Live-cell imaging and sequential measurements of UV intensity changes of freshly harvested and freeze-thawed gametocytes, used as a control for these experiments, was performed in two spatial dimensions using wide-field techniques and time-lapse video microscopy, as previously described12 (link). Briefly, freshly harvested gametocytes (2 × 106) were resuspended in 1 ml of SAC buffer (pH 7) and a drop of gametocyte suspension was placed on a round 35-mm glass-bottom dish. The cells were left to settle for 15 min in a microscope heated chamber. The chamber was pre-warmed to 37 °C and humidified (5% CO2) before imaging on an automated epifluorescence Nikon Eclipse Ti-U inverted microscope (Nikon, Tokyo, Japan) equipped with a 60 X oil objective lens (Plan Apo NA 1.4 aperture) and the Perfect Focus System™ for continuous maintenance of focus. Differential interference (DIC) and the corresponding UV images (excitation: 470/40 nm, emission: 525/50 nm) were acquired every 10 minutes over a 3 h period with a high-speed charge-coupled device (CCD) camera. Each movie was then exported in a separate folder from NIS Elements acquisition software as uncompressed TIFF files and accompanying Excel spreadsheets analysed using GraphPad prism.
Plan apo na 1.4 aperture
Manufactured by Nikon
Sourced in Japan
The Nikon Plan Apo NA 1.4 aperture is a high-performance objective lens designed for laboratory and scientific applications. It features a numerical aperture (NA) of 1.4, which allows for the collection of a large amount of light and enables high-resolution imaging. The lens is optimized for flatfield correction, ensuring a uniform image quality across the entire field of view.
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2 protocols using plan apo na 1.4 aperture
1
Live-Cell Imaging of Gametocyte UV Dynamics
2
Time-lapse Imaging of Aze-induced Cell Death
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Cells were plated into 24 well plates at 60,000 cells/well, allowed to adhere overnight, then stained with Hoechst 33258 nuclei stain for 15 min and treated in duplicate with Aze at 500 µM and 2000 µM in EMEM containing propidium iodide (PI) and placed in a microscope heated chamber warmed to 37 °C with 5% CO2. Images were captured with a high-speed charge-coupled device (CCD) camera using the NIS-Elements acquisition software mounted on a Nikon Ti inverted fluorescence microscope equipped with 20X objective lens (Plan Apo NA 1.4 aperture) and the Perfect Focus System™ for continuous maintenance of focus. Hoechst was monitored with a bandpass 470 emission filter and PI was monitored at emission 636. Time-lapse images were collected every 30 min over a 24 h period with 2 fields of view from each well imaged. Exposure time and brightness/ contrast setting were kept constant for each using the NIS-Elements acquisition software. Images were analysed in FIJI (Schindelin et al. 2012 (link)) following background subtraction with a rolling ball radius. PI staining was measured using the mean grey value and fluorescence was normalised to the number of Hoechst positive nuclei.
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