Biomaterials Tutorial

Histology

Colleen Irvin
University of Washington Engineered Biomaterials

Histology is the preparation of tissue samples in order to evaluate tissue structure and cell organization using light microscopy. The end goal may be to see what cell types or matrix components are present and how they are oriented.  This information can help the researcher evaluate how these constituents contribute to the local anatomy of the tissue and its perceived function, or to help diagnose disease.   When biomaterials are tested in vivo, the final samples are examined through thin sections put onto glass slides.  Using a variety of selective stains, complex tissues can be studied. 

Light microscopy is the method most often thought of when discussing histology and the study of tissues.  For revealing ultra structure of cell interiors or cell surfaces, however, much higher magnifications can be obtained using electron microscopy techniques, which require specialized sample preparation procedures.

Yet, for light microscopy, there are a number of steps involved in processing a tissue sample into paraffin and preparing slides.  These steps include fixation of the tissue, dehydration, infiltration with embedding media, orientation, sectioning, and staining.

Fixation of the Tissue and Dehydration

First the tissue must be obtained and placed in fixative.  The fixative solution prevents the enzymes in the cell structures and tissue architecture from degrading and preserves the relationships of the various components of the sample.   A common fixative is 10% Formalin (4% formaldehyde).  Other fixatives used include methanol-acetic acid solutions or buffered zinc solution.  The fixative choice must be compatible with later uses of the samples, such as the staining methods to be used on the slides.

Infiltration with Embedding Media and Orientation

In order to cut thin slices of tissue, it must be embedded in a support medium.   For light microscopy, this is most often done by replacing the water in the tissue with paraffin.  Following fixation, water is removed from the tissues using a series of rinses with increasing concentrations of ethanol.   Since ethanol is not miscible with wax, it is removed by rinsing with xylene or toluene.  The samples are then immersed in warm liquid paraffin that replaces the xylene. Infiltrated tissue samples are then oriented in a wax block for easy handling, so that the correct aspect of the tissue can be sectioned.

Sectioning

Wax-infiltrated tissues are cut using a rotary microtome and disposable steel blade. The tissue sections are commonly cut 5-7 microns thick, floated on water, and picked up onto glass slides.  Once the section adheres to the slide and the wax is removed, components of the tissue section can be identified with histologic stains or by using antibodies against more specific molecular markers. 

Staining

The most common histological stain is probably hematoxylin and eosin (H&E), which stains the cell nuclei dark purple-blue and the cytoplasm pink.  Another very useful stain is Masson’s trichrome, which colors nuclei with a hematoxylin dye, stains collagen bright blue, and stains muscle and epithelial cells red.  For evaluating the collagen capsule of a foreign body response, Masson’s trichrome is an especially useful stain on samples from biomaterials in vivo tests. 

Some molecular markers are fragile and cannot withstand the solvents or heat of paraffin processing.   Also, some samples cannot wait several hours to process into wax.  For antibody staining of fragile markers, or in cases requiring immediate results such as surgical biopsy, frozen sections can be prepared instead of using paraffin.   This process involves freezing the samples in a simple cryomedia using a dry ice slurry or liquid nitrogen followed by sectioning on a cold cryotome.

There are atlases of images of normal tissues from all tissue types and organs found in the body are available.  These atlases help to evaluate and identify tissue constituents on the microscope.  Images are prepared with H&E or the most relevant histological stain for the tissue type.

Thanks to histologist Kelly Booher for the references to online Histology resources noted below.

References:

http://www.udel.edu/Biology/Wags/histopage/colorpage/colorpage.htm

http://www-medlib.med.utah.edu/WebPath/HISTHTML/HISTOTCH/HISTOTCH.html

Carson FL. Histotechnology, A self-instructional text, 2nd edition. Chicago: ASCP Press; 1997.   

Gartner LP, Hiatt JL. Color atlas of histology, 3rd edition. Baltimore, Maryland: Lippincott Williams &Wilkins; 2000. 

Ratner BD, Hoffman AS, Schoen FJ, and Lemons JE, editors. Biomaterials Science, San Diego, California: Academic Press; 1996.