Autofluorescence from the PC12 cells as a response to excitation of near-infrared femtocecond laser


Elena Perevedentseva, Fu-Jen Kao

Institute of Electro-Optical Engineering, National Sun Yat-sen University

Kaohsiung, 80424 Taiwan


The issue of photodamage induced by near-infrared femtosecond laser beam on living cells and tissues is important in the applications of non-linear optical signals for research on cells/tissues structure and function1. In this presentation we are reporting the use of two-photon fluorescence spectroscopy and microscopy in combination with optical trapping. The same femtosecond NIR laser beam simultaneously traps the targeted cells and excites 2p autofluorescence out of them. Such excitation is considered non-destructive and non-invasive since the cells do not absorb NIR efficiently and the average power of the pulsed laser is low. The autofluorescence is generated by endogenous fluorophores so that injection of exogenous agents into the cells is not needed. However, femtosecond NIR laser may induce invisible biological changes within a living cell1,2. Endogenous fluorophores, either from structural elements of cells and tissues or found in metabolic processes,  may reflect changes in the structure or in the metabolism induced by the laser. In this work the effect of NIR femtosecond Ti:S laser on an individual PC12 cell is investigated with 2p-fluorescence microscopy and spectroscopy, simultaneously with cell trapping.

The cell’s 2p autofluorescence excited with incident laser beam in the spectral range of 735-750 nm is measured as a function of time and the power density of the laser between 0.3x108 to 1.6x108 Wcm2. The autofluorescence from the cells is analyzed and is compared with the cell living conditions. In addition, the power issue will be discussed. Under a certain threshold, the femtosecond NIR laser may serve as a non-destructive trapping and exciting beam.

1 K.König, P.T.C.So, W.W.Mantulin, E.Gratton. Cellular response to near-infrared femtosecond laser pulses in two-photon microscopes. Opt.Lett. 22(2) 135-135 (1997)

2 F.J. Kao, Y.M. Wang, J.C. Chen, P.C. Cheng, R.W. Chen and B.L. Lin, Photobleaching under single photon and multi-photon excitation: chloroplasts in protoplasts from Arabidopsis thaliana, Optics Communications, 201, 85-91 (2002)




Monitoring Transdermal Delivery of Quantum Dots Using Multiphoton Fluorescence Microscopy


Wen Lo1*, Tso Chien-Hsin1, Sun-Jan Lin2, Shiou-Hwa Jee2, Chen-Yuan Dong1

1Department of Physics, National Taiwan University, No.1Roosevelt Road Section 4, Taipei, 106, Taiwan

2National Taiwan University Hosipital and National Taiwan University, College of Medicine.

No.7, Chung San South Road, Taipei, 100, Taiwan and No.1, Ren-Ai Rd, 1st Section, Taipei, 100, Taiwan.


  Luminescent quantum dots (QDs) are attractive fluorescent probes in bioimaging applications. Broad excitation spectra, narrow and tunable emission spectra, and high photochemical stability are among the desirable properties of QDs. Chemically modified QDs have been used in bioimaging and these novel probes have potentials for tissue imaging applications. In this work, we investigate the feasibility of administering QDs through transdermal delivery pathways using multiphoton fluorescence microscopy. Multiphoton fluorescence microscopy is the preferred technique due to its ability to image deep within tissue specimens. Our multiphoton imaging results show that chemical enhancer can be used for transdermal QD delivery.

Our results suggest two exciting applications of transdermal QD delivery. First, chemically modified QDs may be administered to diseased tissues for pathological imaging. Furthermore, the similarity in the sizes of QDs (nanometers) to important biological macromolecules imply that fluorescent QDs may be used to clarified the delivery pathways of these biomolecules across biological barriers.




