BIOQUANT LIFE SCIENCE
Skeletal Muscle Phenotyping


Definition of Muscle Phenotyping

Muscle phenotyping is the quantitative characterization of muscle cells to assess the practical impact of a particular genotype on muscle function. Common analytical end points include: the cross-sectional area of myocytes, the number of myocytes that express different myosin heavy chain isoforms, the distribution of myocytes of different types within the whole muscle, the number of satellite cells associated with each myocyte, and the distribution of satellite cells relative to their associated myocyte.

Staining

Many different staining techniques are used in the analysis of muscle tissue. The preferred method for a project depends on the type of data that must be collected.

ImmunofluorescenCE for morphology

munofluorescent staining. Red: laminin. Green: dystrophin. Blue: satellite cells.
Multi-Parametric MRI at 14T for Muscular Dystrophy Mice Treated with AAV Vector-Mediated Gene Therapy
(
http://dx.doi.org/10.1371/journal.pone.0124914.g005).

Ideally, identification of the borders of individual myocytes can be accomplished by immunostaining of dystrophin or laminin. This can be combined with nuclear staining to visualize satellite cells. Since myocytes frequently appear bundled together, having a specific label to indicate the boundary of each cell greatly increases the automation of cross-sectional area analysis.

Hematoxylin and eosin for morphology

Hematoxylin and eosin staining. Pink: myocytes. Purple: satellite cells.
Effects on Contralateral Muscles after Unilateral Electrical Muscle Stimulation and Exercise
(
http://dx.doi.org/10.1371/journal.pone.0052230).

Routine H&E staining, while fast and inexpensive, lacks the specificity needed for automated cross-sectional area analysis. Manual editing of the boundaries between myocytes will be needed.

ATPase Staining for Myocyte ISOFORM Typing

Alkaline Myosin ATPase Staining. Sections are paired top to bottom: A-E, B-F, C-G, D-H.
Unilateral Muscle Overuse Causes Bilateral Changes in Muscle Fiber Composition and Vascular Supply
(http://dx.doi.org/10.1371/journal.pone.0116455).

ATPase staining performed on serial tissue sections can be used to identify fiber types. Variations in pH control stain specificity to different heavy-chain isoforms. ATPase staining has the benefit of being relatively straightforward and inexpensive but the drawback of requiring multiple serial sections, one for each MyHC type visualized.

Multi-channel Fluorescence for Myocyte Isoforms

Multi-channel fluorescent staining. Red: type IIB. Blue: type I. Green: type IIA. Purple: IIX / IIAX.
Rapid Determination of Myosin Heavy Chain Expression in Rat, Mouse, and Human Skeletal Muscle Using Multicolor Immunofluorescence Analysis
(http://dx.doi.org/10.1371/journal.pone.0035273).

While more expensive and technically complex to perform, multi-channel immunofluorescence requires only a single section physical section and significantly simplifies imaging and automated fiber typing.

Image Your Sample

click image to enlarge

ATPase Staining

ATPase staining identifies muscle fibers by contractile speed allowing them to be categorized by software into Type 1 or Type 2 or "intermediate". Acidic pre-incubation causes Type 1 fibers to stain darkly. Basic pre-incubation causes Type 2 fibers to stain darkly.

Alternatively, immunohistochemical staining with dystrophin can be used to uniformly indicated the borders of individual myocytes.

Section Imaging

Scanning of the section using either a digital pathology core facility or one of the BIOQUANT microscope scanning upgrades is recommended. This preserves the staining of the section and simplifies subsequent analysis. Scanning at 20X is typical, but 40X may be necessary depending on the pathology of the sample.

Define Muscle Area

Collect Muscle Area Data

Measure the boundary of the fascicle at low magnification using BIOQUANT's irregular Region of Interest tool. The software calculates the area of the muscle, and uses this tracing to ensure that cells are counted only within the sampling area when you move to higher magnification.

Zoom in to Cellular Resolution

After measuring the fascicle area at low magnification, zoom in to cellular magnification by using the Large Image Navigator, or by changing objectives. Using the Navigator and Overview windows, BIOQUANT helps you stay within the Tissue Volume even when working at high magnification.

Field 1 Higher Resolution

Measure Cells at High Magnification

Satellite Cell Data

Automatic detection of satellite cells is challenging: the stain may not be strong enough for automatic thresholding. BIOQUANT provides the option to use a small brush to mark each cell manually. BIOQUANT then automatically counts the cells that were marked.

Myocyte Data

The ATPase staining makes it easy for BIOQUANT to automatically detect myocytes. Using color, shape, and size, BIOQUANT traces the contour of each myocyte in the field of view. Manual brush and eraser tools are available to correct minor mistakes or add or remove whole cells. Once the tracings are correct, the myocytes are measured.

Moving to the Next Field of View

Field 2

This is where the unique tools in BIOQUANT really shine. When moving to the next overlapping field of view, BIOQUANT automatically:

  • Ensures that any cells measured are within the contour of the muscle, which was traced earlier.
  • Ensures that only complete cells are counted. Any cell bisected by the edge of the field of view or the muscle boundary is excluded until later when it can be measured correctly.
  • Ensures that no cells are counted twice.

Automatic Cell Counting with Intelligent Filtering

BIOQUANT maps the location of each cell as it is counted. This allows BIOQUANT to automatically filter out cells that have already been counted, even when they appear on adjacent, overlapping fields of view. Cells tracings are mapped in the Overview Window to give you a bird's eye view of everything that has been measured so far.

Repeat the Protocol for Field 2

Computed Data

Muscle Fascicle Measured

Whole Muscle Data

  • Total Area
  • Mean Diameter

Satellite Cell Data

  • Satellite Cell Number
  • Mean Number of Satellite Cells per Myocyte

Myocyte Data

  • Positive Myocyte Number
  • Individual Positive Myocyte Number
  • Mean Positive Myocyte Area
  • Individual Positive Myocyte Diameter
  • Mean Positive Myocyte Diameter
  • Positive Myocyte Area / Muscle Area Ratio
  • Positive Myocyte Optical Density
  • Negative Myocyte Number
  • Individual Negative Myocyte Area
  • Mean Negative Myocyte Area
  • Individual Negative Myocyte Diameter
  • Mean Negative Myocyte Diameter
  • Negative Myocyte Area /  Muscle Area Ratio
  • Negative Myocyte Diameter / Muscle Diameter Ratio
  • Negative Myocyte Optical Density
  • Total Myocyte Area
  • Total Myocyte Area / Muscle Area Ratio