Quasi-Infinite Lightsheet Microscopy

Abstract

Fluorescence microscopy has brought about a paradigm shift in numerous scientific domains, enabling the visualization of subcellular structures with remarkable precision. One of the most intriguing fluorescence imaging techniques is lightsheet microscopy, which combines the rapid imaging capabilities of epi-fluorescence microscopy with the superb optical sectioning abilities of confocal microscopy. The high-speed 3D imaging capabilities of lightsheet microscopy have made it particularly valuable in embryonic development research. Its applications, including clinical use in genetics and drug screening, have rapidly expanded. Nevertheless, this technique encounters a fundamental challenge in overcoming the inverse relationship between FOV and axial resolution, which is influenced by light diffraction. Various strategies have been proposed to overcome the trade-off between FOV and axial resolution in lightsheet microscopy. Structured illumination (SI) techniques extend the FOV while partially preserving axial resolution; however, they are accompanied by the emergence of intense side lobes. Digital scanning techniques employing Bessel beams present a viable solution to minimize the blurring caused by side lobes in extended point spread functions (PSFs). Unfortunately, their application is limited in fast live imaging scenarios owing to their relatively low temporal resolution. Another advanced approach, lattice lightsheet microscopy, aims to achieve an extended FOV while maintaining isotropic high resolution and temporal resolution. However, its implementation necessitates intricate and costly optical systems. In this study, we introduce an fast and simplified lightsheet microscopy method called quasi-infinite lightsheet microscopy (qILM). The qILM has been precisely devised to enable isotropic high-resolution imaging across large FOV while effectively suppressing side lobes through the interference of only four optical modes represented by plane waves. The lightsheet generated by two optical modes may exhibit relatively lower energy efficiency compared to linear Bessel beam illumination, primarily due to the presence of intense side lobes. However, this limitation can be circumvented by incorporating two additional low-frequency modes, thereby enhancing energy efficiency. Consequently, qILM enables high-speed imaging approaching real-time levels by capitalizing on the advantages offered by widefield imaging and a straightforward experimental setup, while ensuring the desired isotropic resolution. Finally, Our qILM fundamentally removes the connection between axial resolution and the achievable FOV and we demonstrate isotropic 3D imaging across large FOVs only limited by the camera pixel number. With its simplified optical configuration and high-speed imaging capabilities, qILM holds great promise for a wide range of biological and clinical applications.