This article describes the structure, histologic features, and ultrastructural features of normal adult human skeletal muscle and addresses the results of improper specimen handling during muscle biopsy. Many of the images show the normal microscopic appearance of muscle biopsy specimens, with some of the basic histological stains that are used in the evaluation of muscle biopsies. Some of the captions mention abnormalities that can be seen with those stains to provide some insight into their practical applications in biopsy diagnosis of neuromuscular disease. This article provides introductory background information to assist the reader in understanding pathologic findings in muscle in various disorders that are presented in a separate article.
The intended audience is any physician who deals with patients with primary neuromuscular disease or neuromuscular complications of systemic disease and who considers obtaining or performing a muscle biopsy for diagnosis. This includes primary care physicians, pediatricians, rheumatologists, neurologists, dermatologists, oncologists, radiologists, and surgeons. These physicians should use this article as the foundation for comparing normal muscle to what is found in the setting of disease and to learn about appropriate care and handling of skeletal muscle tissue. This article can assist physicians who read muscle biopsy reports written by others to comprehend the significance of pathological findings described in muscle biopsies by seeing how they differ from normal skeletal muscle. This article should be useful to those beginning their training in interpretation of muscle biopsies and can serve as a resource for medical students who are learning about muscle structure and function and are being introduced to neuromuscular disorders.
For a detailed discussion of muscle biopsy procedure and an overview of the clinical and laboratory features of neuromuscular disease, see Muscle Biopsy and Clinical and Laboratory Features of Neuromuscular Disease.  For details on pathology in several major categories of neuromuscular disease, see Skeletal Muscle Pathology. 
Basic structure and terminology
A layer of dense connective tissue, which is known as epimysium and is continuous with the tendon, surrounds each muscle (see the image below). A muscle is composed of numerous bundles of muscle fibers, termed fascicles, which are separated from each other by a connective tissue layer termed perimysium. Endomysium is the connective tissue that separates individual muscle fibers from each other. Mature muscle cells are termed muscle fibers or myofibers and they are often simply referred to as fibers. Each myofiber is a multinucleate syncytium formed by fusion of immature muscle cells termed myoblasts.
Sarcoplasm, the cytoplasm of each myofiber, is occupied largely by the contractile apparatus of the cell. This is composed of myofibrils arranged in sarcomeres, which are the contractile units of the cell. The sarcomeres contain a number of proteins, including alpha actinin, which is the major constituent of the Z band, and actin and myosin, which are the major components of the thin and thick filaments, respectively.
The remainder of the sarcoplasm, located between the myofibrils, is termed the intermyofibrillar network and contains mitochondria, lipid, glycogen, T-tubules, and sarcoplasmic reticulum. T-tubules are responsible for conduction of electrical signals from the cell surface to the internal areas of myofibers. The sarcoplasmic reticulum provides the intracellular storage and release of calcium required for contraction to occur. The electrical signals conducted by the T-tubules stimulate the sarcoplasmic reticulum to release calcium.
The two basic myofiber types are type 1 and type 2. The designation of these types is based on their physiologic properties, which are correlated with their cellular structural specializations and are reflected in their histochemical properties (see the following image).
Type 1 myofibers are the slow-twitch fibers. Physiologists refer to them as slow-oxidative (SO) fibers. They have a slow contraction time following electrical stimulation, and they generate less force than type 2 myofibers. If the response of a muscle to the application of gradually increasing loads is measured, the slow fibers are recruited first. They are used for sustained, low-level activity. To accomplish this, they are equipped with numerous large mitochondria and abundant intracellular lipid for oxidative metabolism.
Type 2 myofibers are the fast-twitch fibers. Physiologists call these the fast-glycolytic (FG) fibers. They have a rapid contraction time following stimulation. If the response of a muscle to the application of gradually increasing loads is measured, the fast fibers are recruited late. They are used for brief-duration intense activity and for carrying heavy loads and are specialized for anaerobic metabolism. These fibers contain smaller, less numerous mitochondria, less lipid, and have larger glycogen stores than type 1 fibers. The subgroups of type 2 fibers are not discussed here.
