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WIREs Nanomed Nanobiotechnol
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Engineered viral nanoparticles for flow cytometry and fluorescence microscopy applications

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Abstract Viral nanoparticles (VNPs) are attractive platforms for use in the biotechnology and biomedical fields because of their biological nature. A wide variety of these particles, labeled with fluorescent reporters, have been characterized using flow cytometry and cellular imaging techniques. Fluorescence microscopy allows the direct observation of VNPs on the cell surface or inside the membrane as well as the cellular localization of the nanoparticles while flow cytometry allows the statistical quantification of nanoparticle uptake and targeting specificity. These techniques are essential when characterizing the properties of VNPs and provide information toward the use of VNPs for targeting, imaging, and/or cargo delivery. WIREs Nanomed Nanobiotechnol 2012, 4:511–524. doi: 10.1002/wnan.1177 This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Nanotechnology Approaches to Biology > Nanoscale Systems in Biology

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Examples of some common viral nanoparticle (VNP) labeling chemistries.

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Flow cytometry analyses of the binding of cowpea mosaic virus (CPMV)‐glycans with native cells in a CD22‐ and sialic‐acid‐dependent manner. (a, b) CPMV‐LacNAc (gray filled) or CPMV‐BPCsial (black) incubated with Chinese hamster ovary (CHO) or CHO‐CD22 cells. (c) CPMV bearing LacNAc (gray filled) or BPCsial (thick black solid) incubated with native Raji cells. BPCsial‐bearing viruses were also incubated in the presence of the indicated concentrations of free BPC‐sialoside 3 as inhibitor. (Reprinted with permission from Ref 19. Copyright 2008 American Chemical Society)

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Fluorescently labeled potato virus X (PVX)−cell interactions using confocal microscopy. (a) PVX particles (negative control) overlaid with a differential interference contrast (DIC) image. The cell membrane was stained with A555‐labeled wheat germ agglutinin (WGA‐A555, in red). (b) PVX‐A647 (pseudocolored in green) and WGA‐555 (in red), with DIC image in the background. (c) PVX‐A647 (in green) and WGA‐555 (in red) (2× zoom). (d) PVX‐A647 (in green) and cowpea mosaic virus (CPMV)‐O488 (in red). (a‐d) Images were analyzed using Image J. (e‐i) Z‐sections were recorded at a step size of 0.3 µm. PVX‐A647 is shown in green and WGA‐A555 staining is shown in red. (e) Analysis of the data is done using Image J software, a cross section through the cell is shown, the section comprises four frames and is 1.2 µm deep. (f‐i) Same cell as shown in panel e. The data were analyzed using Imaris Software. (f) WGA‐A555 stain (cell membrane) is shown as solid. (g) Side view, WGA‐A555 (cell membrane) is shown transparent. (h) Top view. (i) Side view. (a‐i) Cell nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) and are shown in blue. The scale bar is 20 µm in all images. Imaging was performed using a Biorad 2100 confocal microscope with a 60× oil objective. (Reprinted with permission from Ref 10. Copyright 2010 American Chemical Society)

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Flow cytometric detection of cell proliferation over time following A546‐T4 nanoparticle (NP) treatment (left) and confocal microscopy images of A549 cells after uptake of Alexa 546‐T4 NPs (2282 dyes/virus) 4',6‐diamidino‐2‐phenylindole (DAPI) (blue), Alexa 546‐T4 (yellow). (Adapted with permission from Ref 11. Copyright 2011 American Chemical Society)

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Cowpea mosaic virus (CPMV) and surface vimentin colocalize on the luminal surface of rat aorta. Freshly isolated rat aorta was incubated with CPMV and vimentin antibodies ex vivo, and then 10 µm cryosections were stained with secondary antibodies for confocal microscopy. (a) Colocalization of CPMV and vimentin on aortic endothelium. * = aorta lumen. (b–d) Individual detection and colocalization of CPMV and vimentin. Green = vimentin (panels a, b, d); red = CPMV (panels a, c, d); colocalization of CPMV and vimentin signal = white (a, d, e), where (e) shows only colocalized signal. Blue = 4',6‐diamidino‐2‐phenylindole (DAPI) stain for nuclei. Bar = 100 µm (a) and 25 µm (b), respectively. (Adapted with permission from Ref 60. Copyright 2009 The Public Library of Science (PLoS) under the terms of the Creative Commons Attribution Licence)

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Packaged green fluorescent protein (GFP)‐dependent analysis of cell binding and internalization. (a) Flow cytometry of CD22‐Chinese hamster ovary (CHO) cells treated with buffer alone (gray) or fluorescently labeled anti‐CD22 antibody (blue); 4°C for 1 h, followed by washing. (b) As in panel (a); cells treated with buffer (gray), 5 (25 µg/mL, 10 nM in particles, pink), 6 (2.5 µg/mL, 1 nM in particles, orange), or 6 (25 µg/mL, 10 nM in particles, blue). (Reprinted with permission from Ref 42. Copyright 2011 American Chemical Society)

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Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
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