PN1 Monte Carlo Simulation Studies of Human Serum Albumin Adsorption on Hydrophobic and Hydrophilic Surfaces

Shu ChienHao-Jen Hsu1, Sheh-Yi Sheu2, and Ruey-Yug Tsay1

1Institute of Biomedical Engineering, 2Department of Life Science, National Yang-Ming University, Taipei 112, Taiwan, R.O.C.

 

Plasma protein adsorption is a crucial determinant in the hemo/ biocompatibility of a biomaterial. Because both of the molecular orientation and the possible conformational change of the adsorbed protein will affect the continuing cellular response, it is very important to know in detail about the adsorption state of these molecules. In this work, we apply Monte Carlo simulations for the adsorption of Human Serum Albumin (HSA) on a hydrophobic and a hydrophilic self-assembled monolayer (SAM) based on an all-atom model to study the preference orientation of the adsorbed molecule. A map giving the minimum interaction energy of the adsorbed molecule at various adsorption orientations is also established by artificial rotation and translation of the molecule, which is consistent with MC results. Simulation results show that at a (20x20) CH3 terminated SAM surface, HSA tends to prefer a “side-plane AC” orientation and posses the lowest interaction energy. On the other hand, HSA tends to resume a “back-lying” orientation on the (20x20) OH terminated SAM surface. The negative correlation between interaction energy and the number of atoms appeared within an interaction distance of 5 Å shows that more residues attaching to the surface may lead to lower interaction energy. The interaction energy for hydrophilic SAMs is higher than that for hydrophobic SAMs suggesting that HSA prefers to adsorb on a hydrophobic surface. This result is consonant with various experimental data shown in the literatures.

 

 

PN2

Water hydrolyzed induced the critical fluctuation and thickening broken on critical swelling of phospholipid bilayers

Y. Y. Chen (陳奕穎), J. H. Tsai(蔡景翰),S. L. Luo(羅順隆),J. H. Yang(楊忠翰), J. W. Shieu謝毅翁and W. C. Hung (洪偉清)

Department of Physics, Chinese Military Academy, Taiwan

 

It has been a puzzle that phospholipid bilayers of fluid phase are critically fluctuated and meantime weakly thickened in full hydration, when temperature T lowers toward the “main” transition [Chen et al., 1997, Phys. Rev. Lett.79, 4026]. Osmotic pressure raises transitional temperature, and their relation defines a transitional line. Exploring the fluid phase along the transition line down to the “main” transition point enables to find out the answer for the puzzle. This study measured dilauroyl-phosphatidylcholine (DLPC) bilayers by lamellar x-ray diffraction along isotherms varying from 1o to 35 oC as a function of Π. The result shows that an intermediate phase of partial ordering in chain to a critical thickness Dl* is emerged from the fluid phase along the transition line. As the transition is close to “main” transition point, partial domain of the intermediate phase is water hydrolyzed so that the bilayer thickness is non-uniform, leading to a significant bilayer fluctuation and a weaker bilayer thickening. This gives a new explanation for the critical swelling of phospholipid bilayers near “main” transition.

* This work was supported by National Science Council (Taiwan) Contract NSC94-2112-M-145-002.

 

 

PN3 Correlation between Lipid’s Spontaneous Curvature and Pore Formation by Antimicrobial Peptides

J. Y. Lin(林建佑),J. S. Lan(藍仲聖),J. W. Chen(陳俊文),J. U. Shiau(蕭智元),W. C. Hung (洪偉清)1 , M. T. Lee (李明道) 2, F. Y. Chen(陳方玉) 3, and H. W. Huang (黃惠文) 4

1Department of Physics, Chinese Military Academy, Fengshan,2National Synchrotron Radiation Research Center, Hsinchu 30076, Taiwan,3 Department of Physics, National Central University, Chung-Li, 32054 Taiwan,4Department of Physics & Astronomy, Rice University, Houston, Texas 77251

 

Recently we have shown that the free energy for pore formation induced by antimicrobial peptides contains a term representing peptide-peptide interactions mediated by membrane thinning. This many-body effect gives rise to the cooperative concentration dependence of peptides’ activities. Here we performed oriented circular dichroism and x-ray diffraction experiments to study the lipid dependence of this many-body effect. In particular we studied the correlation between lipid’s spontaneous curvature and peptide’s threshold concentration for pore formation, by adding PE and lysoPC to PC bilayers. Previously this correlation exhibited by magainin and melittin was argued to support the toroidal model for the pores. Here we found similar correlations exhibited by melittin and alamethicin. We found that the main effect of varying the spontaneous curvature of lipid is changing the degree of membrane thinning, which in turns influences the threshold concentration for pore formation.

