We tested the ability of multiphoton microscopy (MPM) devices to produce label-free images of the virus Sars-Cov-2 responsible for the Covid global health crisis of the years 2020-2022. The virion cultures were grown in our high-security virology laboratory and then optically inactivated so as to preserve the integrity of the virus without its contagious nature. We used magnetic beads for concentrating the particles and increasing the viral quantity. We have determined a spectral range of excitation and emission, combined with adapted energy levels for the non-destructive excitation and efficient collection. We obtained a discriminating signature of these viral particles thanks to the nonlinear optical process involved in MPM and produced an image of virion bundles concentrated around magnetic particles. We have confirmed the origin of the MPM images from the SARS-Cov-2 viral particles thanks to electron microscopy images. This super-resolution imaging solution has allowed to reveal the shape of viral particles of SARS-Cov-2 in their entirety, combined in clusters around magnetic particles, the core of which is clearly identified by its diameter of 200 nm. We attributed the origin of the optical signature from the virions to the presence of a large quantity of genetic material in the free viral particle. This substance, the DNA or RNA, is made up of an assembly of nucleic acids with a known nonlinear optical property. We are confident that the assembly of these formally non-linear elements enabled the creation of the multiphoton microscopy signal.
We are presenting the application of an optical and computational pipeline FAMOUS for revealing the presence of free viral particles named “virions”. The idea of such a protocol is to give rise to images of virions in their environment with a soft solution for recording the native image, contrary to the standard solution of imaging virions with electron microscopy (EM) for visualizing viral particles. The final aim of the current work is to observe free viral particles of SARS-CoV-2, the virions responsible for the worldwide pandemic of Covid-19. But such particles have diameters between 80 and 120 nm, a dimension smaller than the resolution limit of optical-only microscopy solutions. We have chosen to start with the biggest free virions, cytomegalovirus (CMV), a virus from the herpesvirus family also named “Human Herpes Virus 5”. Two kinds of cultures were involved: a fluorescent culture (BAD) and a label-free one (VHLE), both being collected from infected cell culture. VHLE virions were first observed after secondary immunostaining and concentrated with magnetic nanoparticles and then without labelling. The optical protocol rests on a standard solution of multiphoton microscopy combined with a computational strategy based on the point -spread-function (PSF) recordings, its mathematical modeling and the restauration of the image resting on the PSF model. A test with free viral particles of SARS-CoV-2 is led, delivering an optical visualization of the free-viral particles. The visualization of objects aggregates obtained in both situations confirm the relevance of the pipeline FAMOUS for imaging free virions.
KEYWORDS: Human vision and color perception, Particles, Viruses, Visualization, Clouds, Image visualization, Analytical research, Social sciences, Photonics, Visual optics
Viruses are unseen enemies which tend to disarmingly spread from person-to-person, therefore causing health damages and weakens the immunity system. Their invisibility give rise to various representations, projections and imaginations that allow laypeople to tame the unseen and intangible nature of viruses. For the first time to our knowledge, we are proposing a novel and uncommon scientific approach resting on the synergy between two scientific field: Technical Sciences & Medicine (TSM) with Social Sciences and Humanities (SSH). Therefore, we present the results of our investigations concerning the evaluation of the social impact of the scientific image of free virions on a specific population, particularly affected by the Covid-19 pandemic: people over 60 years old. For this research, we have implemented two scientific imaging solutions to visualize the free viral particles of SARS-CoV-2. The first one is a standard solution of electron microscopy and the second one is an optical and computational solution of microscopy. The scientific representations of SARS-CoV-2 that we have proposed is in fact highly different from the mass media image that we can see everywhere. Concerning the targeted population, we have demonstrated that the scientific image has a negative impact on the population. Thus, the socially constructed representations of these invisible enemies have a preponderant role in driving laypeople’s emotional reactions and health-related behaviors. Therefore, imaging viruses remains a critical scientific effort that contributes irrevocably to alleviate laypeople’s misrepresentations of these invisible enemies.
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.
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