Observation of SARS-CoV-2 viral particles using electron microscopy-guided multiphoton microscopy

Abstract

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 highsecurity 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 superresolution 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.