly to ergosterol, showed a significant decrease (approximate to 47%) in intensity at elevated temperatures above 35 degrees C. In contrast to this high temperature sensitivity of the ergosterol Raman band, the phospholipid and protein Raman bands did not vary much with increasing culture temperature in the 2638 degrees C range. This finding agrees with a previous biochemical study that showed that the initial stages of ergosterol biosynthesis in yeast are hindered by temperature elevation. Moreover, our result demonstrates that Raman microspectroscopy holds promise for elucidation of temperature-dependent cellular activities in living cells, with a high molecular specificity that the commonly used fluorescence microscopy cannot offer.
Probing the binding kinetics of proinflammatory cytokine-antibody interactions using dual color fluorescence cross correlation spectroscopy
(2011)
Wu, Chia-Yan
;
Huang, Chuan-Keng
;
Chung, Chao-Yu
;
Huang, I-Ping
;
Hwu, Yeukuang
Dual color fluorescence cross correlation spectroscopy (FCCS) was used to investigate quantitatively the binding kinetics of tumor necrosis factor (TNF alpha) with TNF alpha antibody (anti-TNF alpha) following fluorescent labeling. Through the analysis of the auto correlation curves of fluorescence correlation spectroscopy (FCS), diffusion coefficients of 100.06 +/- 4.9 mu m(2) s(-1) and 48.96 +/- 2.52 mu m(2) s(-1) for Alexa488-TNF alpha and Atto647N-anti-TNF alpha were obtained. In addition, the calculated hydrodynamic diameters of the Alexa488-TNF alpha and Atto647N-anti-TNF alpha were approximately 4.89 +/- 0.24 nm and 9.99 +/- 0.52 nm, respectively, which agrees with the values of 5.20 +/- 1.23 nm and 9.28 +/- 0.86 nm for the native TNF alpha and the anti-TNF alpha as determined from dynamic light scattering measurements. For the binding kinetics, association (k(on)) and dissociation (k(off)) rate constants were (1.13 +/- 0.08) x 10(4) M(-1) s(-1) and (1.53 +/- 0.19) x 10(-3) s(-1) while the corresponding dissociation constant (K(d)) at 25 degrees C was (1.36 +/- 0.10) x 10(-7) M. We believe this is the first report on the binding kinetics for TNF alpha-antibody recognition in the homogeneous phase. Using this technology, we have shown that controlled experiments can be performed to gain insight into molecular mechanisms involved in the immune response.