HOW WE DO ARRAY TOMOGRAPHY

Fixing and embedding tissue for Array TomographyTissue Fixation and Embedding

Most tissues from a variety of species can be prepared for array tomography, including brain, skin, and other soft tissues. Tissues are typically chemically fixed by immersion or whole-animal perfusion, dissected, dehydrated and embedded in resin. Fluorescent proteins typically retain their fluorescence.

 

Coating Coverslips for Array TomographyCoverslip Preparation

In order to maximize imaging resolution, we use high-tolerance coverslips, 170 µm +/- 5 µm thick to match the specifications of high numerical aperture microscope objectives. Furthermore, coverslips are chemically cleaned, coated with a proprietary layer and finally carbon-coated in order to tightly retain ultrathin tissue sections through multiple rounds of staining, imaging, and de-staining.

 

Array Tomography Slice and Mount ArrayCutting Serial Sections

Embedded tissue blocks are faced and cut on an ultramicrotome to produce arrays of up to 60 serial ultrathin sections each. Sections are usually cut at 70 nm thickness and adhered to coated coverslips. Many arrays can be cut from a single block, and can be stored indefinitely.

 

Array Tomography Multicolor StainMulticolor Staining and Destaining

We stain arrays using up to four channels, using a variety of stains, antibodies and lectins, followed by appropriate fluorophore-labeled secondary reagents. These can be removed with a destaining protocol, allowing the array to be re-stained and re-imaged. This cycle can be repeated many times, building multidimensional datasets.

 

Array Tomography Fluorescence ImagingFluorescence Imaging

We image arrays using up to 63x/1.4NA oil-immersion objectives on automated epifluorescence microscopes controlled by proprietary control and acquisition software. Imaging includes a low-magnification scan of the entire array followed by high-resolution imaging of either the entire array, or specific regions of interest. Precise mapping allows us to reliably image the same regions through multiple staining cycles.

 

Merge, Stitch, and Align for Array TomographyArray Tomography AnalysisImage Processing and Analysis

Images are deconvolved (from the microscope’s point-spread function) to maximize X/Y resolution. Image tiles from each tissue section are stitched, then the whole stack of sections is aligned to form a three-dimensional volume. Data from additional stain cycles, either epifluorecence or electron microscopy, can be merged into the stack at this point, and the 3-D volume visualized. Regions of interest can be selected, and progressed to analysis routines such as synapse counting.

 

Electron Microscopy Imaging for Array TomographySEM Staining and Processing

For samples requiring ultrastructure information, samples can also be imaged by electron microscopy. After epifluorescence cycles, arrays are de-stained once more, then treated with heavy metals and imaged by field-emission scanning electron microscopy. Samples can also be prepared for immunoelectron microscopy by incubation with primary and gold bead-labeled secondary antibodies. Electron microscopy images can be merged with epifluorescence data for correlative microscopy.