• Title/Summary/Keyword: fluorescence microscopy.

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High-speed Three-dimensional Surface Profile Measurement with the HiLo Optical Imaging Technique

  • Kang, Sewon;Ryu, Inkeon;Kim, Daekeun;Kauh, Sang Ken
    • Current Optics and Photonics
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    • v.2 no.6
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    • pp.568-575
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    • 2018
  • Various techniques to measure the three-dimensional (3D) surface profile of a 3D micro- or nanostructure have been proposed. However, it is difficult to apply such techniques directly to industrial uses because most of them are relatively slow, unreliable, and expensive. The HiLo optical imaging technique, which was recently introduced in the field of fluorescence imaging, is a promising wide-field imaging technique capable of high-speed imaging with a simple optical configuration. It has not been used in measuring a 3D surface profile although confocal microscopy originally developed for fluorescence imaging has been adapted to the field of 3D optical measurement for a long time. In this paper, to the best of our knowledge, the HiLo optical imaging technique for measuring a 3D surface profile is proposed for the first time. Its optical configuration and algorithm for a precisely detecting surface position are designed, optimized, and implemented. Optical performance for several 3D microscale structures is evaluated, and it is confirmed that the capability of measuring a 3D surface profile with HiLo optical imaging technique is comparable to that with confocal microscopy.

Construction and Patterning of the biospecific affinity surfaces on gold using dendrimer

  • Hong, Mi-Yeong;Yun, Hyeon-Cheol;Kim, Hak-Seong
    • 한국생물공학회:학술대회논문집
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    • 2000.11a
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    • pp.703-706
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    • 2000
  • We constructed a biospecific affinity surface using hyper-branched dendrimers on gold for biospecific recognition, and characterized the resulting surfaces by using confocal fluorescence microscopy. The dendrimer monolayer was firstly constructed on the mercaptoundecanoic acid SAM/Au with pentafluorophenyl ester activation and further functionalized with sulfo-NHS-biotin, an activated ester of biotin. To confirm the formation of biospecific affinity surface, FITC(fluorescein isothiocyanate)-labeled avidin was loaded onto the biotinylated dendrimer monolayer, and fluorescence images of the bound avidins were investigated with a confocal microscope. The constructed biospecific affinity surface showed a much more dense and uniform fluorescence compared to those from poly-L-lysine- and cystamine SAM-based affinity surfaces. For the dependency on the concentration of added FITC-labeled avidin on the affinity surface, derived fluorescence could be detectable from as low as $1{\mu}g/ml$, and intensified up to $50{\mu}g/ml$. Further reaction of FITC-labeled avidin layer with TMR(tetramethylrhodamine)-biocytins resulted in the efficient FRET(fluorescence resonance energy transfer) phenomenon. As an extension of the study, we attempted a patterning of the affinity surfaces on gold by microcontact printing. Fluorescence of the patterned surface demonstrated that FITC-labeled avidin molecules were specifically bound to the biotinylated patches.

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Synthesis of a Novel Near-Infrared Fluorescent Dye: Applications for Fluorescence Imaging in Living Cells and Animals

  • Chen, Tongbin;Lai, Yijun;Huang, Suisheng
    • Bulletin of the Korean Chemical Society
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    • v.34 no.10
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    • pp.2937-2941
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    • 2013
  • Fluorescence imaging is considered as one of the most powerful techniques for monitoring biomolecule activities in living systems. Near-infrared (NIR) light is advantageous for minimum photodamage, deep tissue penetration, and minimum background autofluorescence interference. Herein, we have developed a new NIR fluorescent dye, namely, RB-1, based on the Rhodamine B scaffold. RB-1 exhibits excellent photophysical properties including large absorption extinction coefficients, high fluorescence quantum yields, and high photostability. In particular, RB-1 displays both absorption and emission in the NIR region of the "biological window" (650-900 nm) for imaging in biological samples. RB-1 shows absorption maximum at 614 nm (500-725 nm) and emission maximum at 712 nm (650-825 nm) in ethanol, which is superior to those of traditional rhodamine B in the selected spectral region. Furthermore, applications of RB-1 for fluorescence imaging in living cells and small animals were investigated using confocal fluorescence microscopy and in vivo imaging system with a high signal-to-noise ratio (SNR = 10.1).

