Browsing by Author "Deka, Gitanjal"

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Dynamic photopatterning of cells in situ by Q-switched neodymium-doped yttrium ortho-vanadate laser
(2014-01-01)
Deka, Gitanjal
;
Okano, Kazunori
;
Kao, Fu-Jen
;
前瞻跨領域基礎科學中心
;
Center for Interdisciplinary Science
Cellular micropattering has been increasingly adopted in quantitative biological experiments. A Q-switched pulsed neodymium-doped yttrium ortho-vanadate (Nd:YVO4) laser directed in-situ microfabrication technique for cell patterning is presented. A platform is designed uniquely to achieve laser ablation. The platform is comprised of thin gold coating over a glass surface that functions as a thermal transducer and is over-layered by a cell repellant polymer layer. Micropatterns are engraved on the platform, subsequently exposing specific cell adhesive micro-domains by ablating the gold-polymer coating photothermally. Experimental results indicate that the proposed approach is applicable under culture conditions, viable toward cells, and has a higher engraving speed. Possible uses in arraying isolated single cells on the platform are also shown. Additionally, based on those micro-patterns, dynamic cellular morphological changes and migrational speed in response to geometrical barriers are studied to demonstrate the potential applications of the proposed approach. Our results further demonstrate that cells in narrower geometry had elongated shapes and higher migrational speed than those in wider geometry. Importantly, the proposed approach will provide a valuable reference for efforts to study single cell dynamics and cellular migration related processes for areas such as cell division, wound healing, and cancer invasion. (C) The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.
Metabolic variation of HeLa cells migrating on microfabricated cytophilic channels studied by the fluorescence lifetime of NADH
(2014-01-01)
Deka, Gitanjal
;
Okano, Kazunori
;
Masuhara, Hiroshi
;
Li, Yaw-Kuen
;
Kao, Fu-Jen
We report a novel method for studying cellular migration in vitro. Cytophilic microdomains were formed on a cytophobic substrate by laser ablation. HeLa cells were grown on those domains until confluence, and then channels were formed to guide cellular migration. Two-photon excitation fluorescence-lifetime imaging of NADH revealed metabolic variation among migrating and nonmigrating cells.
Multiphoton microscopy for skin wound healing study in terms of cellular metabolism and collagen regeneration
(2014-01-01)
Deka, Gitanjal
;
Okano, Kazunori
;
Wu, Wei-Wen
;
Kao, Fu-Jen
;
前瞻跨領域基礎科學中心
Multiphoton microscopy was employed to study normal skin wound healing in live rats noninvasively. Wound healing is a process involving series of biochemical events. This study evaluates the regeneration of collagen and change in cellular metabolic activity during wound healing in rats, with second harmonic generation (SHG) and fluorescence lifetime imaging microscopy (FLIM), respectively. In eukaryotic cells ATP is the molecule that holds the energy for cellular functioning. Whereas NADH is an electron donor in the metabolic pathways, required to generate ATP. Fluorescence lifetime of NADH free to protein bound ratio was evaluated to determine the relative metabolic activity. The FLIM data were acquired by a TCSPC system using SPCM software and analyzed by SPCImage software. Additionally, polarization resolved SHG signals were also collected to observe the changes in optical birefringence and hence the anisotropy of regenerated collagens from rat wound biopsy samples. Mat lab programming was used to process the data to construct the anisotropy images. Results indicated that, cells involved in healing had higher metabolic activity during the first week of healing, which decreases gradually and become equivalent to normal skin upon healing completes. A net degradation of collagen during the inflammatory phase and net regeneration starting from day 5 were observed in terms of SHG signal intensity change. Polarization resolved SHG imaging of the wound biopsy sample indicates higher value of anisotropy in proliferative phase, from day 4th to 8th, of wound formation; however the anisotropy decreases upon healing.