Each muscle has a characteristic ratio of type 1 to type 2 myofibers. For example, in the vastus lateralis, the most commonly biopsied muscle, more than 50% of the fibers (as many as two thirds) are expected to be type 2 myofibers. In the deltoid muscle, another commonly biopsied muscle, the balance typically favors type 1 myofibers. In normal human muscle, the two myofiber types are interspersed in a random interdigitating pattern. The type 1 myofibers are normally similar in size to the type 2 fibers.
Different pathologic processes alter the ratio of the myofiber types and their distributions in the muscle and may selectively affect the size of one type or the other or of both equally. Therefore, a change in the arrangement and/or sizes of the myofiber types in a muscle biopsy often provides a significant diagnostic clue about the underlying disorder.
The innervation of a particular muscle fiber determines whether it is type 1 or type 2. Therefore, if the type of motor neuron innervating a myofiber is changed, that myofiber acquires a new phenotype from its new innervation. Pathologists use this fact to evaluate for evidence of neurogenic disease in a muscle biopsy specimen. In a muscle in which denervation has been followed by reinnervation due to sprouting of residual viable motor neuron terminals, groups of myofibers of a single type are present instead of the random interdigitation normally found.
Histology of Normal Skeletal Muscle
This section presents the normal histology of skeletal muscle as seen with some of the tissue stains that are commonly used for the evaluation of muscle biopsy specimens. Some abnormalities that can be seen with these stains and some of the diagnostic utility of some of the preparations are mentioned in the discussion below. Please see Skeletal Muscle Pathology for more extensive and detailed information about the pathologic features of skeletal muscle in a variety of disorders and to see more stains used in the processing of muscle biopsy specimens.
Cryostat (frozen) sections
Hematoxylin and eosin stain
On a cryostat (frozen) hematoxylin and eosin (H&E) section, a cross-section of a frozen sample of normal skeletal muscle stained with H&E (see the image below) shows several fascicles surrounded by and separated from each other by a thin layer of perimysium. The muscle fibers are of relatively uniform size and shape, with nuclei located at the periphery of the individual muscle cells (myofibers). In normal muscle, fewer than 3% of myofibers should have internal nuclei, which are nuclei that are located in the center of the myofiber. The myofibers fit together in a mosaic pattern.
At high power (see the following image), the endomysium separating the myofibers can be observed as normally so thin and delicate it is almost invisible, and the contiguous myofibers appear to have almost no space between them. The sarcoplasm is relatively uniform throughout the cell.
On the frozen section stained with the nicotinamide adenine dinucleotide tetrazolium reductase stain (NADH) (see the first image below), which stains predominantly, but not exclusively, mitochondria in the intermyofibrillar network, the type 1 myofibers are darker than the type 2 myofibers. In normal muscle, the stain appears to be distributed fairly uniformly throughout the sarcoplasm. High power (see the second image below) allows observation that the stain is actually distributed in a punctate pattern, because it is localized mostly to the mitochondria in the intermyofibrillar network.
Myosin ATPase stain
On the frozen section for fiber-typing in the image below, which is treated with the stain for myosin ATPase at pH 10.5 (the actual pH used varies among laboratories), type 2 myofibers are stained brown, and type 1 fibers are stained pink with an eosin counterstain to make them visible. This section demonstrates the normal, random, almost checkerboard distribution of the two types of myofibers. The same stain, performed at a pH of 4.3, would demonstrate staining of the type 1 myofibers, such that the section would show exactly the reverse or complementary pattern of that seen in the image here. Many laboratories no longer perform this stain because it is labor-intensive and they rely on the less time-consuming myosin heavy chain fiber-typing stains, which are presented below.
Myosin heavy chain immunohistochemical stain
An alternative to the relatively technically difficult myosin ATPase stain is the immunohistochemical stain for myosin heavy chain. The first image below shows the stain for myosin heavy chain slow type, which stains the type 1 myofibers. In the second image below, a serial section from the same biopsy is stained for myosin heavy chain fast type, which stains the type 2 myofibers. The positive staining is brown. The pink in the sections is an eosin counterstain; without a counterstain, the negative myofibers would be invisible. Currently, hematoxylin, which is a blue stain, is more commonly used as a counterstain than eosin.
