Analysis of inclusions in muscle biopsies from an LOPD patient (NBSL9a) and a GAA-KO mouse. (a) Confocal images of a muscle fiber from a LOPD biopsy with excitation at 405, 488, and 568 nm respectively. The last panel shows the sum of the three images. Autofluorescent particles are excited by each of the wavelengths while a Hoechst-stained nucleus (asterisk) is only excited at 405 nm. An arrowhead points to a small normal-looking lysosome at a pole of the nucleus while an arrow points to the end of the particle row with a small brighter area. (b) Two-photon excited fluorescence of LOPD and GAA-KO fibers recorded in spectral mode on a confocal microscope. Fluorescence emission spectra from 460 to 660 nm were displayed for the areas within colored boxes and plotted in Excel. There are minor differences between the human and mouse samples - LOPD fibers have particles that stand out in brightness and are slightly red-shifted (purple box and spectrum); these particles are commonly found at the end of the row of inclusions (see also arrows in panel a). Background autofluorescence corresponds to mitochondria in I bands . (c) FLIM analysis confirms the heterogeneity of autofluorescent particles in both GAA-KO and LOPD fibers. Left panels show the intensity of fluorescence emission while right panels are pseudo-colored to represent average lifetimes. The bright particles in the LOPD fiber (arrows) are similar to those in the purple box shown in b; their average lifetime is shorter (blue color). The wide spectra (a & b) support the notion that the inclusions consist of lipofuscin; FLIM analysis suggests that the particles may mature as the disease progresses. Bars: 10 μm (a); 50 μm (b).