Muscle Fiber Phenotyping
High-speed Multi-channel Fluorescence Imaging
The fastest way to collect this data is to scan the entire muscle area using an automated slide scanner or automated microscope. 20X or 10X is most common depending on the animal model.
For multi-channel fluorescent labeling, it is recommended to use a multi-band fluorescent filter cube and a color digital camera for imaging. The relative intensity of labels can be adjusted within the camera controls.
This speeds up imaging since you only have to capture one image per field of view and you don't have to apply false coloring to monochrome images and the merge them to create the same color image.
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.
Automated 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.
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.
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.