2 μl of purified A. algerae spores obtained from H. zea (6x107 spores/ml) or purified E. hellem and E. intestinalis spores from tissue culture (~108 spores/ml) were mixed with 10 μl of germination buffer. The reaction was placed on ice to prevent PT firing prior to imaging. 2 μl was placed on a poly-L-lysine-coated glass slide (Fisher Scientific, catalog #12-545-78) and sealed with a #1.5 18 x 18 mm coverslip (Fisher Scientific, catalog #12-519-21A). Polar tube firing typically occurred ~2–5 minutes after mixing the spores with the germination buffer. PT firing was imaged using a Nikon Eclipse Ti microscope with a Nikon 60x N.A. 1.4 oil immersion Plan Apochromat Ph3 phase-contrast objective lens. An Andor Zyla 5.5 megapixel sCMOS camera was used, which provided a wide field of view at 14–50 frames per second with 3–35 ms exposure time, no binning was applied. The microscope was equipped with an environmental chamber which was set at 30°C for A. algerae[24 (link)] and 37°C for Encephalitozoon species[54 (link)].
Plan apochromat ph3
Manufactured by Nikon
The Plan Apochromat Ph3 is a high-quality microscope objective lens designed for phase contrast microscopy. It features an apochromatic correction for improved color reproduction and a plan-corrected flat field for sharp images across the entire field of view. The lens has a numerical aperture of 0.95 and a working distance of 0.17 mm, making it suitable for a variety of applications that require high-resolution imaging.
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3 protocols using plan apochromat ph3
1
Imaging Polar Tube Firing in Microsporidia
2
Live-cell Imaging of Polar Tube Firing
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Live-cell imaging of PT firing 2 μl of purified spores (~10 8 spores/ml) were mixed with 10 ul of germination buffer. The reaction was placed on ice to prevent PT firing prior to imaging. 2 ul was placed on a poly-L-lysine-coated glass slide (Fisher Scientific, catalog #12-545-78) and sealed with a #1.5 18 x 18 mm coverslip (Fisher Scientific, catalog #12-519-21A). Polar tube firing typically occurred ~2-5 minutes after mixing the spores with the germination buffer. PT firing was imaged using a Nikon Eclipse Ti microscope with a Nikon 60x N.A. 1.4 oil immersion Plan Apochromat Ph3 phase-contrast objective lens. An Andor Zyla 5.5 megapixel sCMOS camera was used, which provided a wide field of view at 14-50 frames per second with 3-35 ms exposure time, no binning was applied. The microscope was equipped with an environmental chamber which was set at 30°C for these experiments.
3
Germination and Live Staining of A. algerea Spores
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4 μl of A. algerea spores (10 8 spores/ml) was incubated with 20 μl of germination buffer at 30°C for 30 min. 40 ul of NucBlue™ Live ReadyProbes™ Reagent (Invitrogen, catalog #R37605) was added and the reaction was incubated at 25°C for 20 min. Spores were pelleted by centrifugation at 1,000 g for 1 min at room temperature and the supernatant was removed. Spores were resuspended in 6 ul of fresh germination buffer. 2 μl of the reaction was placed onto a glass slide and sealed with a #1.5 18 x 18 mm coverglass. Spores were imaged using a Nikon Eclipse Ti microscope with a Nikon 60x N.A. 1.4 oil immersion Plan Apochromat Ph3 phase-contrast objective lens. A Zyla 5.5 megapixel sCMOS camera was used at 126 ms exposure time, and no binning was applied.
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