Project Summary
Life is a complex and dynamics process that relies on intricate spatiotemporal interactions among molecules and
subcellular organelles within cells and multicellular organisms. Recent advances in light sheet fluorescence
microscopy (LSFM), have greatly expanded our capability to visualize, track and quantify these dynamic
interactions with high-resolution across all four dimensions (4D) of spacetime with minimum invasiveness and
photobleaching. Notable among the LSFM technologies is the lattice light sheet microscopy (LLSM), which
excels at 4D subcellular dynamics imaging with resolution, speed, and non-invasiveness superior to the widely
used confocal microscopy. However, the adoption of LSFM in biomedical research remains limited due to its
incompatibility with widely used sample holders such as multi-well plates or microfluidic chips. LSFM, when
operated in an inverted open-top configuration with two orthogonal high numerical aperture (NA) objectives,
experiences complex optical aberrations that severely degrade image quality. Significant efforts, including
specialized sample holder, single-objective architecture, and adaptive optics, have been devoted, but they are
either technically demanding or suffer from various performance drawbacks. A compact, high-performance, cost-
effective, and user-friendly LSFM system that suits the widely used sample holders has yet to be developed.
This proposal aims to address this crucial incompatibility between LSFM and high-throughput imaging by
leveraging recent advances in optical metasurfaces. These microfabricated 2D metasurfaces consisting of
nanoscale plasmonic arrays with subwavelength periodicity are capable of shaping light with an unprecedented
level of complexity and precision. We will develop an inverted open-top LLSM system with integrated optical
metasurfaces that is compatible with conventional high-throughput sample holders for imaging of molecular and
subcellular dynamics within live cells and multicellular organisms. In addition, we will create an open-source
computational pipeline to facilitate the design and fabrication of optical metasurfaces for user-defined open-top
LSFM configurations. Our proposed system will provide a compact, cost-effective, and high-performance solution
to overcome the limitations of existing LSFM systems, enabling biomedical researchers to visualize subcellular
dynamics in a high-throughput and noninvasive manner. To validate the effectiveness and applicability of our
system, we will demonstrate its performance through high-content molecular dynamic mapping of chimeric
antigen receptor T (CAR-T) cells and organelle profiling of zebrafish development. While the implementation of
our metasurface-integrated open-top LSFM is on LLSM, the metasurface design pipeline and integration protocol
can be readily adapted and extended to a wide range of existing LSFM systems. By combining the power of
LSFM with metasurfaces, our proposed technology will provide an accessible and high-performance solution for
high-resolution 4D imaging of physiological and pathological processes within live cells and organisms.
