Electron microscopic observation

Quantitative TEM was performed using a Zeiss 902 analytical electron microscope coupled with a Pro-Scan digital camera. Several tissue overview images were captured and evaluated for possible damage induced by the freezing procedure. From the overview, higher magnification images were selected randomly and areas within the captured images of the electron micrographs were measured using the Metamorph tracing tool for semi-automatic counting. In addition to autophagosomes, each mitochondrion and rough endoplasmic reticulum (RER) structure was carefully outlined and the area within the traced region was assessed using Analysis 3.2 Software. All areas were calibrated to scale bars on the micrographs and expressed as area in nm² and as the percentage of the entire tissue area measured, and were digitally evaluated in 4 or 2 animals per group in every other field of 100 randomly selected fields per animal as described recently.10,12

Since every captured image was calibrated with a scale bar, software such as ImageJ or Adobe Photoshop is also suitable for measuring the area of the organelle profile and can be used to calculate the volume fraction of the organelle of interest.