• Title/Summary/Keyword: single cell imaging

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Single-molecule fluorescence in situ hybridization: Quantitative imaging of single RNA molecules

  • Kwon, Sunjong
    • BMB Reports
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    • v.46 no.2
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    • pp.65-72
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    • 2013
  • In situ detection of RNAs is becoming increasingly important for analysis of gene expression within and between intact cells in tissues. International genomics efforts are now cataloging patterns of RNA transcription that play roles in cell function, differentiation, and disease formation, and they are demon-strating the importance of coding and noncoding RNA transcripts in these processes. However, these techniques typically provide ensemble averages of transcription across many cells. In situ hybridization-based analysis methods complement these studies by providing information about how expression levels change between cells within normal and diseased tissues, and they provide information about the localization of transcripts within cells, which is important in understanding mechanisms of gene regulation. Multi-color, single-molecule fluorescence in situ hybridization (smFISH) is particularly useful since it enables analysis of several different transcripts simultaneously. Combining smFISH with immunofluorescent protein detection provides additional information about the association between transcription level, cellular localization, and protein expression in individual cells.

A Study on Single Cell Polarized Signals Using Polydimethylsiloxane-based Micropatterned Channel System (폴리디메틸실록산 기반 마이크로패턴 채널 시스템을 이용한 단일 세포의 극성 신호에 관한 연구)

  • Suh, Jung-Soo;Lee, Chanbin;Pan, Yijia;Wang, Yingxiao;Jung, Youngmi;Kim, Tae-Jin
    • Korean Chemical Engineering Research
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    • v.58 no.1
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    • pp.122-126
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    • 2020
  • In this study, we produced the micropatterned channel system using polydimethylsiloxane (PDMS) and micromolding in capillaries (MIMIC) technology and evaluated cellular polarity signals through high-resolved imaging at the single-cell level. In cells treated with platelet-derived growth factor (PDGF), three types of key signals in cell migration; phosphoinositide 3-kinase (PI3 K), Rac, and Actin, were strongly activated in the front area compared to the rear region, whereas myosin light chain (MLC) showed no notable activity in the front and rear areas. Our results will, therefore, provide important information and methodology for studying the correlation between cell polarity signals and cell migration under the newly defined microenvironment.

Preclinical evaluation using functional SPECT imaging of 123I-metaiodobenzylguanidine (mIBG) for adrenal medulla in normal mice

  • Yiseul Choi;Hye Kyung Chung;Sang Keun Woo;Kyo Chul Lee;Seowon Kang;Seowon Kang;Joo Hyun Kang;Iljung Lee
    • Journal of Radiopharmaceuticals and Molecular Probes
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    • v.7 no.2
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    • pp.93-98
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    • 2021
  • meta-iodobenzylguanidine is one of the norepinephrine analogs and reuptakes together with norepinephrine with norepinephrine transporter. The radioiodinated ligand, 123I-meta-iodobenzylguanidine, is the most widely used for single photon emission computed tomography imaging to diagnose functional abnormalities and tumors of the sympathetic nervous system. In this study, we performed cellular uptake studies of 123I-meta-iodobenzylguanidine in positive- and negative-norepinephrine transporter cells in vitro to verify the uptake activity for norepinephrine transporter. After 123I-meta-iodobenzylguanidine was injected via a tail vein into normal mice, Single photon emission computed tomography/computed tomography images were acquired at 1 h, 4 h, and 24 h post-injection, and quantified the distribution in each organ including the adrenal medulla as a norepinephrine transporter expressing organ. In vitro cell study showed that 123I-meta-iodobenzylguanidine specifically uptaked via norepinephrine transporter, and significant uptake of 123I-meta-iodobenzylguanidine in the adrenal medulla in vivo single photon emission computed tomography images. These results demonstrated that single photon emission computed tomography imaging with 123I-meta-iodobenzylguanidine were able to quantify the biodistribution in vivo in the adrenal medulla in normal mice.

SIMS Protein imaging with nanoparticle tagged antibody for simultaneous omic imaging

  • Lee, Seon-Yeong;Mun, Dae-Won
    • Proceedings of the Korean Vacuum Society Conference
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    • 2015.08a
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    • pp.230.1-230.1
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    • 2015
  • One of the major problems of biological ToF-SIMS imaging is the lack of protein and peptide imaging. Most of biological story telling is mianly based on proteins. The biological implication of lipid ToF-SIMS imaging would be much higher if protein imaging is provided together. Utilizing high secondary ion yields of metals, proteins can be ToF-SIMS imaged with nanoparticle tagged proteins. Nanoparticles such as Fe3O4, SiO2, PbS were used for imaing NeuN, MCH, Orexin A, ${\alpha}$ synucline, TH(Tryosine Hydroxylase) in mouse tissues with a spatial resolution of ${\sim}2{\mu}m$ using a TOF-SIMS. Lipids and neurotransmitters images obtained simultaneously with protein images were overlayed for more deeper understanding of neurobiology, which is not allowed by any other bioimaging technqiues. The protein images from TOF-SIMS were compared with confocal fluorescence microscopy and NanoSIMS images. A new sample preparation method for imaging single cell membranes in a tissue using the vibrotome technique to prepare a tissue slice without any fixation and freeze drying will be also presented briefly for Hippocampus and Hypothalamus tissues.

