Immunohistochemical Muscle Fibertyping

Imaging

Immunohistochemical staining indicating fibertype. Click image to enlarge.

The fastest way to collect this data is to scan the entire muscle area using an automated slide scanner or automated microscope. 20X and 10X are the most common, but it depends on the animal model.

Define the Muscle Boundary

Irregular Region of Interest

At low magnification, the boundary of the muscle is traced using the irregular ROI (region of interest) tool. This allows BIOQUANT to analyze only myofibers within the boundary when working at higher magnification.

Automated Detection of Unlabeled Myofibers

Automated analysis of myofibers depends on a high-contrast label applied to the boundary of each myofiber. Generally this requires a label specific to dystrophin or laminin.

Automatic color thresholding uses this boundary stain to identify the myofibers. Manual editing with a brush and eraser makes it simple to correct mistakes.

Intelligent filters remove previously measured myofibers and myofibers that are not entirely visible within the field of view.

Measuring Unlabeled Myofibers

BIOQUANT simultaneously collects the following data from each myofiber:

  • Myofiber Cross-sectional Area
  • Myofiber Shortest Diameter
  • Myofiber Perimeter
  • Myofiber Location
  • Myofiber Circularity
  • Myofiber Fibertype
  • Number of Myofibers
  • Number of Central Nuclei per Myofiber

Automatic Detection of Labeled Myofibers

Automated analysis of myofibers depends on a high-contrast label applied to the boundary of each myofiber. Generally this requires a label specific to dystrophin or laminin.

In some cases, automatic color thresholding can use the labeling in the myofiber to identify the myofibers. Separation of clusters of myofibers can be labor intensive. Future work is planned to automate this task.

Intelligent filters remove previously measured myofibers and myofibers that are not entirely visible within the field of view.

Measuring Labeled Myofibers

BIOQUANT simultaneously collects the following data from each myofiber:

  • Myofiber Cross-sectional Area
  • Myofiber Shortest Diameter
  • Myofiber Perimeter
  • Myofiber Location
  • Myofiber Circularity
  • Myofiber Fibertype
  • Number of Myofibers
  • Number of Central Nuclei per Myofiber

Subsequent Fields of View

The Large Image Navigator in BIOQUANT makes it simple to move sequentially through a large section in consecutive, overlapping fields of view. BIOQUANT automatically tracks the boundary of the muscle and skips fields of view which are outside the muscle boundary.

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