Application of Epifluorescence, Microscopy for Measurement of Bacterial Population in Water Supplies (용수중(用水中) 세균계수(細菌計數)를 위한 형광검경법(螢光檢鏡法)의 응용(應用))

  • Rhee, Young-Hwan;Shin, Seung-Yee
    • Korean Journal of Environmental Agriculture
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    • v.1 no.1
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    • pp.48-52
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    • 1982
  • Methods for the measurement of aquatic bacteria can be divided into two groups. The first group of these methods is based on the 'replicon' concept that live bacterial cells, when diluted and transferred to a suitable medium, produce colonies. These methods distinguish living from dead bacteria, but they massively underestimate bacterial numbers. The second group of enumeration methods uses visual counting technique using specific apparatus such as a microscope. These methods are generally direct and simple, but it is very hard to distinguish between live and dead bacteria and between small particle and bacteria. Recently developed technique in staining methods has provided a reliable method of visual determination of aquatic bacteria. This uses epifluorescence microscopy to measure the total bacterial population. In order to present the fluorescence microscopy as a new methodology for the determination of bacterial numbers in water supplies, data were obtained from chlorine and monochloramine doses added to samples. Total counts by fluorescence microscopy were compared with standard plate count method. The total number of bacteria in water supplies can be determined with fluorescence microscopy. This technique allows better resolution of small bacteria and differentiation of particle from bacteria. Chloramine was found to persist longer in natural waters and prevent bacterial regrowth.

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Detection of Mycoplasma-like Organisms in Some Trees by Fluorescence Microscopy with Berberine Sulphate (Berberine Sulphate를 이용(利用)한 형광현미경기법(螢光顯微鏡技法)에 의(依)한 수목(樹木)마이코플라스마검정(檢定))

  • Bak, Won Chull;La, Yong Joon
    • Journal of Korean Society of Forest Science
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    • v.80 no.2
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    • pp.232-236
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    • 1991
  • The efficacy of berberine sulphate, a fluorochrome having binding properties with both DNA and RNA, was investigated for the detection of mycoplasma-like organisms(MLOs) in jujube(Zizyphus jujuba), paulownia(Paulownia tomentosa), mulberry(Morus alba) trees and periwinkle (Catharanthus roseus) plant. When examined under fluorescence microscope, berberine sulphate-stained sections of diseased samples showed distinct MLO-specific fluorescent particles in the phloem area, while such fluorescence was absent in the healthy ones. This staining technique was proved to be a very accurate method for the diagnosis of MLO infections in woody and herbaceous plants. Furthermore, the cheap and easy procedure could be used to test a great number of samples on MLO infections with reliability and rapidity.

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The Inactivation Effects of UV Light on Bacteriophage f2 (박테리오파지 f2에 대한 자외광선의 살균효과)

  • Kim, Chi-Kyung;Quae Chae
    • Microbiology and Biotechnology Letters
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    • v.11 no.3
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    • pp.155-161
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    • 1983
  • The effects of ultraviolet light on bacteriophage f2 were investigated to determine the inactivation kinetics and its mechanism. The 260nm light showed a little higher inactivation rate than the one of 300 nm. In this work, our main concern was whether structural and/or conformational changes in the protein capsid could occur by UV irradiation. The inactivation for the first 20 minutes irradiation was rapid with a loss of about 4 logs and followed by a slower rate during the next 40 minutes with no survival noted in the samples irradiated for 90 minutes or longer. The structural change of the protein capsid was examined by optical spectroscopic techniques and electron microscopy. The absorption spectra of the UV irradiated phages showed no detectable differences in terms of the spectral shape and intensity from the control phage. However, the fluorescence emission spectroscopic data, i.e. 1) fluorescence quenching of tryptophan residues upon irradiation of 300 nm light, 2) enhancement of fluorescence emission of ANS (8-aniline-1-naphthalene sulfonate) bound to the intact phages compared to the one in the UV-treated phages, and 3) decrease of energy transfer efficiency from tryptophan to ANS in the UV-treated samples, presented remarkable differences between the intact and UV-treated phages. Such a structural alteration was also observed by electron microscopy The UV-treated phages appeared to be broken and empty capsids. Therefore, the inactivation of the bacteriophage f2 by UV irradiation is thought to be attributed to the structural change in the protein capsid as well as damage in the viral RNA by UV irradiation.

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