Fluoview fv10i confocal laser scanning microscopy

SVNV accumulation in guts and salivary glands were examined by Olympus Fluoview^® FV10i Confocal Laser Scanning Microscopy (Olympus, Tokyo, Japan) using a B-2A filter and a 60X oil-immersion objective with variable scan zoom. Images were exported using Olympus FV10-ASW Version 3.1 and further processed with ImageJ Version 1.45. The pinhole aperture was fixed at ×2.0 μm for all three channels. The laser intensity and sensitivity of light absorption for specific wavelengths were set as follow: a 405 nm laser diode at 30% of sensitivity and 30% of laser intensity for detecting blue DAPI fluorescence; a 473 nm laser diode at 45% of sensitivity and 60% of laser intensity for detecting green FITC fluorescence; and a 559 nm laser diode at 45% of sensitivity and 55% of laser intensity for detecting red Alexa Fluor 594. Z-stacks were taken for every image for all three channels with an automatic calculated optimum of 1.00 μm per slide. The depth of each insect tissue for z-stack imaging was manually adjusted and set to the highest and lowest points of the observed feature, which were determined when one of the extremes was barely perceptible. Gain settings for green channel were fixed as mentioned above to compare the intensity of SVNV signal between images. Gain for the other two channels was adjusted accordingly to obtain the best detail of nuclei and actin in each image.