• 한국가시화정보학회지
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• 제21권3호
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• pp.65-74
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• 2023

Compared to epifluorescence(EPI) microscopy which captures fluorescence from the entire depth of sample, total internal reflection fluorescence(TIRF) can selectively visualize only a single surface of it. TIRF uses a thin evanescent field generated by the total internal reflection of laser light on surface. However, conventional TIRF system are designed for total internal reflection to occur at the upper surface of sample, making them unsuitable for sessile droplet imaging. We designed a TIRF system suitable for a sessile droplet imaging by utilizing slide glass as a lightguide. We presented the details for constructing the TIRF system using a prism, slide glass, air slit, and optical trap. Then, we compared the TIRF with EPI by imaging the droplet with fluorescent particles during its drying process. As a result, TIRF allows us to distinctly visualize the drying pattern on the bottom surface of droplet.

• Bulletin of the Korean Chemical Society
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• 제34권9호
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• pp.2725-2730
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• 2013

Single C-reactive protein (CRP) molecules, which are non-specific acute phase markers and products of the innate immune system, were quantitatively detected on a gold-nanopatterned biochip using evanescent field-enhanced fluorescence imaging. The $4{\times}5$ gold-nanopatterned biochip (spot diameter of 500 nm) was fabricated by electron beam nanolithography. Unlabeled CRP molecules in human serum were identified with single-molecule sandwich immunoassay by detecting secondary fluorescence generated by total internal reflection fluorescence (TIRF) microscopy. With decreased standard CRP concentrations, relative fluorescence intensities reduced in the range of 33.3 zM-800 pM. To enhance fluorescence intensities in TIRF images, the distance between biochip surface and CRP molecules was optimally adjusted by considering the quenching effect of gold and the evanescent field intensity. As a result, TIRF only detected one single-CRP molecule on the biochip the first time.

• 전기학회논문지
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• 제56권9호
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• pp.1694-1698
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• 2007

This study suggests a new fluorescence microscope to observe micro-samples within fluorophore in a variety of biomedical fields including the fluorescence analysis of a biochip, such as a DNA micro-array. A fluorescence microscope is a device for irradiating light onto a micro-object, executing an excitation and fluorescence emission process. In this study, it adopts a total internal reflection fluorescence(TIRF) method to excite a whole micro-sample substrate different from an existing way which uses an evanescent wave resulting from a total internal reflection on the micro-sample surface. Suggested TIRF microscope can reduce optical noise and obtain images with higher sensitivity thus obtain precise information about the density, quantity, location, etc. of a flurophore, and can simultaneously process separate images even when plurality of fluorophores having different excitation and fluorescent wavelength ranges is distributed, thus easily obtain information about the fluorophores.

• 대한기계학회논문집A
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• 제31권3호
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• pp.380-387
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• 2007

We carried out an image analysis of living cells forming their contacts at the bottom of the cell culturing substrate. In order to visualize the contact area selectively, we adopted total-internal-reflection-fluorescence (TIRF) method, which can illuminate the specimen volume within only several hundred nano-meters above the substrate. From the fluorescent intensity of the TRF image, we could calculate the distance of the cell surface from the substrate. As a result, we visualized the origin of cell contacts, their movements, and the change of cell-contact type from the close-contact into focal-contact with information of its vertical displacement representing the temporal evolution process of the three-dimensional cell-surface-profile near the contact area during this metamorphosis.

• BMB Reports
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• 제50권2호
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• pp.55-57
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• 2017

Toll-like receptor 4 (TLR4) together with MD2, one of the key pattern recognition receptors for a pathogen-associated molecular pattern, activates innate immunity by recognizing lipopolysaccharide (LPS) of Gram-negative bacteria. Although LBP and CD14 catalyze LPS transfer to the TLR4/MD2 complex, the detail mechanisms underlying this dynamic LPS transfer remain elusive. Using negative-stain electron microscopy, we visualized the dynamic intermediate complexes during LPS transfer-LBP/LPS micelles and ternary CD14/LBP/LPS micelle complexes. We also reconstituted the entire cascade of LPS transfer to TLR4/MD2 in a total internal reflection fluorescence (TIRF) microscope for a single molecule fluorescence analysis. These analyses reveal longitudinal LBP binding to the surface of LPS micelles and multi-round binding/unbinding of CD14 to single LBP/LPS micelles via key charged residues on LBP and CD14. Finally, we reveal that a single LPS molecule bound to CD14 is transferred to TLR4/MD2 in a TLR4-dependent manner. These discoveries, which clarify the molecular mechanism of dynamic LPS transfer to TLR4/MD2 via LBP and CD14, provide novel insights into the initiation of innate immune responses.

• Bulletin of the Korean Chemical Society
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• 제28권5호
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• pp.783-790
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• 2007

The immobilization of proteins and their molecular interactions on various polymer-modified glass substrates [i.e. 3-aminopropyltriethoxysilane (APTS), 3-glycidoxypropyltrimethoxysilane (GPTS), poly (ethylene glycol) diacrylate (PEG-DA), chitosan (CHI), glutaraldehyde (GA), 3-(trichlorosilyl)propyl methacrylate (TPM), 3'-mercaptopropyltrimethoxysilane (MPTMS), glycidyl methacrylate (GMA) and poly-l-lysine (PL).] for potential applications in a nanoarray protein chip at the single-molecule level was evaluated using prismtype dual-color total internal reflection fluorescence microscopy (dual-color TIRFM). A dual-color TIRF microscope, which contained two individual laser beams and a single high-sensitivity camera, was used for the rapid and simultaneous dual-color detection of the interactions and colocalization of different proteins labeled with different fluorescent dyes such as Alexa Fluor® 488, Qdot® 525 and Alexa Fluor® 633. Most of the polymer-modified glass substrates showed good stability and a relative high signal-to-noise (S/N) ratio over a 40-day period after making the substrates. The GPTS/CHI/GA-modified glass substrate showed a 13.5-56.3% higher relative S/N ratio than the other substrates. 1% Top-Block in 10 mM phosphate buffered saline (pH 7.4) showed a 99.2% increase in the blocking effect of non-specific adsorption. These results show that dual-color TIRFM is a powerful methodology for detecting proteins at the single-molecule level with potential applications in nanoarray chips or nano-biosensors.