Images of shed virus particles collected with Nikon TI2 confocal microscopy system using a 60x, 1.40NA objective were segmented and quantified to determine the major and minor axis length and HA intensity measured by fluorescent non-competing antibodies as previously described (62 (link)).
Ti2 confocal microscopy system
Manufactured by Nikon
The TI2 confocal microscopy system is a high-resolution imaging platform designed for advanced microscopy applications. It features a modular design that allows for customization and integration of various components, enabling researchers to configure the system to meet their specific needs. The core function of the TI2is to provide precise, high-quality imaging of samples at the cellular and subcellular level, facilitating a wide range of scientific investigations and research endeavors.
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4 protocols using ti2 confocal microscopy system
1
Quantifying Viral Particle Morphology
2
Imaging and Quantifying Shed Virions
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Infected cells (MOI ≈ 0.003) were imaged with a Nikon Ti2 confocal microscopy system using a 40×, 1.30-NA objective at 16 h.p.i. Infected cells were selected based on surface expression of HA (using 9 nM CR9114 Fab for H1N1 strains and 19 nM FI6v3 plus 19 nM H3v-47 [71 (link)] for H3N2 strains) and M2 (using a Fab fragment derived from mAb148 [72 (link)]). Confocal z-stacks spanning a range of 15 μm were collected for the channel corresponding to labeled HA. Image analysis was performed on Nikon NIS Element software 5.21. Briefly, a maximum intensity projection was generated from confocal z-stacks, from which the body of the infected cell was identified by size and intensity and stored (Mask A). This mask was dilated by ~1 cell diameter to capture a concentric region surrounding the infected cell (Mask B). Peak detection was performed within the region between Masks A and B, where bright spots corresponding to shed virions were identified and counted.
3
Characterizing Shed Viral Particles
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Images of shed virus particles collected with Nikon TI2 confocal microscopy system using a 60×, 1.40-NA objective were segmented and quantified to determine the major and minor axis length and HA intensity measured by fluorescent non-competing antibodies as previously described (55 (link)).
4
Measuring Influenza Virus Binding Avidity
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To measure virion binding avidity, A549 cells with different Sia abundance were prepared by treatment with different concentrations of CpNA (Roche) for 30 min at 37°C. Simultaneously, viruses were labeled with fluorescent Fab fragments (18 nM CR9114 for H1N1, 19 nM FI6v3 plus 19 nM H3v-47 for H3N2) for 20 min at room temperature. Following CpNA treatment, the cells were washed with PBS twice and incubated with 100 μL virus-containing Opti-MEM at 4°C. After incubating for 30 min, virus-containing medium was removed, and the cells were washed and supplemented with cold Opti-MEM. Viruses attached to the cell surface or endocytosed were imaged by the Nikon Ti2 confocal microscopy system using a 40×, 1.30-NA objective.
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