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Brightfield Köhler illumination was provided via a fiber illuminator (model OSL1, Thorlabs, USA) coupled through a 50/50 beam splitter.
eGFP was exited using a HG fiber illuminator with filter cube (Excitation Filter EX450-490, DM505oic Mirror DM505, Barrier Filter BA520, Tokyo, Japan) and images were acquired using a high-resolution color camera head DS-Ri2 (Nikon, Tokyo, Japan).
The epifluorescence images were captured using the Precentered Fiber Illuminator (Intensilight, C-HGFI, Nikon), the ND-filter (64-fold in total), the adequate fluorescence filter set, and 40× objective lens (Nikon Plan Fluor, NA 0.60).
Images were taken on a Nikon epifluorescence microscope equipped with a Nikon Intensilight C-HGFIE fiber illuminator.
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The camera was mounted on a heavy tripod, and a subject flower was held immobile in a darkroom laboratory under controlled, directional, invariant illumination provided from a fiber optic illuminator (Schott KL1500 Electronic Light Source).
A fiber optics illuminator was used as the light source.
To provide total darkness in the module, the fiber optic illuminator is switched off and a light stop is put in the excitation filter box to block light entering the module via leaks in the illuminator.
An MI-150 fiber optic illuminator (Edmund Optics Inc., Barrington, NJ, USA) with a 150-W EKE halogen light bulb produced a high intensity bundle of light (450 to 800 nm), covering our wavelength range of interest for fluorescence imaging.
A focused light from an additional microscope fiber optic illuminator was used to increase the fly's temperature from room temperature to 30°C.
To activate bPAC, sperm samples were illuminated for 90 min in suspension in a reaction tube using a halogen fiber optics illuminator (Olympus, Tokio, Japan).
To induce capacitation by light, sperm were illuminated with a halogen fiber-optics illuminator (150 W, Intralux 6000-1; Volpi) at 37°C for 90 min. The acrosome reaction was induced by incubating sperm with 2 µM ionomycin for 10 min, incubation with 1% DMSO served as control.
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