 

 

PN4 Differentiation of basal cell carcinoma from the adjacent normal tissue by of non-linear microscopy : implication for surgical guidance

Chien-Jui Kuoa, Sung-Jan Linb, Wen Loa, Shiou-Hwa Jeeb, Chen-Yuan Donga

aDepartment of Physics, National Taiwan University, Taipei, Taiwan
bDermatology, Taipei Campus and Yun-Lin Branch, National Taiwan University Hospital, Taiwan

 

Basal cell carcinoma is the most common skin cancer in the Asian population. It usually develops in the sun-exposed skin. Conventionally, the cancer is removed by wide excision, aimed at preventing local recurrence. When a wide safety margin can not be obtained in areas, including eyelids, periorbital area and nasal alae, Mohs’ surgery is employed to ensure complete elimination of cancer cells while preserving these anatomically important structures. However, it takes time to process the tissue for microscopical examination. In this work, we use multiphoton fluorescence and second harmonic generation microscopy to examine basal cell carcinoma specimens. The autofluorescence and second harmonic generation signals are compared with routine histological examinations using H&E staining. Our results show that cancer cells and cancer stroma can be differentiated from the adjacent normal skin using multiphoton microscopy and this technique may be used in the clinical settings.

 

 

PN5 Multiphoton Characterization of Infection Pathogens

Chien-Jui Kuoa ,Sung-Jan Linb,Ruei-Jr Wua ,Yee-Chun Chen c ,Shiou-Han Wang e ,Hsin-Yuan Tand,Jun-Ling Wang c , Feng-Chieh Lia, Chun-Chin Wanga, Wen Loa,Chen-Yuan Donga

aDepartment of Physics, National Taiwan University, Taipei, Taiwan
bDepartment of Dermatology, Taipei Campus and Yun-Lin Branch, National Taiwan University Hospital, Taiwan
cDepartment of Internal Medicine, National Taiwan University Hospital, Taiwan
dDepartment of Ophthalmology, Chang Gung Memorial Hospital, Linko, Taiwan
eDepartment of Dermatology, National Taiwan University Hospital and National Taiwan University College Medicine, Taipei, Taiwan

 

We performed multiphoton imaging on four fungi and four Candida of medical significance. Intense autofluoresence can be detected from spores and hyphae, especially the cell wall and septum. We analyze the emission spectrum and it shows that fungi and candida of various species have different characteristic fingerprint emission spectrum: Microsporum canis has intense emission in the blue light, Trichophyton mentagrophytes shows more intense emission in the yellow light, Aspergillus flavus has a preferential emission in the red light, and Fusarium solani has a characteristic autofluorescence in the blue light. Our images also show that the Candida tropicalis has an elongated shape, the Candida albicans and Candida krusei has a circular shape, and the Candida glabrata has an elliptical shape. Our result shows that multiphoton microscopy can be used as a noninvasive tool for differentiating fungal and Candida species.

 

 

PN6 The formation and detection of tight-turn in pyrimidine/purine/pyrimidine type DNA triplexes – new findings on motifs CC and TT by fluorescence spectroscopy at pH 7

Ming-Tsai Wey a,b, Lou-sing Kan b*

a College of Life Science, National Tsinghua University, Hsinchu 300, Taiwan
b Institute of Chemistry, Academia Sinica, Taipei 11529, Taiwan

 

DNA triplexes with ‘tight turns’, i.e., sharp turns without any intervening bases, are studied by fluorescence resonance energy transfer process (FRET). The particular turning motifs under consideration are CC and TT imbedded in triplexes (5’-F-(TC)3(CT)3-D + 5’-AGAGAG) and (5’-F-(CT)3(TC)3-D + 5’-GAGAGA), where F and D are 6-fluorescein and 4-(4-dimethylaminophenylazo) benzoic acid, respectively. The association equilibrium constants of the above two triplexes ranged from 1.7x107 to 2.9x104 and 0.75x107 to 1.8x104 from 10 to 35°C, respectively. The thermodynamic parameters of these two triplexes are comparable (⊿H: -46.0 vs. –45.0 Kcal/mol; ⊿S: -129 vs -127 cal/mol K; ⊿G: -7.5 vs. –7.2 Kcal/mol, respectively). Thus, the stability of the tight-turn triplex with a CC motif is comparable to that with a TT motif, but slightly favored over the latter. This is the first report of a py/pu/py type triplex containing such tight turns studied under pH at 7. The importance is manifested in their stabilities maintained under physiological neutral condition, as opposed to previous studies only obtainable in acidic conditions.

