Software for muscle fibre type classification and analysis

Abstract

Fibre type determination requires a large series of differently stained muscle sections. The manual identification of individual fibres through the series is tedious and time consuming. This paper presents a software that enables (i) adjusting the position of individual fibres through a series of differently stained sections (image registration) and identification of individual fibres through the series as well as (ii) muscle fibre classification and (iii) quantitative analysis. The data output of the system is the following: numerical and areal proportions of fibre types, fibre type size and optical density (grey level) of the final reaction product in every fibre. The muscle fibre type can be determined stepwise, based on one set of stained sections while further, newly stained sections can be added to the already defined muscle fibre profile. Several advantages of the presented software application in skeletal muscle research are presented. The system is semiquantitative, flexible, and user friendly.

Figure 1

Muscle fibre registration applying MuscRegM programme (Figure 1a) and muscle fibre type classification applying FibReg programme (Figure 1b) in serial sections of human biceps femoris muscle. In Figure 1a the original images are arranged in the first two columns and the registered images in the third and fourth column. The first image of the first column and the first image of the third column are ‘the master’ images (the first column presents immunohistochemical demonstration of myosin heavy chain isoforms MyHC-1, MyHC-2a, MyHC-2x whilst the second column presents the corresponding mRNA transcripts for MyHC-1, MyHC-2a, MyHC-2x). Figure 1b shows the thresholding of the registered images, which is the essential step in the fibre classification.

Figure 2

Mismatch between the MyHC isoforms and mRNA transcripts expression in human biceps femoris muscle. Immunohistochemical demonstration of MyHC-1 by BA-D5 (a), MyHC-2a (intensively) and -2x (moderately) by SC-71 (b), MyHC-2x by 6H1 (c) isoforms and β-slow (d), 2a (e), 2x (f) MyHC transcripts expression revealed by in situ hybridisation technique. Note the mismatched fibre No. 48, expressing MyHC-1 (a) and - 2a (b), thus classified as 1/2a fibre but expressing 2a MyHC transcripts only. Another mismatched fibre is No. 35, histochemicaly classified as type 1, but hardly expressing corresponding β-slow MyHC transcripts.