Periodic acid-Schiff stain
On the frozen periodic acid-Schiff (PAS) section, PAS-positive material, most of which is glycogen, is distributed fairly uniformly across the normal myofibers (see the following image). It is located mostly in the intermyofibrillar network, which contains much of the intracellular glycogen. Normally, the type 2 myofibers stain darker with this stain than type 1 fibers, because the type 2 fibers use glycolysis more than type 1 fibers. (Glycolysis relies largely on glucose phosphate derived from glycogen as a substrate.) The exact staining in a given case is dependent upon recent carbohydrate ingestion and exercise, so this stain cannot be used to reliably identify myofiber types.
Modified Gomori trichrome stain
With the modified Gomori trichrome stain performed on a frozen section (see the image below), the myofibers and connective tissue stain slightly different shades of blue-green. Nuclei are normally red-purple. The intermyofibrillar network exhibits punctate staining of mitochondria, which stain red, which is usually inconspicuous.
Sudan Black stain
With the Sudan Black stain for lipid, performed on a frozen section (see the image below), intracellular lipid appears blue-black and is distributed throughout the intermyofibrillar network. Type 1 myofibers stain darker than the others because of their high reliance on oxidative metabolism, which can use lipid as a substrate. For this reason, type 1 fibers have a greater lipid content than the type 2 myofibers, which rely more on anaerobic than oxidative metabolism. Oil-red-O is another stain for lipid that many laboratories use as their routine lipid stain.
Major Histocompatibility Complex Class I or Human Leucocyte Antigen Class ABC (or type I) Immunohistochemistry (HLA Class I)
The image below shows HLA Class I immunohistochemistry study as it appears in normal muscle or in a muscle that does not have an immune-mediated disorder. The capillaries exhibit strong labeling, indicated by the brown staining, and the myofibers are unstained, or negative. This study is helpful in the diagnosis of some autoimmune disorders that have little to no inflammation, in which a strongly positive HLA Class I immunohistochemistry study provides evidence of an immune-mediated process, despite the lack of inflammation. As with every procedure, this stain must be interpreted with caution because nonspecific myofiber labeling can occur in the setting of myofiber necrosis and because some disorders are not classic inflammatory myopathies, such as certain muscular dystrophies, and can be positive. No individual stain or histological finding is specifically diagnostic by itself but must be interpreted within the clinical context and in view of the composite of histopathological findings in an individual case.
Hematoxylin and eosin stain
The paraffin section is stained with H&E. In a low-power view of the paraffin section (see the first image below), the fibers are seen in longitudinal section, forming an array of fibers lined up in parallel. At high power (see the second image below) in normal myofibers, the striations, which are formed by the sarcomeres, are readily demonstrated. One of the earliest changes in myofiber necrosis is loss of the striations. On occasion, this subtle but important finding may be the only pathologic change in a sample.
Ultrastructure of Normal Skeletal Muscle
At the ultrastructural level, as seen by electron microscopy (EM), normal muscle in longitudinal section (see the following image) exhibits a remarkable architectural order. The myofibrils are the contractile machinery of the cell and are arranged in units, the sarcomeres. The boundary of each is a thin dark line, the Z disk or Z band. This is the anchor for the thin filaments, of which actin is the major constituent.
The thin filaments are best seen in the pale gray zones of the sarcomere, known as the I band, adjacent to each Z disk. The broad darker gray central region of each sarcomere is the A band, formed mostly by the overlap of the thick myosin filaments and the thin filaments. In the center of each sarcomere is a thin dark band termed the M band, flanked by thin pale H zones, where the thick and thin filaments do not overlap.
Between the myofibrils, the sarcoplasm contains the intermyofibrillar network. Mitochondria are the moderately dense oval structures located adjacent to the I bands (pale gray zones). At high power (see the image below), glycogen in the intermyofibrillar network can be seen as dark granular material distributed diffusely through this area. The triads are also visible. Each triad is formed by a segment of the T-tubule flanked on either side by the lateral sacs of the sarcoplasmic reticulum.
