Figure 1
Two-photon (2P) microscopy by SHG and desmin 2P immunofluorescence in single fibers from DesR349P mice. (a) Optical beam path in 2P imaging of intrinsic myosin-derived SHG signals or 2P-desmin immunofluorescence confined to a tiny excitation volume of about 1 µm3 within the tissue (left part) in comparison to conventional 1P-confocal imaging. Although off-plane emission signals (marked as ②) are blocked from detection by the pinhole, confocal imaging suffers from Gaussian beam profiles during excitation of the whole z-depth during point-scanning. This gives rise to photo-bleaching and photo-damage while obtaining XYZ stacks in thick samples, like muscle fibers. Also, 1P-imaging requires external labels at all times. In contrast, non-linear 2P-imaging takes advantage of signals from selected intrinsic proteins, such as myosin II in muscle. This allows to minimal-invasively obtain detailed 3D views of sarcomere ultrastructure within XYZ stacks through single fibers. This 3D visualization of myofibrillar geometry is not possible from trans-illumination imaging or in histology sections. Examples from single plane SHG images as well as a 3D rendered XYZ volume from a SOL single fiber of a hom DesR349P mouse are shown (left lower panel) as well as desmin 2P-signals from wt mice. (b) Image processing algorithms applied to obtain SHG 3D volumes allow a quantitative morphometry approach using cosine angle sums (CAS) and vernier densities (VD) as morphological parameters to describe disturbances in muscle architecture as a morphological correlate for muscle weakness.
