Analyzing Myofibers with Central Nuclei
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
Click image to enlarge.
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.
Measuring 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
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.