The T-tubule is continuous with the sarcolemma, which is the plasma membrane of the myofiber, from which it rapidly transmits the muscle cell action potential throughout the cell. When the muscle cell is at rest, calcium is sequestered from the myofibrils by the sarcoplasmic reticulum. Excitation transmitted from the myofiber surface by the T-tubule to the sarcoplasmic reticulum is responsible for the intracellular release of calcium required for contraction.
Distinguishing type 1 and type 2 myofibers is often possible based on their ultrastructural appearances. The ultrastructural specializations are correlated with their functional roles. Type 1 fibers (see the first image below), the highly oxidative myofibers, contain abundant, fairly large, prominent mitochondria and abundant lipid. The mitochondria are the ovoid structures, and the fat is contained in pale homogeneous round structures. Type 2 fibers (see the second image below) relying heavily on anaerobic metabolism contain smaller, less abundant, less prominent mitochondria. Glycogen is abundant, and lipid is more difficult to find in these myofibers than in type 1 myofibers. These two images are from two adjacent myofibers in one muscle biopsy.
Results of Improper Handling
The preceding presentation of the histologic and ultrastructural features of normal skeletal muscle has been illustrated by images taken of muscle biopsies that are technically excellent. The biopsies were performed properly and the performance of the laboratory technologists was excellent in all of these cases. If either of the biopsy or processing procedure is technically deficient, the histologic and ultrastructural features of the sample might not be maintained, which could interfere with diagnosis.
A muscle biopsy should be performed carefully to avoid excessive mechanical trauma to the muscle. The orientation of the myofibers should be maintained. Contraction of the muscle should not be permitted. Electrocautery should be avoided.
For those planning a muscle biopsy, there are 2 very important points: (1) A muscle biopsy should be performed by an individual who has knowledge of what is required and (2) the specimen should be sent to a pathology laboratory specialized for the processing of muscle biopsy specimens.
For information on the proper performance of a muscle biopsy, please see Muscle Biopsy and Clinical and Laboratory Features of Neuromuscular Disease.
Examples of suboptimal handling
The following biopsies were performed by individuals who did not follow the optimal procedures. Compare the appearances of improperly handled specimens with those of properly handled specimens shown in the preceding sections.
The specimen shown in the following image arrived at the laboratory stuck to dry ice. This improper handling caused uneven freezing of the specimen and freeze artifact, resulting in disruption of the sarcoplasmic features and a loss of information about the state of the myofibers.
The image below is from a case in which the muscle specimen was immersed in cold fixative without prior immobilization by a clamp. This allowed the muscle to hypercontract, producing the appearance of contraction bands, a finding that can be associated with myofiber necrosis. However, in this situation, this finding is an artifact and has no bearing on the diagnosis.
The following image is an electron micrograph of a specimen of muscle in which the surgeon was instructed to mince the muscle sample before submitting it in glutaraldehyde. The photograph demonstrates the serious disruption of the normally orderly ultrastructural architecture of the myofiber caused by this procedure.
In these 3 examples, improper handling of the muscle specimen at the time of biopsy in the operating room could have made diagnosis impossible. Fortunately, in each of these examples, arriving at a diagnosis was still possible.
This article has presented the normal structure, histology and ultrastructure of skeletal muscle, as seen in biopsies that have been performed to evaluate for possible neuromuscular disease. These preparations illustrate excellent examples of biopsies that have been obtained and processed optimally. This article also includes some examples of biopsies that have not been optimally obtained (but nonetheless were diagnostically useful) to contrast with the ideal appearance of skeletal muscle. This also helps to emphasize the importance of proper handling and technique and its relationship to the quality of a muscle biopsy, which is directed at those physicians who will be involved in planning or performing muscle biopsies.
In addition, this article provides a limited introduction to the correlation of muscle structure with function to assist with comprehension of basic muscle physiology needed for assessment of the various stains and preparations used in neuromuscular pathology and for understanding the clinical manifestations of some neuromuscular disorders. Finally, the article serves as a foundation to permit learning about the clinical presentation of neuromuscular disease and the pathology of skeletal muscle in neuromuscular diseases, as well as serves to demonstrate the need for proper performance of muscle biopsies.
To read further on these topics, please see Muscle Biopsy and Clinical and Laboratory Features of Neuromuscular Disease and Skeletal Muscle Pathology.