 

 

PN7 Intravital Multiphoton Microscopy of Dynamics Hepatic Process

Yuan Liua, Hsiao-Ching Chenb, Wen Loa, Ling-Ling Chioub, Guan-Tarn Huangb, Chen-Yuan Donga, Hsuan-Shu Leeb

aDepartment of Physics, National Taiwan University, Taipei 106, Taiwan, R.O.C.
bDepartment of Internal Medicine, National Taiwan University Hospital and National Taiwan University, College of Medicine, Taipei 100, Taiwan, R.O.C.

 

Multiphoton microscopy is a newly developed nonlinear optical technique from the early 1990s. The minimally invasiveness and optical sectioning ability of two-photon fluorescence and second harmonic generation enable us to investigate the tissue in depth. Intravital and real-time imaging of liver is vital for elucidating hepatic dynamics. Here we describe a novel method to observe through a chronic window device by multiphoton microscopy the optical liver histology to show the intravital dynamics of uptake and excretion in the hepatobiliary system. Some fluorophores were used to identify the structures within the liver. Our images show the sequential uptake of carboxyfluorescein diacetate from hepatocytes and its subsequent excretion into bile canaliculi. The potential of multiphoton microscopy in intravital liver observation is demonstrated in this work. By this technique, various hepatic diseases and mechanisms can be studied.

 

 

PN8 Pathological Investigation of Cornea by Use of Multiphoton Microscopy

Wen Lo1, Hsin-Yuan Tan2,3, Shu-Wen Teng1, Yen Sun1, Chen-Yuan Dong1

1National Taiwan University, Department of Physics
2Department of ophthalmology, Chang Gung Memorial Hospital, Linko
3 Institute of Biomedical engineering, college of medicine and engineering, National Taiwan University

 

In this work, multiphoton microscopy was used to obtain pathological images of several human corneal diseases, including fungal keratitis, bacterial keratitis, corneal amebiasis, corneal scar and keratoconus.

For a normal corneal specimen, the multiphoton images show that the lamellar structure in corneal stroma can be visualized by second harmonic generation (SHG) signal while the multiphoton autofluorescence reveals morphology of epithelial cells. In diseased corneal stroma, degeneration and altered alignment can be visualized by SHG images. Inflammatory cells and activated keratocytes in diseased stroma can be also highlighted by increased cytoplasmic autofluorescence.
Our results suggest that multiphoton microscopy can be a powerful tool in corneal researches and be potentially developed into an in vivo image modality for corneal pathology.

 

 

PN9 The Combination of Mutiphoton and Reflected Confocal Microscopy for Cornea Imaging

Wei-Liang Chena, Yen Suna, Wen Loa, Hsin-Yuan Tanb,c, Chen-Yuan Donga*

aDepartment of Physics, National Taiwan University, Taipei, Taiwan.
bInstitute of Medical Engineering, College of Medicine and College of Engineering, Taipei 100, Taiwan
cDepartment of Ophthalmology, Chang Gung Memorial Hospital, Linko 333, Taiwan

 

The difficulty of optically imaging the highly translucent cornea has prevented the development of an effective non-invasive system for the clinical monitoring of the cornea. We combine reflective confocal microscopy with multiphoton microscopy to form a minimally invasive technique to observe the cornea. The two imaging modalities allow detection of complementary information from the cornea. The autofluorescence signal shows the cell plasma of epithelial cell, while leaving the cell walls and the cell nucleus dark. The second harmonic generation signal is used to detect collagen found mostly in the stroma of the cornea. The reflective confocal imaging allows detection of epithelial cell walls and keratocye in the stroma. The system is first tested on bovine cornea. Assessment of the result on the bovine eye will be used to evaluate the potential of the system as a technique for in vivo clinical application.

