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

Introduction In vivo imaging provides a window into the spatially complex, rapidly evolving physiology of the cell that structural imaging alone cannot. However, observing this physiology directly involves inevitable tradeoffs of spatial resolution, temporal resolution, and phototoxicity. This is especially true when imaging in three dimensions, which is essential to obtain a complete picture of many dynamic subcellular processes. Although traditional in vivo imaging tools, such as widefield and confocal microscopy, and newer ones, such as light-sheet microscopy, can image in three dimensions, they sacrifice substantial spatiotemporal resolution to do so and, even then, can often be used for only very limited durations before altering the physiological state of the specimen. Lattice light-sheet microscopy. An ultrathin structured light sheet (blue-green, center) excites fluorescence (orange) in successive planes as it sweeps through a specimen (gray) to generate a 3D image. The speed, noninvasiveness, and high spatial resolution of this approach make it a promising tool for in vivo 3D imaging of fast dynamic processes in cells and embryos, as shown here in five surrounding examples. Lattice light-sheet microscopy. An ultrathin structured light sheet (blue-green, center) excites fluorescence (orange) in successive planes as it sweeps through a specimen (gray) to generate a 3D image. The speed, noninvasiveness, and high spatial resolution of this approach make it a promising tool for in vivo 3D imaging of fast dynamic processes in cells and embryos, as shown here in five surrounding examples. Rationale To address these limitations, we developed a new microscope using ultrathin light sheets derived from two-dimensional (2D) optical lattices. These are scanned plane-by-plane through the specimen to generate a 3D image. The thinness of the sheet leads to high axial resolution and negligible photobleaching and background outside of the focal plane, while its simultaneous illumination of the entire field of view permits imaging at hundreds of planes per second even at extremely low peak excitation intensities. By implementing either superresolution structured illumination or by dithering the lattice to create a uniform light sheet, we imaged cells and small embryos in three dimensions, often at subsecond intervals, for hundreds to thousands of time points at the diffraction limit and beyond. Results We demonstrated the technique on 20 different biological processes spanning four orders of magnitude in space and time, including the binding kinetics of single Sox2 transcription factor molecules, 3D superresolution photoactivated localization microscopy of nuclear lamins, dynamic organelle rearrangements and 3D tracking of microtubule plus ends during mitosis, neutrophil motility in a collagen mesh, and subcellular protein localization and dynamics during embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. Throughout, we established the performance advantages of lattice light-sheet microscopy compared with previous techniques and highlighted phenomena that, when seen at increased spatiotemporal detail, may hint at previously unknown biological mechanisms. Conclusion Photobleaching and phototoxicity are typically reduced by one to two orders of magnitude relative to that seen with a 1D scanned Bessel beam or the point array scanned excitation of spinning disk confocal microscopy. This suggests that the instantaneous peak power delivered to the specimen may be an even more important metric of cell health than the total photon dose and should enable extended 3D observation of endogenous levels of even sparsely expressed proteins produced by genome editing. Improvements of similar magnitude in imaging speed and a twofold gain in axial resolution relative to confocal microscopy yield 4D spatiotemporal resolution high enough to follow fast, nanoscale dynamic processes that would otherwise be obscured by poor resolution along one or more axes of spacetime. Last, the negligible background makes lattice light-sheet microscopy a promising platform for the extension of all methods of superresolution to larger and more densely fluorescent specimens and enables the study of signaling, transport, and stochastic self-assembly in complex environments with single-molecule sensitivity. From single molecules to embryos in living color Animation defines life, and the three-dimensional (3D) imaging of dynamic biological processes occurring within living specimens is essential to understand life. However, in vivo imaging, especially in 3D, involves inevitable tradeoffs of resolution, speed, and phototoxicity. Chen et al. describe a microscope that can address these concerns. They used a class of nondiffracting beams, known as 2D optical lattices, which spread the excitation energy across the entire field of view while simultaneously eliminating out-of-focus excitation. Lattice light sheets increase the speed of image acquisition and reduce phototoxicity, which expands the range of biological problems that can be investigated. The authors illustrate the power of their approach using 20 distinct biological systems ranging from single-molecule binding kinetics to cell migration and division, immunology, and embryonic development. Science, this issue 10.1126/science.1257998 A new microscope allows three-dimensional imaging of living systems at very high resolution in real time. Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond 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 the complexity of living systems.