Array Tomography Core

Array Tomography Overview

• Physical sectioning at 70 nm
• Optical imaging with lateral resolution of <1 micron
• Synergistic with tissue preparation for EM
• Fluorescent antibodies are more flexible than EM/immunogoldlocalization
• Tissue can be probed, stripped and re-probed with antibodies
• 3D reconstructions with extremely high spatial resolution

Array tomography is a light microscopic method of analysis that allows increased structural detail to be achieved at the light microscope level such as that associated with individual synapses. Preparation for array tomography requires the same tissue embedding equipment and ultramicratome used for TEM preparation.

Tissue is embedded in a classical EM ultrastuctural media, cut into ribbons of 70 nanometer serial sections, then stained with multicolor immunofluorescence methods to distinguish subcellular epitopes. Images are acquired using a Zeiss Axio Imager Z2 microscope with coupled Photometrics CoolSNAP HQ2 CCD camera and a high resolution objective (63x 1.4NA Plan Apochromatic oil DIC).

Images from the same position on each section of the ribbon are collected making a stack for 3-dimensional reconstruction of the subjects of interest.

The image analysis is performed in a semi-automated fashion using NIH software Image J, Matlab, and a series of simple programs written and made freely available by the inventor of the method.