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: Fast algorithm for 3D multiphoton microscopy of biomedical structures. This pipeline rests on a multiphoton microscopy (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 3 dimensions 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.

An instrumental and computational pipeline for visualizing free viral particles

“…Figure 2 illustrates the raw recordings and the images resulting from the run of the pipeline FAMOUS. A constant average PSF have been taken into consideration for this run, taking into accompt that the PSF variation initially highlighted into [11] were observed exclusively into thick biological samples along the optical axis. Here the thickness of the sample is about few microns, not deep enough for producing a variation of the PSF into the optical axis.…”

“…We have recently demonstrated the high interest of merging an instrumental solution of optical characterization, the multiphoton microscopy (MPM), with the most recent computational solution for image restauration resting on the estimation of the Point-Spread-Function (PSF) of the instrument, its mathematical modeling, and the image restauration considering the instrumental response function mathematically described all along the 3D view point of the image. The technical details of the instrumental and computational pipeline FAMOUS have been published in 2021 in [11]. But the question of the object dimensions remains unsettled.…”

An optical visualization of free virions for revealing the first public enemy

“…Our pipeline FAMOUS proposes to record and to estimate mathematically the specific instrumental contribution contained into the image. The whole technical description of FAMOUS is proposed in [9]. Figure 1 represents the instrumental solution proposed for delivering an optical visualization of the free viral particles of SARS-CoV-2.…”

Application of Multiphoton Microscopic Imaging in Study of Gastric Cancer

“… 24 Single-photon excited microscopy demands excitation light in the visible wavelength range, and the excitation pulses for MPM center at much longer wavelength in the infrared range. Although different tissues exhibit varied scattering properties, 25 longer excitation wavelength in general experiences less scattering loss and hence MPM features much larger imaging depth into the tissue. 10 , 23 Ramos-Gomes applied both single-photon and 2-photon methods to imaging human micrometastatic and spreading tumor cells.…”