Reviewed by Lexie CornerOct 18 2024
A collaboration between the Centre de Biologie Intégrative at Toulouse University and the Institut Fresnel at Aix Marseille University in France has led to the development of a novel microscope called Extended Depth-of-Field Random Illumination Microscopy (EDF-RIM). This research was published in Light: Science & Applications.
Working Principle of Extended Depth-of-Field Random Illumination Microscopy (EDF-RIM). Image Credit: Light: Science & Applications
Fluorescence microscopy is a powerful tool in biology, allowing researchers to observe cells and tissues at the molecular level. Although this method has completely changed how we understand biological processes, imaging large and complex 3D structures like organoids or embryos remains challenging, particularly when using structured illumination microscopy (SIM) to study fine details beyond the optical resolution limit.
SIM creates super-resolved images by reconstructing multiple images taken under structured illumination. However, this process requires numerous images for each 3D slice, leading to long acquisition times and increased light exposure to living samples.
The new method overcomes these limitations by capturing entire volumes in a single image, combining super-resolution microscopy with an extended depth-of-field detection scheme.
RIM, a super-resolution method that uses random speckle patterns for illumination, is the basis for EDF-RIM. Since these patterns are unaffected by aberrations or scattering, RIM is particularly suited for 3D imaging. By analyzing the variance in images captured under various speckle patterns, a numerical algorithm reconstructs a super-resolved image.
EDF-RIM enhances RIM by incorporating an EDF detection system. This allows the projection of an entire 3D volume onto a single image by quickly scanning the focal plane, drastically reducing light exposure and acquisition time compared to traditional methods.
EDF-RIM outperforms conventional EDF microscopy by offering enhanced contrast and a two-fold increase in resolution. However, because of its projection-based nature, some axial information is lost. To address this, the researchers developed a method for estimating surface topography, such as in the case of drosophila epithelium, where fluorescence is dispersed over a curved surface, enabling the reconstruction of a super-resolved 3D image.
This breakthrough offers a more efficient and powerful approach to imaging large and complex 3D structures. It has significant potential to advance biological research where low light exposure, speed, and high resolution are essential.
Funding Information
Institut Carnot star (3D-RIM); Agence Nationale de la Recherche (ANR-18-CE13-028, ANR-20-CE45-0024, ANR-22-CE13-0039, ANR-22-CE42-0010, ANR-22-CE42-0026). This project is supported by the French National Research Agency's "France 2030" investment plan (ANR-16-CONV-0001, ANR-21-ESRE-0002) and the Aix-Marseille University Excellence Initiative (A*MIDEX).
Journal Reference:
Mazzella, L. et. al. (2024) Extended-depth of field random illumination microscopy, EDF-RIM, provides super-resolved projective imaging. Light: Science & Applications. doi.org/10.1038/s41377-024-01612-0