sCMOS camera powers light-sheet microscopy techniques

Researchers in Europe and the US have developed microscopes that employ a scientific CMOS (sCMOS) camera to enable imaging rapid biological processes in thick samples in unprecedented detail. What's more, the microscopes enable long-term study.

Philipp J. Keller designed the microscope used by the group from the Howard Hughes Medical Institute (Chevy Chase, MD), while Lars Hufnagel led the team from the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany). Called SiMView by Keller and Multi-View SPIM (MuVi-SPIM) by Hufnagel, the new 4-axis light-sheet microscopes build upon the selective-plane illumination microscopy (SPIM) technology developed by Keller and Stelzer at EMBL and feature a sCMOS camera (the 5.5 Mpixel, 100 fps Neo sCMOS camera from Andor Technology) at their cores.

The SiMView light sheet microscope in Philipp Keller's laboratory
The SiMView light sheet microscope in Philipp Keller's laboratory.

Like SPIM, the new microscopes shine a thin sheet of light on the sample, illuminating one layer at a time to obtain an image of the whole sample with minimal light-induced damage. However, SiMView/MuVi-SPIM uses two diagonally opposed light sources and two cameras to capture four images from different angles, eliminating the need to rotate the sample. This speeds up imaging and enables the four images to merge into a single high-quality, three-dimensional (3D) image for the first time.

"For global measurements of the dynamic behavior and structural changes of all cells in a developing organism, data acquisition must occur at speeds that match the timescales of the fastest processes of interest and with minimal time shifts between complementary views," says Keller. "SiMView/MuVi-SPIM overcomes the limitations of sequential multiview strategies and enables quantitative systems-level imaging of fast dynamic events in large living specimens under physiological conditions."

MuVi-SPIM microscope setup and data processing pipeline
MuVi-SPIM microscope setup and data processing pipeline. (a) MuVi-SPIM setup, consisting of two illumination and two detection arms arranged along two perpendicular axes. (b) The water-filled experimental chamber containing the agarose-mounted specimen at the intersection of the illumination and detection axes of the four objectives. (c) Specimen mounted in a cylindrical block of agarose and illuminated with a light sheet from one of the illumination arms. (d) The two illumination arms (light sheets 1 and 2) and the associated detection arms (camera 1 and 2). Orange arrows indicate the illumination direction and red arrows indicate the direction of the collected emission.

Both teams used the sCMOS cameras to capture a complete view of an embryo of Drosophila melanogaster within a few seconds. Hufnagel focused on the biomechanics of development using one-photon microscopy and recorded the movements of every nucleus in the embryo throughout the first three hours of life when nuclei divide very rapidly. He also produced a 20-hour video showing the fruit fly embryo from approximately two and a half hours to the stage it walked away from the microscope as a larva. Keller used both one- and two-photon microscopy, penetrating deeper inside the embryo with multiphoton microscopy to look at subcellular events happening throughout the whole embryo development until eventual hatching.

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