 

 

PN10 Investigating Mechanisms of Collagen Thermal Denaturation by High Resolution Second-Harmonic Generation Imaging

Yen Sun1, Wei-Liang Chen1, Sung-Jan Lin2,3, Shiou-Hwa Jee2, Yang-Fang Chen1, Ling-Chih Lin1, Peter T. C. So4, Chen-Yuan Dong1

1Department of Physics, National Taiwan University, Taipei 106, Taiwan
2Department of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei 100, Taiwan
3Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei 100, Taiwan
4Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

 

We apply the technique of second-harmonic generation (SHG) microscopy to obtain large area submicron resolution image of type I collagen from rat tail tendon as it is heated from 40℃ to 70℃ for 0 to 180 minutes. The change in the collagen structure as reflected in its SHG image is observed at length scales from submicron to hundreds of micron. We observed that heating the tendon below the temperature of 54℃ does not produce any change in the averaged SHG intensity. At the heating temperature of 54℃ and above, we find that increasing the heating temperature and time leads to decreasing SHG intensity. As the tendon is heated above 54℃, the regions where the SHG signal vanishes form a tiger-tail like pattern, but a decrease in the SHG signal occurs uniformly throughout the tendon. By comparing the relative SHG intensities in small and large areas, we found that the denaturation process responsible for forming the tiger-tail like pattern occurs at a higher rate than the global denaturation process occurring throughout the tendon. We also measured the fibril spacing and found that it remains constant at 1.61 ± 0.04 micron for all heating temperature and times. The constant fibril density shows that the global denaturation process occurs at a length scale smaller than the size of the fibril. Our results show that second-harmonic generation microscopy is effective in monitoring the thermal damage to collagen and has potential applications in biomedicine.

 

 

PN11 Characterization of Skin Dermal Thermal Damage by Multiphoton Microscopy

Ming-Gu Lina , Tsung-Lin Yanga, Jin-Ning Leea, Cheng-Tien Chianga, Hsien-Ching Kaoa, Wen Loa, Sung-Jan Linb, Shiou-Hwa Jeeb, Yang-Fang Chena, Chen-Yuan Donga

aDepartment of Physics, National Taiwan University, Taipei, Taiwan
bDepartment of Dermatology, National Taiwan University Hospital and College of Medicine, Taipei, Taiwan

 

In recent years multiphoton fluorescence microscopy has gained significant interest in bioimaging applications. Since skin tissue injury due to thermal damage is common in daily day, we attempted to characterize the degree of dermal thermal damage by the use of multiphoton microscopy. We characterize the autofluorescence and SHG signal for skin dermis treated at different temperatures. The intensity of collagen decreased with increasing temperature but the autofluorescence intensity did not decrease significantly with increasing temperatures. We were able to locate dermal collagen by autofluorescence and SHG imaging. The elastin fibers aggregated starting at 65 ℃ and can be observed by the autofluorescence imaging. Also we found that the ratio of autofluorescence to SHG intensity is almost identical before 60 ℃ and sharply increases when the temperature is further risen. This study demonstrated that multiphoton microscopy can provide effective qualitative and quantitative information for skin dermal damage.

 

 

PN12 Concerted Experimental Approach for Sequential Mapping of Peptides and Phosphopeptides Using C18-Functionalized Magnetic Nanoparticles

He-Hsuan Hsiao, Hsin-Yu Hsieh, Shu-Yu Lin, Chi-Chi Chou, Andrew H.J. Wang, and Kay-Hooi Khoo*

National Core Facilities for Proteomics Research and §The Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.

 

An integrated analytical approach for the enrichment, detection and sequencing of phosphopeptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS) was developed. Based on C18-functionalized Fe3O4 nanoparticles, the enrichment method was designed not only to specifically trap phosphopeptides but also non-phosphorylated peptides, both of which can be subsequently desorbed selectively and directly for MALDI-MS analysis without an elution step. Peptide binding is afforded by the C18-derivatization whereas the highly selective capture of phosphopeptides is based on higher binding affinity afforded by additional metal chelating interaction between the Fe3O4 nanoparticles and the phosphate groups. Upon binding, the initial aqueous wash allows desalting while a second wash with high acetonitrile content coupled with diluted sulfuric acid will remove most of the bound non-phosphorylated peptides. Selective or sequential mapping of the peptides and phosphopeptides can thus be effected by spotting the washed nanoparticles onto the MALDI target plate along with judicious choice of matrices. The inclusion of phosphoric acid in a dihydroxybenzoic acid matrix allows the desorption and detection of phosphopeptides whereas an a-cyano-4-hydroxy-cinnamic acid matrix with formic acid allows only the desorption of non-phosphorylated peptides. The method is versatile enough in that for more complicated mixtures, the peptides and phosphopeptides can also be sequentially eluted off the nanoparticles for additional liquid chromatography separations prior to MS applications. We have demonstrated the applicability of C18-functionalized Fe3O4 nanoparticles in the detection of in vitro phosphorylation sites on the myelin basic protein and at least thirteen phosphopeptides were identified, including one previously uncharacterized site.