FAMOUS: a fast instrumental and computational pipeline for multiphoton microscopy applied to 3D imaging of muscle ultrastructure

Abstract We present a new instrumental and computational pipeline named FAMOUS: f ast a lgorithm for three-dimensional (3D) m ultiphoton microscopy of bi o medical str u cture s . This pipeline rests on a MPM strategy combined with an original 3D post-processing computational approach. In the present work, FAMOUS approach is devoted to the 3D imaging of the myosin assembly of the ultrastructure of a whole striated skeletal muscle unsliced. Raw recordings of second harmonic generation (SHG) from myosin and instrumental point-spread functions (PSF) are led simultaneously all along the unsliced muscle depth. This procedure highlights a space-variant distortion of the PSF and the SHG signals, and an optical degradation of the axial resolution increasing with imaging depth resulting from the optical heterogeneity of the muscle structure. A 3D mathematical modelling of the PSF, relying on the recent FIGARO method, evaluates and models the depth-variant evolution of the optical distortions. Then, the fast image deblurring algorithm BD3MG is employed to correct those non-stationary distortions all along the sample, thanks to a sounded regularized inverse problem methodology. This leads to the pipeline called FAMOUS, whose performance are highlighted for the optimization of the axial information of myosin structure, whose dimensions are close to the axial resolution limit. For the first time, the 3D organization of the myosin in skeletal muscle is visually shown from an unsliced whole muscle, starting with a solution of optical microscopy. The axial visualization of this organization presently disclosed were never shown until now without a preliminary procedure of sample slicing and labelling. Our original solution FAMOUS delivers a new point of view of this biological structure in the 3D and especially in the optical axis. Image information theoretically expected are now revealed visually in the optical axis for the first time in a whole organ unsliced and label free.