In a medical use, ultrastructure of muscle is currently revealed by images of resected samples achieved thanks to Electron Microscopy (EM), requiring freezing and paraffin sections, with a set of histological, molecular and biochemical analyses. The resection, slicing and labelling steps cause an alteration of the phenotypic and volumetric information compared to their initial integrity. Starting from this statement, we have developed an original pipeline resting on the imbrication of an optical and computational strategy for imaging 3D biomedical structures without resorting to slicing, freezing and labelling steps. The assembly of myosin of a whole muscle is revealed thanks to the second harmonic generation (SHG) recorded with a multiphoton microscope, all along the entire 180 μm of thickness of the Extensor digitorum longus (EDL) of a wild mouse. During the SHG recording, the Point-Spread-Function (PSF) of the multiphoton microscope is recorded all along the imaging depth. This step highlights an axial broadening of the PSF while maintaining a constant planar PSF throughout the whole depth of the recordings. Then, a fitting algorithm estimated a mathematical model of the PSF, highlighting its variability into the whole image depth. Finally, a computational image restauration is led thanks to the fast image deblurring algorithm BD3MG accounting for the depth variant PSF. The non-stationary distortions all along the recorded image is employed thus correcting accurately the image distortion. The axial organization of the myosin is revealed for the first time, highlighting tubular organization of myosin into the myofibrils.
Three-dimensional (3D), in vivo and in live imaging of living samples with a sub-micrometer resolution is a current necessity for biomedical researches which corresponds to a hot topic for microscopy engineers. It is likely that the role of computational approaches is yet underestimated and underused in 3D-microscopy. This paper is an illustration of the fundamental role that could be played by such strategies. Usual imaging depths in biomedical microscopy reach few hundreds of micrometers in optimal conditions when multiphoton approaches are favored. However, light scattering and absorption still damage image quality, especially as imaging depth increases. Our approach rests on the multi-parametric 3D-PSF estimation of the true 3DGaussian model of the PSF along multi-millimeter depth. Image acquisition in MPM generates stacks of 2D images constituting an overall 3D-image. 3D-volumes of images containing a single object are selected and isolated all along the depth using automatic morphological tools. Finally, our computational 3DGaussian shape-fitting algorithm named FIGARO is applied on each individual PSF and quantify PSF full-width at half maximum (FWHM) in the 3 dimensions simultaneously with PSF tilt angles. FWHM evolution in the 3 dimensions along the 2 mm depth highlights a highly significant effect of spherical aberrations. Starting from standard values of PSF measured at sample top compared to what expected in MPM, we show an increase with a factor three of the PSF FWHM in axial plane when the sample bottom is reached, and no modification of PSF dimensions in lateral plane all along the depth. The main direction of the PSF shows a convergence toward a focal point that will be discussed
Multiphoton microscopy (MPM) is an approach now well established in biomedical sciences, especially thanks to its excitation spectrum in the near infrared range (NIR). The simultaneous imaging of numerous of these substances imposes the use of a wideband excitation spectrum, indispensable in the case of in vivo and in live imaging or for detecting phenomena at video rates. A unique spectral bandwidth, covering the range between 750 and 1000 nm has been recently demonstrated and has made emerging a simplification in MPM: the excitation system is now no longer an lock for generating multiphoton images of numerous fluorophores. But such a solution might be highly sensitive to chromatic distortions and diffraction limit which might result in detrimental effects on image quality and especially on resolution performance. This question is at the core of the current presentation. A point-spread function (PSF) estimation is realized with a standard computational tool. Our experimental strategy has shown two interesting points. First, the resolution is preserved in the lateral plan (xy) regardless of the excitation procedure chosen. Second, a significant deterioration of the resolution is observed in the axial direction (z), with a factor 4 between the best resolution obtained with a standard imaging procedure and the worst one obtained with the wider spectral bandwidth. Starting with this result, the role of a computational solution of image reconstruction is highlighted for reducing the gap observed in axial resolution between standard and wideband excitation solution of MPM. The illustration of the interest of a large spectral bandwidth of excitation is then shown on a mouse muscle sample presenting 3 fluorophores having a spectral bandwidth of excitation spread along 300 nm. This set of experiments illustrates the impact of chromatic distortions and diffraction limit on the deterioration of resolution. As a conclusion, a basic protocol for image reconstruction is used in order to highlight the interesting level of improvement of the visual image quality generated by a standard computational image restoration.
Multiphoton microscopy (MPM) is a method for characterizing biological samples, becoming more and more established in life sciences labs thanks to its label-free imaging ability. In MPM, few biological substances have been highlighted as endogenously fluorescent, such as elastin, myosin, keratin, redox indicators, collagen or amino acids. Tryptophan, an amino acid fundamental in the synthesis of proteins, is known for its endogenous fluorescence. In this article, we propose to show an original solution specifically dedicated to multiphoton microscopy with an ultrawide band laser system. The specificity stands into the filtering system based on a prisms-line allowing spatial shaping of the spectrum. Our custom-designed multiphoton microscope, coupled with a picosecond ultrawide band laser correctly filtered spectrally, is adapted for charactering the two- and three-photon absorption ranges and imaging of tryptophan. This highlights in one hand that the use of a picosecond ultrawide band laser spectrally filtered does allow to reach both the two- and three-photon abortion (TPA and ThPA) ranges of this substance. In another hand, a quantitative comparison of the resulting images shows high differences in the image quality where the three-photon image looks better contrasted and better resolved than the two-photon one. An explanation of this highly interesting phenomena can be proposed with a study of the probability of presence of multiphoton processes involved and the cross section values of ThPA and TPA. This initiating work, cumulating an innovative multiphoton setup and interesting results, plays a crucial role for extending the label-free imaging ability of MPM.
The ability of a low repetition rate pulsed-nanosecond supercontinuum source (SC) have been recently demonstrated as able to generate multiphoton images with a quality similar as those obtained with a standard titanium sapphire source. In this publication, the phenomenon involved is theoretically and experimentally studied and detailed. First, the interest of a SC source for multiphoton microscopy (MPM) is reminded and this point is illustrated with a numerical comparison of the effect of the combination of pulse duration and repetition rate of the source on the TPA of photons by a fluorophore. Then, a theoretical comparison of excitation sources used in MPM is presented. Finally, an experimental focus on the effect of the repetition rate on the image quality and the axial and lateral resolutions are highlighted. The results pave the way for simultaneous multiplex MPM.
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