AO-LLSM Microscope Achieves Aberration-Free Imaging

Scientists have combined lattice light-sheet microscopy (LLSM) with adaptive optics (AO) to capture high-resolution 3D movies of cells deep within living systems. The AO-LLSM microscope enables cells to be viewed in their native multicellular environments, under as gentle illumination as possible, with minimal external perturbation. The microscope could create new opportunities to study the diversity of intracellular dynamics, extracellular communication, and collective cell behavior across different cell types, organisms, and developmental stages.

Inside the spinal cord of a zebrafish embryo, new neurons light up in different colors, letting scientists track nerve circuit development. Courtesy of T. Liu et al./Science 2018.

The microscope uses LLSM to sweep an ultrathin sheet of light through a volume of interest while acquiring a series of images for building a high-resolution 3D movie of the dynamics within the cell. The illumination is confined to a thin plane, ensuring that regions outside the volume of interest remain unexposed, while the parallel collection of fluorescence from across the plane permits low, less perturbative intensities to be used.

An AO system maintains the thin illumination of a lattice light sheet as it penetrates within the cell, and a second AO system creates distortion-free images when researchers look down on the illuminated plane from above.

By shining a laser through either of the AO system pathways, researchers can create a bright point of light — a “guide star” — within the region they want to image. AO measures sample-induced distortions to the image of this fluorescent guide star created within the volume, and compensates for these distortions by changing the shape of a mirror to create an equal but opposite distortion.

Over large volumes, the distortions change as the light traverses different tissues. Large 3D images can be assembled from a series of subvolumes, each with its own independent excitation and detection corrections.

Using the new microscopy method, the research team from Howard Hughes Medical Institute’s (HHMI) Janelia Research Campus studied a variety of subcellular events in vivo, including organelle remodeling during mitosis and growth cone dynamics during spinal cord development. Clear delineation of cell membranes allowed researchers to isolate for individual study any cell within the multicellular environment of the intact organism. By doing so, they could compare specific processes across different cell types.

According to group leader Eric Betzig, the clarity level provided by the new system allows researchers to computationally disassemble cells in the tissue to focus on the dynamics within a single cell, such as the remodeling of internal organelles during cell division.

This level of detail is hard to see without adaptive optics, Betzig said. In his view, AO is one of the most important areas in microscopy research today, and the lattice light-sheet microscope, which excels at 3D live imaging, provides a suitable platform for its use.

The next step for the team will be to make the technology affordable and user-friendly. Researchers are working on a next-generation version that should fit on a small desk at a cost within reach of individual labs. The first such instrument will go to Janelia’s Advanced Imaging Center, where scientists from around the world could apply to use it. A guide for researchers who want to create their own copies of the AO-LLSM microscope will be made available.

Ultimately, Betzig hopes that the microscope will be commercialized, bringing adaptive optics into the mainstream.

“If you really want to understand the cell in vivo, and image it with the quality possible in vitro, this is the price of admission,” he said.

The research was published in Science (doi:10.1126/science.aaq1392).

An immune cell migrates inside a zebrafish's inner ear while scooping up particles of sugar (blue) along the way. Courtesy of T. Liu et al./Science 2018.