Characterizing the Effects of Spherical Aberration and Scattering on Multi-Photon Image Formation


Tsung-Kai Chiu1*, Chin-Kuan Tung1, Wen Lo1, Sun-Jan Lin2, Shiou-Hwa Jee2,3, Chen-Yuan Dong1

1Department of Physics, National Taiwan University, Taipei106, Taiwan 2Department of Dermatology, National Taiwan University Hospital, Taipei 100, Taiwan  3National Taiwan University Collage of Medicine, Taipei 100, Taiwan


Compared to confocal microscopy, imaging using multi-photon fluorescence microscopy (MPFM) allows deeper penetration depths, reduced photodamage and improved contrast in tissue imaging. As a result, MPFM is an especially powerful tool for in-depth imaging of tissues such as the skin, brain and cornea. However, refractive index mismatch induced spherical aberration and scattering in samples can degrade multi-photon images. In order to optimize multi-photon imaging for biological and medical application, it is important to characterize the effects of index mismatch induced spherical aberration and scattering in image formation. In this work, we investigate these issues by imaging fluorescent specimens of different refractive indices and scattering parameters. For the spherical aberration studies, we use four types of immersion objectives (air, water, glycerin and oil) to examine samples with different refractive index properties (water, glycerin, oil and the skin). To investigate the effect of scattering, we measure in-depth fluorescence intensity profiles acquired using different excitation wavelengths in the excitation of fluorescent molecules with different emission properties. In particular, fluorescent specimens containing 1% or 2% intralipid were used to mimic tissue-like scattering conditions. We hope that our work can elucidate the mechanisms of multi-photon image formation in tissues.




Electron Microscopic Single Particle Analysis with

a Calcium Channel



Kuniaki Nagayama*, Tomoharu Matsumoto, Yuji Hara and Yasuo Mori

Center for Integrative Bioscience, Okazaki National Research Institutes,

 Myodaiji-cho, Okazaki, 444-8585 JAPAN (nagayama@nips.ac.jp)

Myodaiji-cho, Okazaki, 444-8585 JAPAN


TRPM 2 (renamed from LTRPC21)) is a calcium channel postulated to have an in vivo function related to the redox-state depending apoptosis.  The plasmid containing the gene (1503 AA equivalent) reconstructed from the cDNA library of human brains was cloned in cells of silkworms as the infectious form of baculovirus, leading to a large scale of gene products.  Purification of gene products with column chromatography resulted in a SDS single-banded entity, of which electron micrographs had shown an image of uniformly sized particles.  For the sample after negatively stained, a single particle analysis was performed with a transmission electron microscope of 100kV acceleration.  Our experiments are summarized as;

Materials: ABv baculovirus – silkworm system (Katakura), solubilization reagent: pentadecafluorooctanoic acid.

Purfication: Ni-chelate affinity column chromatography, gel permeation chromatography.

Identification: amino acid analysis to confirm native polypeptides: confirmed within experimental errors.

Single particle analysis: clearly recognized tilted image series of nine channel particles, total of particle images: 45.

Resolution: differential phase residual analysis to determine the spatial resolution: 3.2 nm.

3D structure: Low density central area was surrounded by four or five high density domains.

1.  Mori Y, Wakamori M, Miyakawa T, Hermosura M, Hara Y, Nishida M, Hirose K, Mizushima A, Kurosaki M, Mori E, et al: Transient receptor potential 1 regulates capacitative Ca2+ entry and Ca2+ release from endoplasmic reticulum in B lymphocytes. J. Exp Med 195, 673-681 (2002).




Single molecule imaging reveals oligomerization of chemoattractant receptors

Keiichi Kinowaki1*, Silvia Iskandar1, Tadashi Tojo1, Daisuke Aoki1, Hisashi Tadakuma1, Takashi Funatsu1.

1Department of Physics, School of Science & Engineering, Waseda University,

3-4-1 Okubo Shinjuku-ku Tokyo 169-8555, Japan


GPCRs(G-Protein-Coupled Receptors) are thought to be targets for 50~70% of the medicines for clinical treatments. GPCRs play critical roles on homeostasis in mammals as receptors for neurotransmitters, hormones, chemokines and so on. GPCRs were considered to exist as a monomer on plasma membrane. However, recent increasing evidence obtained by biochemical analyses implies that GPCRs form homo and hetero-dimers or oligomers. To examine this, receptor fused with EGFP at the C terminus (Receptor-EGFP), was expressed in CHO cells and observed in real time using an objective type total internal reflection fluorescence microscopy (TIRFM). As the representation of GPCR family, we used CC-chemokine receptor 5 (CCR5) and CXC-chemokine receptor 4 (CXCR4) which are the most physiologically relevant and widely used coreceptors for human immunodeficiency virus type 1 (HIV-1). We observed Receptor-EGFP as individual fluorescent spots that diffuse laterally on the plasma membrane of living cells, and the distribution of the fluorescent intensity of them indicates that the major population of Receptor-EGFP forms oligomers of various sizes, revealing that these receptors constitutively oligomerize independent of ligand binding.

