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)].
5.5 megapixel scmos camera
Manufactured by Oxford Instruments
The 5.5 megapixel sCMOS camera is a high-resolution imaging device designed for scientific applications. It features a large sensor size and low noise characteristics, providing high-quality images for a variety of research and analysis tasks.
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4 protocols using 5.5 megapixel scmos camera
1
Imaging Polar Tube Firing in Microsporidia
2
Microscopic Visualization of Fungal Spores
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4 μl of A. algerae spores purified from infected H. zea (108 spores/ml) was incubated with 20 μl of germination buffer at 30°C for 30 min. 40 μl 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 μl 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 coverslip. 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.
3
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.
4
Microscopic Analysis of Spore Nuclear Dynamics
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4 μl of purified spores (6x10 7 spores/ml) were mixed with 40 μl of NucBlue (Invitrogen, catalog #R37605) and incubated at 25°C for 40 min. Spores were then centrifuged at 5,000 g for 1 min at room temperature and the supernatant was removed. 10 μl of germination buffer was added to the pre-stained spores and stored on ice prior to imaging. 2 ul of this reaction was added to a poly-L-lysine coated glass slide and covered with a #1.5 18 x 18 mm coverslip.
To image nuclear movement inside the spore coat prior to translocation into the PT, imaging was performed on a Nikon Eclipse Ti microscope with Nikon 60x N.A. 1.4 oil immersion Plan Apochromat Ph3 objective lens. Intensity of fluorescent excitation and intensity of transmitted light were balanced to allow simultaneous single channel single camera imaging (Duo-detection). A Zyla 5.5 megapixel sCMOS camera was used, providing a wide field of view at 28 frames per second with 30 ms exposure time, no binning was applied.
To observe nuclear translocation through the PT, imaging was performed on a Zeiss AxioObserver Z1 with 40x N.A. 1.3 EC Plan-Neofluar oil immersion objective lens. An Axiocam 503 Monochrome CCD camera was used, yielding 20 frames per second with 45 ms exposure time, and 3x3 binning was applied.
To image nuclear movement inside the spore coat prior to translocation into the PT, imaging was performed on a Nikon Eclipse Ti microscope with Nikon 60x N.A. 1.4 oil immersion Plan Apochromat Ph3 objective lens. Intensity of fluorescent excitation and intensity of transmitted light were balanced to allow simultaneous single channel single camera imaging (Duo-detection). A Zyla 5.5 megapixel sCMOS camera was used, providing a wide field of view at 28 frames per second with 30 ms exposure time, no binning was applied.
To observe nuclear translocation through the PT, imaging was performed on a Zeiss AxioObserver Z1 with 40x N.A. 1.3 EC Plan-Neofluar oil immersion objective lens. An Axiocam 503 Monochrome CCD camera was used, yielding 20 frames per second with 45 ms exposure time, and 3x3 binning was applied.
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