Lattice light-sheet microscopy: Imaging molecules to embryos at high spatiotemporal resolution

Abstract

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems.

Transcription factors in eukaryotic cells can functionally regulate gene expression by acting in oligomeric assemblies formed from an intrinsically disordered protein phase transition enabled by molecular crowding

“…AFM cantilever lightsheet (by Gebhardt, J.C. et al [11]), lattice light-sheet (by Chen B.C. et al [56]), multi-focus (by Abrahamsson S. et al [57]. ), remote focusing (by Yang et al [58].…”

Unleashing Optics and Optoacoustics for Developmental Biology

“…In Figure 3F, 3D-SIM images of DNA, nuclear lamina, and nuclear pore complex (NPC) epitopes are compared to an image from a confocal laser scanning microscope showing how 3D-SIM offers superior resolution while still allowing in vivo imaging. Another implementation of SIM is shown in Figure 3A-D [82], where the sample is illuminated with a structured (lattice) light sheet, enabling fast imaging at resolutions beyond the diffraction limit, a technique which can also be combined with PALM ( Figure 3C,D).…”

Developing bioimaging and quantitative methods to study 3D genome

“…LLSM was used to image TagRFP-H2B highlighted chromosomes, at five different stages during the division of a single HeLa cell [86]. The super-resolution imaging of chromosomes, mitochondria and ER in dividing LLC-PK1 cells indicates that LLSM is a very promising tool to image, in 3D fashion, the fast dynamic processes in vivo in cell nuclei, due to its noninvasiveness, speed, and high spatial resolution.…”

“…To address the limitation that traditional SR microscope can only be used for very limited temporal durations, a new microscope named Lattice Light-sheet Microscope (LLSM) was developed which uses ultrathin light sheets derived from two-dimensional (2D) optical lattices ( Figure 4) [86]. For LLSM, the high axial resolution, negligible photobleaching and negligible background outside of the focal plane were obtained by the thinness of the sheet, while the simultaneous illumination of the entire field of view renders imaging at the rate of hundreds of planes per second even with the extremely low peak excitation intensities.…”