Use of an Automated Image Processing Program to Quantify Recombinant Adenovirus Particles

Obenauer-Kutner, Linda; Halperin, Rebecca F.; Ihnát, Peter; Tully, Christopher P.; Bordens, Ronald; Grace, Michael J.

doi:10.1017/s1431927605050038

Cited by 8 publications

(5 citation statements)

References 12 publications

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“…This binary mask was then applied to the corresponding DHE image by a logical AND operation giving an image with DHE only in the defined ROI ( 5). Intensity of DHE in those ROI was measured using ImagePro Plus (Media Cybernetics, Silver Spring, MD) allowing for statistical assessment of ROI properties including object size, shape, and pixel intensity ± standard deviation (SD) as well as for automatic ranking of defined ROI properties (see Results section and for other examples (25, 26). In the second approach, the corresponding bright field image of DHE labeled cells was used for defining ROI (in this case for lipid droplets, see Results section).…”

Section: Methodsmentioning

confidence: 99%

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Wüstner

Færgeman

2008

Cytometry Pt A

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Intrinsically fluorescent sterols, like dehydroergosterol (DHE), mimic cholesterol closely and are therefore suitable to determine cholesterol transport by fluorescence microscopy. Disadvantages of DHE are its low quantum yield, rapid bleaching, and the fact that its excitation and emission is in the UV region of the spectrum. Thus, one has to deal with chromatic aberration and low signal-to-noise ratio. We developed a method to correct for chromatic aberration between the UV channel and the red/green channel in multicolor imaging of DHE compared with the lipid droplet marker Nile Red in living macrophage foam cells and in adipocytes. We used deconvolution microscopy and developed image segmentation techniques to assess the DHE content of lipid droplets in both cell types in an automated manner. Pulse-chase studies and colocalization analysis were performed to monitor the redistribution of DHE upon adipocyte differentiation. DHE is targeted to transferrin-positive recycling endosomes in preadipocytes but associates with droplets in mature adipocytes. Only in adipocytes but not in foam cells fluorescent sterol was confined to the droplet-limiting membrane. We developed an approach to visualize and quantify sterol content of lipid droplets in living cells with potential for automated high content screening of cellular sterol transport. ' 2008 International Society for Advancement of Cytometry Key terms cholesterol; fluorescence; deconvolution; Nile Red; adipocyte; macrophage CHOLESTEROL is an essential lipid constituent of cellular membranes. It plays an important role in membrane trafficking and signal transduction (1). These functions depend on the tightly controlled intracellular distribution of cholesterol. Determination of cholesterol's intracellular distribution and transport dynamics rely either on labeling cells with radioactive cholesterol isotopes and membrane fractionation or on imaging-based approaches. While radioactive cholesterol isotopes monitor the behavior of cholesterol in cells, membrane fractionation is a crude and time-consuming method during which the sterol can redistribute between isolated organelles. Fluorescence microscopy provides deepened insight into cellular architecture and function but requires accurate reporter molecules. Fluorescent analogs of cholesterol have been designed bearing a chemically linked extrinsic reporter moiety whose properties are optimized for detection of the molecule by fluorescence spectroscopy or microscopy (2). A disadvantage of those analogs is the fact that the fluorophore has a large impact on the physico-chemical properties of the labeled sterol due to its charge, polarity and size. A way to circumvent the labeling problem is to use sterols with intrinsic fluorescence like the natural sterol dehydroergosterol (DHE). DHE differs from cholesterol in having three additional double bounds and an extra methyl group in the sterol side chain; it is closely related to ergosterol, a sterol found in yeast cells and other fungi. DHE emits light in the UV ...

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Section: Methodsmentioning

confidence: 99%

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Wüstner

Færgeman

2008

Cytometry Pt A

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“…Excellent work has been done in this field in recent years [46][47][48] . Some researchers write custom software or macros for non-high-throughput image analysis programs, but their general applicability is limited 8,13,24,40,42,[49][50][51][52][53][54][55][56][57] . A more user-friendly solution is proprietary commercial software, typically bundled with an expensive image acquisition instrument and targeted to the pharmaceutical drug screening market 58 (for screening multiwell plates) or to clinical pathology laboratories 59 (for histological slides).…”

Section: Image Analysismentioning

confidence: 99%

Cell microarrays and RNA interference chip away at gene function

2005

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S25 P E R S P E C T I V EThe recent development of cell microarrays offers the potential to accelerate high-throughput functional genetic studies. The widespread use of RNA interference (RNAi) has prompted several groups to fabricate RNAi cell microarrays that make possible discrete, in-parallel transfection with thousands of RNAi reagents on a microarray slide. Though still a budding technology, RNAi cell microarrays promise to increase the efficiency, economy and ease of genome-wide RNAi screens in metazoan cells.

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“…Gold particles (80 ± 5 nm, BB International, Golden Gate, UK), an internal control for sizing of virus particles, were prepared in a similar manner on separate grids. Digitized images prepared from sample grids were then rapidly processed using an automated macro program within Image-Pro Plus, IPP, v.4.1 (Media Cybernetics, Silver Spring, MD) as described previously (Obenauer-Kutner, 2005).…”

Section: Virus Preparationmentioning

confidence: 99%

Controlled inactivation of recombinant viruses with vitamin B2

Callahan

Wonganan

Obenauer-Kutner³

et al. 2008

Journal of Virological Methods

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Inactivated viruses are important tools for vaccine development and gene transfer. 8-methoxypsoralen (8-MOP) and long-wavelength ultraviolet irradiation (LWUVI) inactivates many viruses. Toxicity limits its use in animals and humans. Toxicological and photosensitizing properties of riboflavin make it suitable for virus inactivation in preparations for biological use. Viruses expressing beta-galactosidase were mixed with either 8-MOP (1.5 mM) or riboflavin (50 μM) and exposed to LWUVI (365 nm) for 2 hours. Virus activity was determined by limiting dilution. The half-life of the adenovirus preparation treated with 8-MOP was 8.28 nanoseconds −1 (ns −1 ) and 36.5 ns −1 after treatment with riboflavin. Despite the difference in half-life, both preparations were completely inactivated within 45 minutes. In contrast, the half-lives for adeno-associated virus (AAV) preparations were similar (63 ns −1 8-MOP vs. 67 ns −1 riboflavin). Each AAV preparation was fully inactivated within 90 minutes. The half-life of lentivirus was 193.4 ns −1 after treatment with 8-MOP and 208 ns −1 after exposure to riboflavin. Virus treated with riboflavin was inactivated within 20 minutes. Virus exposed to 8-MOP was inactivated in 90 minutes. DNA and RNA viruses can be inactivated by riboflavin and LWUVI and used in physiological systems sensitive to other photochemicals.

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Use of an Automated Image Processing Program to Quantify Recombinant Adenovirus Particles

Cited by 8 publications

References 12 publications

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets

Cell microarrays and RNA interference chip away at gene function

Controlled inactivation of recombinant viruses with vitamin B2

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