Myofibers with Central Nuclei
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.
Defining the Muscle Boundary
At low magnification, the boundary of the muscle is traced using the irregular ROI (region of interest) tool. This allows BIOQUANT to count only myofibers within the muscle when working at higher magnification.
Detecting Central Nuclei
The counterstained nuclei appear purple. They can automatically thresholded, with some manual editing. BIOQUANT will automatically reject any nuclei not centrally located.
Automatic Myofiber Detection
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 H&E or VVG staining of muscle, it can be difficult to separate clustered myofibers since there's no clear indication of the boundary of an individual fiber. Manual editing with brush and eraser tools can help. Further work is planned to develop tools to automatically separate clustered myofibers.
Intelligent filters remove previously measured myofibers and myofibers that are not entirely visible within the field of view.
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
Certain types of stains are required for fibertype determination.
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.