Biomedical Engineering Reference
In-Depth Information
4.4.2 Preparation
Proper sample preparation is an integral and often underestimated step in
imaging analysis. It should be noted that achieving ideal probe and stain
concentrations takes a considerable amount of optimization and will often
require a signifi cant amount of time and number of trials.
Histological preparation
Histological sample preparation provides a technique that facilitates imag-
ing of tissue sections in a light microscope. Ideally, samples (i.e. tissue, scaf-
fold) to be imaged are extracted from the source in a way that does not
create damage or deform the specimen. The sample then typically under-
goes an immersion fi xation via formalin (10% neutral buffered formalin),
or aldehydes such as formaldehyde or glutaraldehyde, which disrupt the
activity of metabolic enzymes and keep the sample intact for sectioning.
Additionally, a decalcifi cation step is often necessary for musculoskeletal
tissues using a decalcifying agent such as an acid (picric, acetic) or chelating
agent (EDTA). Special steps may also be required for particular scaffolding
materials (Holy and Yakubovich, 2000). An alternative protocol that does
not require a decalcifi cation step is provided at the end of the section.
Next, the samples are dehydrated, typically with a series of ethanol washes
of increasing concentration followed by a clearing agent such as Xylene or
Xylene substitute for scaffolds containing polyesters to eliminate alcohol
residue. The samples are then embedded in paraffi n, for light microscopy,
or stiffer resins for thin sections used in electron microscopy such as epoxy
and acrylic. External embedding is performed with a mold and uses a gel or
wax, which is hardened before sectioning. The sectioning process involves a
microtome for optical microscopy that provides a slice thickness in the range
of 5-10 um and an ultramicrotome for electron microscopy, especially TEM,
which uses a diamond blade capable of cutting sections from 50 to 100 nm.
Staining methods
A variety of staining methods are utilized to introduce contrast to structures
in light microscopy. An important component of most staining processes is
the use of a counterstain, a stain of a contrasting color, designed to make
the primary stain stand out and enhance the fi nal image. A compilation of
protocols for three of the most commonly used stains in histology that are
capable of visualizing proteins and cellular components is provided below.
All provided concentrations and times are merely a guide and should be
optimized for each specifi c application. The ideal values will differ with both
preparation method and sample type. Unless otherwise specifi ed all stain-
ing protocols will use sectioned tissue as a starting point, refer to section
