Lifetime of autofluorescence: A novel method to determine murine skeletal muscle fiber types

Abstract

This work describes a simple way to identify fiber types in living muscles by fluorescence lifetime imaging microscopy (FLIM). We quantified the mean values of lifetimes derived from a two-exponential fit (τ1 and τ2) in freshly dissected mouse FDB and soleus muscles. While τ1 values did not change between muscles, the distribution of τ2 shifted to higher values in FDB. To understand the origin of this difference, we obtained maps of autofluorescence lifetimes in cryosections of both muscles and paired them with immunofluorescence images of myosin heavy chain isoforms (MHC), which allow identification of fiber types. In soleus, τ2 was 3.1 ns for type I (SEM = 0.009, n = 49), 3.4 ns for type IIA (SEM = 0.01, n = 30), and 3.3 ns for type IIX (SEM = 0.01, n = 21). In FDB muscle, τ2 was 3.17 ns for type I (SEM = 0.04, n = 18), 3.5 ns for type IIA (SEM = 0.03, n = 27), and 3.62 ns for type IIX (SEM = 0.03, n = 22). From the distribution of measures, it follows that an FDB fiber with τ2 >3.3 ns is expected to be of type II, and of type I otherwise. This simple classification method has first- and second-class errors estimated at 0.06 and 0.27, respectively. Studies in progress aim at further elucidating the reasons for the different lifetimes, not just among fiber types but between fibers of the same type in the two muscles. Preliminary results point at differences in both the oxidation-reduction and protein-bound versus free states of flavins as causes for the observed divergence of fluorescence lifetimes. Lifetime maps of autofluorescence therefore constitute a tool to identify fiber type that, being practical, fast, and noninvasive, can be applied in living tissue without compromising other experimental interventions.