in a fixed position, providing a three-dimensional sampling of the tissue being examined. This is commonly known as “optical sectioning” wherein thick tissue sections are employed and utilizing the optical ability of the confocal microscope as an optical “microtome,” structural details of the tissue can be revealed in three-dimensions.
The selection of microscope objective for imaging can have serious impacts on quality of images obtained. For most applications, air objectives work effectively; the highest numerical aperture (NA) available at the magnification required should be selected, as higher NA results in more light entering the objective, which yields higher contrast and signal. For deep tissue imaging (> 2mm), the use of glycerol or water immersion objectives is preferred.
To increase the achievable depth at which confocal microscopes can be utilized to obtain images within tissues, clearing techniques (e.g. Visikol HISTO, CLARITY, 3DISCO) can by employed to render tissue specimens optically transparent. In conjunction with fluorescent labeling techniques, tissue clearing dramatically increases the depths at which confocal microscopy can image into tissue, allowing for large scale reconstructions of tissue histology and morphology on large and thick pieces of tissue (> 1 mm).
Loading and Processing Image Stacks in ImageJ (or Fiji)
Three-dimensional confocal imaging results in a collection of images as planes incrementing along the Z axis, known as an “image stack” (also known as a “z-stack”). Depending on the type of instrument and settings used during acquisition, the image may be a sequence of individual images (typically as .TIF files) where each image is a single z-plane in a single color or as a single file containing all images in the stack (e.g. TIF hyperstack, proprietary formats such as Leica .lif file or Zeiss .czi image file).