To clarify that the tendency of EGFP to dimerize doesn’t influence oligomerization of Receptor-EGFP, we also fused receptor with an EGFP mutant “A206K”, which has less tendency to dimerize than EGFP, and obtained results similar to that of EGFP. These results show that Receptor-EGFP oligomerization is not caused by EGFP dimerization, but a truly intrinsic character of these receptors.

Similarly, we revealed that formylpeptide receptor 1 (FPR1) and CXC-chemokine receptor 1 (CXCR1) oligomerize on the plasma membrane of a living cell and the oligomerization is independent of ligand binding. These results suggest that the feature of oligomerization is independent of ligand binding and is common to chemoattractant receptors belonging to GPCR family.




Applying Two-Photon Polarization Microscopy in the Imaging of Skin's Epidermal and Dermal Layers


Yen Sun1*, Wen Lo1, Jiun-Wen Su1, Sun-Jan Lin2, Shion-Hwa Jee2, Chen-Yuan Dong1

1Department of Physics, National Taiwan University, Taiwan and

 2National Taiwan University College of Medicine, Taiwan

1 No.1, Sec.4, Roosevelt Road, Taipei, Taiwan 106 and  2No7, Chung San South Road, Taipei, Taiwan.


Two-photon microscopy (TPM) is a three-dimensional imaging technology with a number of advantages. For example, TPM can image tissues up to a depth of a few hundred micrometers, at high resolution and with improved contrast. Polarization imaging can also lead to enhanced image contrast. In this work, we propose to apply two-photon polarization microscopy in bioimaging applications. Both the excitation and emission polarizations will be varied to optimize image quality. We will apply this methodology to the imaging of skin specimens and we hope to be able to demonstrate potential contrast enhancement by examining different layers of the skin, from the epidermis to the dermal layer.




Schlieren Shutter Phase Retrieval for Strong Objects in Transmission Electron Microscopy: Theory and Simulation


Kuniaki Nagayama1*, Radostion Danev and Tadao Tsuruta2

1Center for Integrative Bioscience, Okazaki National Research Institutes, Myodaiji-cho, Okazaki, 444-8585 JAPAN (nagayama@nips.ac.jp) and 2NICON Corporation., 1-6-3, Nishi-Ooi, Shinagawa, Tokyo, 140-8601 JAPAN


In traditional transmission electron microscopy (TEM) two phase-retrieval approaches are known; off-axis electron holography1) and numerical iteration using real and diffraction images.2)  Recently two alternatives have been proposed with a different view to the issue as how to retrieve complex wave functions exited from objects.  One alternative is the complex observation (COBS) based on the combination of two observation schemes, the conventional bright-field and the Zernike phase contrast.3)  Another is a non-interference scheme theoretically based on the transport of intensity equation (TIE).4)  Though TIE demands higher S/N ratio of images for the complex wave reconstruction, it is superior to COBS by its applicability to strong objects.  To overcome the limitation of COBS, the Zernike scheme has to be replaced by a scheme not relying on the weak object assumption.  Schlieren method using a knife edge, which partially intercepts electrons, was found to give a clue to the problem as it could extract the spatial derivative of phase components irrespective of the strength of objects.  The drawback of the schlieren scheme inducing long tails in images was removed by adapting the knife edge for a shutter and this dynamical filtering seems to be successful to extract phase components of exit wave functions without any deformation.  Theory and simulated results will be shown by drawing a particular comparison with the differential interference contrast, which was recently proposed for electron microscopy.5)

