Whole larva and pupa and adult drosophila were fixed in 2.5% glutaraldehyde solution for 2 hour on a shaker.
First the water was exchanged with ethanol in a graded series (30%,50%,70%,95%,100%). The ethanol wet sample was putted into CPD basket and dried using procedure at the CPD dryer in Wilmot 206a.
The water was exchanged with ethanol in a graded series (30%,50%,70%,95%,100%). The ethanol wet sample was putted into HMDS solution for 15 minutes and air dried over night.
The sputter coater is used to coat non-metallic samples with a thin layer of heavy metal. The samples were coated with 30nm of gold by the sputter coater. The system was pumped to approximately 50mTorr and 15mA run through the cathode for 6min.
For the secondary electron image collection acceleration voltage of 15Kv, long working distance and SE2 detector. Long working distance increase the depth of field but the amount of electrons that reaches the detector is lower this is the reason of the high acceleration voltage.
At this technique the chamber at the electron gun is maintained at high vacuum while a controlled amount of gas is allowed into the sample chamber. Higher pressures in the SEM sample chamber offer two primary benefits compared to traditional high vacuum chambers. First, a higher pressure minimizes the outgassing from volatile samples. Secondly, by allowing a controlled amount of gas into the chamber, charging is diminished on nonconductive samples. The samples were taken at 30 Pascal.
Adobe Photoshop was used for processing of the micrographs and colorized them. The images were converted from gray to RGB color, the magnetic lasso, fill layers, brightness/contrast adjustments and hue/saturation filters.
Anaglyphs provide a way of seeing three dimensional data using two dimensional micrographs. Two images are recorded with 3 degrees stage tilt between them. The images were processed in Photoshop in the following way. Zero tilt image were colorized to red and the tilted image were colorized to cyan colore. Then , the images were layered one on top of the other and the opacity of the top image is adjusted to 50% so that the images are superimposed.