FS1 The Biological Application of Synchrotron Radiation-based Fourier Transform Microspectroscopy

Pei-Yu Huang , Ching-Iue Chen and Yao-Chang Lee

National Synchrotron Radiation Research Center

 

Synchrotron-radiation-based Fourier transform infrared (SR-FTIR) microspectroscopy is a newly emerging bioanalytical and imaging tool. The advantage of infrared synchrotron radiation is high throughput at high spatial resolution compared to a conventional thermal source. And the beam size of the radiation after employing 32¡Ñ confocal Schwarzshild objective is about 10 ¡Ñ 13 £gm2, however, the conventional infrared light source is about 50 ¡Ñ 30 £gm2. SR-FTIR microspectroscopy is the combination of FTIR microspectroscopy and high brightness infrared synchrotron radiation produces the highest signal-to-noise ratio spectrum with ultrahigh spatial resolution as fine as 8 £gm. This unique technique provides mid-infrared spectra based on the vibrations of the nuclei of molecule, and the vibration frequencies of chemical component utilized for production of chemical images according to their characteristic frequencies for indicating each component to the exclusion of others within the sample. Thus it enables researchers to locate, identify, and track specific chemical events within an individual biological cell. Mid-IR photons are too low in energy (0.05-0.5 eV) to either break bonds or to cause ionization. In this study, human scalp hair sections, malignant human colorectal tissue sections and butterfly wing scale were investigated utilizing reflection detection at spatial resolution of 10 £gm. The end result is two dimension infrared spectral images of the sample, enabling constituents and composition to be visualized easily, especially for protein, lipid, carbohydrate, and PO2- residual of DNA. Keywords: Fourier transform infrared microspectroscopy, infrared synchrotron radiation, colorectal tissue, and butterfly wing scale.

 

 

FS2 The qualitative x-ray phase imaging with sub 100 nanometer resolution

Gung-Chian Yin 1,2, Fu-Rong Chen 1, Yeukuang Hwu 3, Han-Ping D. Shieh 2, Keng. S. Liang 1

1 National Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu 30076, Taiwan.
2 Department of Photonics and Display Institute, National Chiao Tung University, Hsinchu 30076, Taiwan.
3 Institute of physic, Academic sinica, Taipei 115, Taiwan

 

In x-ray imaging, the phase contrast is generally 100 to 1000 times higher than the absorption contrast in soft material. However, the quantitative phase information is required for the phase tomography in order to fully utilize the capability of the x-ray imaging. In this work, we demonstrate that the quantitative phase retrieval with a sub-100nm resolution can be achieved from micrographs of zone plate based transmission x-ray microscope (TXM). In this work, a zone plate made of plastic containing objects of sizes from µm down to tens of nano-meter is used as a test sample to quantify the retrieved phase. Using the focal serial images in the image plane, the phase information is retrieved quantitatively across the entire range of sizes by combining the transport intensity equation (TIE) and self-consistent wave propagation methods (SCWP) in this partial coherence system. The results show a good agreement with simulation. The study demonstrates a solution to retrieve the phase in the TXM and overcome the deficiency encountered in the two phase retrieval approaches of TIE and SCWP. This technique can be applied to the micro/nano tomography for living cell observation.

 

 

FS3 Biology sample observed by transmission hard x-ray microscopy

Pao-Yueh Hsiao1,2

1 National Synchrotron Radiation Research Center, 101 Hsin-Ann Road, Hsinchu 30076, Taiwan; 2Institute of Molecular and Cellular Biology, National Tsing Hua University , 101, Section 2 Kuang Fu Road, Hsinchu, 30013 Taiwan

 

X-ray imaging has the advantage of high resolution and high pentration depth. However, the contrast is relatively low compare to transmission electron microscope (TEM) and optical microscope (OM). In this research we demonstrate the high contrast cell image by staining uranyl acetate and zernike¡¦s phase contrast method in transmission x-ray microscope (TXM). Furthermore, the TXM is capable of determining the 3D structure of cell by performing the tomography. The other problem is, due to the loss of water during exposing, cell¡¦s 3D structure varies during the tomography process of cells. We tried to find the resolution of this problem by embedding it in some resin. The result of 3D structure of cell will be demonstrated in this paper.

 

 

FS4 Bio-membrane Structure Probed by the Lamellar X-ray Diffraction and the Small Angle X-ray Scattering at NSRRC

Ming-Tao Lee , Yu-Shan Huang, U-Ser Jeng, Ying-Huang Lai, and Ya-Sen Sun

National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan

 

The bio-membrane structure has long been an interesting issue in life science. Various techniques, including X-ray diffraction, small-angle scattering and nuclear magnetic resonance (NMR), have often been introduced in revealing bio-membrane structures under various humilities and temperatures as well as interactions with proteins. In this study, the lamellar X-ray diffraction (LXD) and small angle X-ray scattering (SAXS) are used to determine the membrane structures under various environments. Temperature- and humility- dependent LXD experiments for a multilamellar membrane on a plane substrate were conducted with 12keV photons of the BL13A beamline at the National Synchrotron Radiation Research Center (NSRRC). The electron density profile and thickness of membrane are extracted from the data fitting. Furthermore, temperature-dependent SAXS experiments for unilamellar vesicles in an aqueous solution were measured with 10.5keV photon of the BL17B3 beamline, NSRRC. Combining model fitting and SAXS data, the membrane thickness of vesicles in the solution has been determined.

 

 

FS5 A XAS-SAXS Study of the Local and Global Structure Changes of Cytochrome c ¡V an Unfolding Process Induced by Urea

I-Jui Hsu1, Ying-Jen Shiu2, U-Ser Jeng3, Tung-Ho Chen3, Yu-Shan Huang3, Ying-Huang Lai3, Ling-Na Tsai4, Ling-Yun Jang3, Jyh-Fu Lee3, Li-Jiaun Lin4, Sheng-Hsien Lin2, Yu Wang1

1Department of Chemistry, National Taiwan University, Taipei, 106, Taiwan
2Institute of Atomic and Molecular Sciences, Academia Sinica, P. O. Box 23-166, 106 Taipei, Taiwan
3National Synchrotron Radiation Research Center, Hsinchu, Taiwan 300
4Industrial Technology Research Institute. Hsinchu, Taiwan 310

 

The local and global structural changes of cytochrome c induced by urea in aqueous solution have been studied using X-ray absorption spectroscopy (XAS) and small angle X-ray scattering (SAXS). According to the XAS result, both the native (folded) protein and the unfolded protein exhibit the same pre-edge features taken at Fe K-edge, indicating that the Fe(III) in the heme group of the protein maintains a six-coordinated local structure in both folded and unfolded states. Furthermore, the discernable differences in the X-ray absorption near-edge structure (XANES) of these two states are attributed to a possible spin transition of the Fe(III) from a low spin state to a high spin state during the unfolding process. The perseverance of six-coordination and the spin transition of the iron are reconciled by a proposed ligand exchange¡Vwith urea and water molecules replacing the Methionine-80 and Histidine-18 axial ligands, respectively. The SAXS result reveals a significant morphology change of cytochrome-c from a globular shape of a radius of gyration Rg = 12.8Å of the native protein to an elongated ellipsoid shape of Rg = 29.7Å for the unfolded protein in the presence of concentrated urea. The extended X-ray absorption fine structure (EXAFS) data unveil the coordination geometries of Fe(III) in both folded and unfolded state of cytochrome c. An initial spin transition of Fe(III) followed by an axial ligand exchange, accompanied with the change in global envelope, is proposed for what happened in the protein unfolding process of cytochrome c.