Using the Stacks FAQ

What is the image quality like?
The quality that you need will depend on what is being measured or observed.
Best quality has sufficient signal-to-noise and resolution to resolve mitochondrial cristae and membranes, but not sufficient to see membrane bilayers or tight junctions.
Image quality is mainly a function of the area to be imaged (µm), the magnification in x,y (nm/pixel), the scan speed (µsec/pixel), the image size (pixels), and cut thickness (nm), and the slice thickness (determined by the kV). The main constraint though is staining density: well stained tissue with lits of membranes in it will image well. Sparse tissue, or insufficiently stained material images poorly, and suffers from charging and beam damage issues that limit image quality and magnification. These are somewhat nonlinear effects.

Do I need special software to view data?
No – ImageJ/FIJI is freely available and free.  It runs on all platforms and will allow you to view your data as a stack of slices, or as a 3D view that can be rotated.  Tiff image stacks  open readily into Amira, Imaris, and a wide variety of other software.  Many of these may  be in use already for confocal imaging.  Individual images plug into GIMP, Photoshop and similar graphics programs.  Just be sure to get them in a non-proprietary version.  That said, there are also free viewers that support native Gatan and Themofisher image and storage formats.  You may also export surfaces from ImageJ to Blender or similar 3D modeling software.

How will I know what I am looking at?
The images look like conventional transmission EM images in textbooks and online, and so it is relatively easy to immerse yourself in the EM appearance of your cells of interest. Further, the image stacks themselves are a great learning tool for understanding the cellular ultrastructure and tissue morphology:  where some features will always be unclear in a given image, they will usually have a more classical appearance in adjacent sections.

How can I make publication images?
Make models etc in ImageJ and then transfer them to your favorite compositing software – Photoshop or Illustrator, or my favorite, GIMP (open source, multiplatform)

Do I need a computer with superpowers?
Datasets can be opened in ImageJ/FIJI on any modern laptop – if you have enough RAM then open them directly, and if not open as Virtual Stacks (which are not stored in RAM).  Virtual stacks can be scaled down to something that you can use on your computer – by (a) downsampling to a scaled version – eg 25%, (b) duplicating a small region of interest (ROI) at full resolution, (c) opening fewer slices (again by duplicating the subset you need).  However, if you plan to do extensive analysis, then it may help to have a workstation that can open 32-256 GB RAM.