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Behavior of Bacteria on the Porous Substrates: Diffusion Effect (다공성 매질 표면에서 박테리아의 거동: 확산의 영향)

  • Cho, Myoung-Ock;Cho, Ji-Yong;Park, Eun-Jung;Lee, Dong-Hee;Lee, Jeong-Hoon;Kim, Jung-Kyung
    • Journal of the Korean Society of Visualization
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    • v.6 no.2
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    • pp.45-50
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    • 2008
  • It has been found that the colony size of bacteria grown on an agar plate decreases with increasing agar gel concentration. Evidenc from recent studies suggests that the bacterial colony dynamics is closely related with the mechanical properties of the substrate. We investigate whether bacterial growth on the agar substrate is controlled mostly by the nutrients' diffusion which is hindered more in porous medium than in solution. The number of bacterial cells in single colonies is found to be inversely correlated with agar concentration. High-resolution live cell imaging at the single bacterium level confirms that the bacterial growth rate is reduced with increasing agar concentration. There is a strong correlation between the slowed diffusion and the reduced number of cells in a high concentration of agar medium.

MS2 Labeling of Endogenous Beta-Actin mRNA Does Not Result in Stabilization of Degradation Intermediates

  • Kim, Songhee H.;Vieira, Melissa;Kim, Hye-Jin;Kesawat, Mahipal Singh;Park, Hye Yoon
    • Molecules and Cells
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    • v.42 no.4
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    • pp.356-362
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    • 2019
  • The binding of MS2 bacteriophage coat protein (MCP) to MS2 binding site (MBS) RNA stem-loop sequences has been widely used to label mRNA for live-cell imaging at single-molecule resolution. However, concerns have been raised recently from studies with budding yeast showing aberrant mRNA metabolism following the MS2-GFP labeling. To investigate the degradation pattern of MS2-GFP-labeled mRNA in mammalian cells and tissues, we used Northern blot analysis of ${\beta}$-actin mRNA extracted from the Actb-MBS knock-in and $MBS{\times}MCP$ hybrid mouse models. In the immortalized mouse embryonic cell lines and various organ tissues derived from the mouse models, we found no noticeable accumulation of decay products of ${\beta}$-actin mRNA compared with the wild-type mice. Our results suggest that accumulation of MBS RNA decay fragments does not always happen depending on the mRNA species and the model organisms used.

A High-Lateral Resolution MALDI Microprobe Imaging Mass Spectrometer Utilizing an Aspherical Singlet Lens

  • Han, Sang Yun;Kim, Hwan Jin;Ha, Tae Kyung
    • Bulletin of the Korean Chemical Society
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    • v.34 no.1
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    • pp.207-210
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    • 2013
  • We report the construction of a MALDI imaging mass spectrometer equipped with a specially designed laser focusing lens, a compact aspherical singlet lens, that obtains a high-lateral imaging resolution in the microprobe mode. The lens is specially designed to focus the ionization laser (${\lambda}$ = 355 nm) down to a $1{\mu}m$ diameter with a long working distance of 34.5 mm. With the lens being perpendicular to the sample surface and sharing the optical axis with the ion path, the imaging mass spectrometer achieved an imaging resolution of as good as $5{\mu}m$ along with a high detection sensitivity of 100 fmol for peptides. The mass resolution was about 900 (m/${\Delta}m$) in the linear TOF mode. The high-resolution capability of this instrument will provide a new research opportunity for label-free imaging studies of various samples including tissues and biochips, even for the study at a single cell level in the future.

A Study on an Automatic Multi-Focus System for Cell Observation

  • Park, Jaeyoung;Lee, Sangjoon
    • Journal of Information Processing Systems
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    • v.15 no.1
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    • pp.47-54
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    • 2019
  • This study is concerned with the mechanism and structure of an optical microscope and an automatic multi-focus algorithm for automatically selecting sharp images from multiple foci of a cell. To obtain precise cell images quickly, a z-axis actuator with a resolution of $0.1{\mu}m$ was designed to control an optical microscope Moreover, a lighting control system was constructed to select the color and brightness of light that best suit the object being viewed. Cell images are captured by the instrument and the sharpness of each image is determined using Gaussian and Laplacian filters. Next, cubic spline interpolation and peak detection algorithms are applied to automatically find the most vivid points among multiple images of a single object. A cancer cell imaging experiment using propidium iodide staining confirmed that a sharp multipoint image can be obtained using this microscope. The proposed system is expected to save time and effort required to extract suitable cell images and increase the convenience of cell analysis.