[1] H. Lichte, “Electron holography approaching atomic resolution”, Ultramicroscopy 20 (1986) 293-304;  [2] W. O. Saxton, Computer Techniques for Image Processing in Electron Microscopy, Academic Press, N.Y., 1978;  [3] R. Danev and K. Nagayama, “Complex Observation in Electron Microsocpy. II. Direct Visualization of Phases and Amplitudes of Exit Wave Functions”, J. Phys. Soc. Jpn. 70 (2001) 696-702;  [4] S. Bajt, A. Barty, K. A. Nugent, M. McCartney, M. Wall and D. Paganin, “Quantitative phase-sensitive imaging in a transmission electron microscope”, Ultramicroscopy 83 (2000) 67-73.  [5] R. Danev, H. Okawara, N. Usuda, K. Kametani and K. Nagayama, “A Novel Phase-contrast Transmission Electron Microscopy Producing High-contrast Topographic Images of Weak Objects”, J. Biol. Phys. 28 (2002) 627-635.




The high resolution NMR investigation of typical MRI contrast agents: possible significance to nanoscale magnetic resonance imaging

Kai-Chao Yu*1,2, Guoping Wang1,3 , Shangwu Ding1

1Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung,

Taiwan 80424, Republic of China;

2Department of Chemistry, Central-China University of Science and Technology, Wuhan, Hubei 430074,China;

3Department of Chemistry, Zhejiang University, Hangzhou, Zhejiang 320027, China.



A number of typical contrast agents for magnetic resonance imaging (MRI) are studied with liquid state nuclear magnetic resonance (NMR) spectroscopy. The contrast agents are added to the a number of solutions with different concentrations and the change of chemical shift and relaxation rates caused by the paramagnetic agents are subsequently measured. For solutions of small molecules, the changes in both the chemical shift and relaxation rate follow precisely the proportional relationship to the concentration of the contrast agents. For solutions of macromolecules, however, the function between the changes in chemical shift or relaxation and the concentration of contrast agents deviate evidently from the linear relationship. Theoretical analysis is given and possible applications to nanoscale magnetic resonance imaging are explored.




A denoising technique for nanoscale magnetic resonance imaging 

Shangwu Ding*1, Lian-Pin Hwang2

1Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung,

Taiwan 80424, Republic of China;

2Department of Chemistry, National Taiwan University, No.1, Sec. 4, Roosevelt Road, Taipei,

Taiwan 106, Republic of China;


The pursuit to achieving magnetic resonance imaging (MRI) with nanometer scale resolution has been vigorous among the MR researchers and biophysicists over the past few years. One of the most challenging obstacles to such high resolution is the extremely low sensitivity even with the most sensitive nucleus (proton). New designs for both hardware and software are needed to realize this feast of molecular imaging. Based on the correlation characteristics of NMR or MRI signals in the time domain, we propose a novel, general denoising technique applicable to enhancing the signal-to-noise ratio of the solid and liquid state NMR spectra or the MRI images. The algorithm and its corresponding code written in Fortran have been completed. The method is verified with a number of real NMR spectra and MRI images. It is found that, among other features, this method shows unique advantages over previously proposed denoising methods; it can also be incorporated with other approaches to further improve the spectral or image quality; in particular, it offers best image processing capacity for MRI at nanoscale.




Design of RF and gradient field microcoils for nanoscale magnetic

resonance imaging 

Shangwu Ding*, Wen-Yan Liang, Zhen Wu, Li-Chiang Chen,Yan-Chi Chen

Department of Chemistry, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung,

Taiwan 80424, Republic of China.


Magnetic resonance imaging (MRI) at high spatial resolution (~<10 μm) has been a vigorous pursuit for magnetic resonance scientists and biophysicists as well. This lab has been conducting research aiming at pushing the MRI resolution to the nanometer scale. Among the challenges for such high resolution are (i) the requirement of high magnetic field gradient and (ii) the high sensitivity for radio-frequency (RF) signal detection of the receiving coils. The use of microcoils for both RF excitation pulses or signal detection and gradient magnetic field helps overcome the aforementioned obstacles. In this work, we give a detailed analysis of the problems related to the design of MRI microcoils with emphasis placed on the new problems for imaging at nanometer scale. We also present the spectral and imaging results obtained with our newly designed and manufactured macrocoil systems.