Acquisition of a 3i Cleared Tissue LightSheet Microscope

The Core Facility for Integrated Microscopy (CFIM) is an open access microscopy facility that offers state-of-the-art microscopy technologies to life scientists at the faculty of Health and Medical Sciences, from other faculties and for other academic and none-academic research institutions. CFIM is part of the Danish BioImaging network and the Danish node of the ERIC Euro BioImaging.

Light Sheet microscopy is the technology of choice to image large multicellular systems live and cleared. CFIM users are in high need to cover imaging of models such as embryos, spheroids, whole organs and others. CFIM will purchase two light sheets to cover a wide range of sample sizes and applications from live to fixed and cleared samples. The 3i CTLS is designed to image large cleared samples. Equipped with a large incubation chamber, it allows for flexible mounting of a big or several small cleared samples.

In order to allow flexible and stable mounting of large samples and several small samples, it is optimal to have an upright system to glue/fix samples in the chamber base. The CTLS allows for flexible mounting through magnet-based podiums.

One of the limitations of imaging large volumes with LS is acquisition speed and size of data generated. To overcome this, 3i has used a unique solution, the 3i CTLS is a Tiling light sheet selective plane illumination microscopy thanks to the Spatial Light Modulator we use to sweep the light sheet focus across the field of view in a synchronized fashion with the field of view imaged In TLS-SPIM, a large field of view (FOV) is imaged by tiling a short but thin light sheet at multiple positions within the detection focal plane and taking a camera exposure at each light sheet tiling position. It has been demonstrated that TLS-SPIM is capable of imaging large multicellular specimens of different sizes, ranging from live embryonic specimens to cleared tissue specimens, at high spatial resolution. In addition, TLS-SPIM allows the optimization of the tiling light sheet intensity profile and the tiling position in milliseconds, so that the 3D imaging ability of the microscope can be optimized in real-time based on the biological specimen and process being imaged.

In addition, for CFIM it is essential that the purchased light sheet is user friendly and imaging experiments can be automatized as much as possible and upgraded to high throughput when possible. For this, the 3i CTLS fulfills our needs very well as it allows for:

- chromatically corrected multiple channels acquisition in each plane before moving on to the next optical section of the z-stack

- acquisition of an overview montage of the chamber to facilitate navigation and to define one or several imaging volume(s).

- montage and visualization of the acquired data inside the software SlideBook.

Patent number: WO 2016/138003 A1 - Discontinuous tiling light sheets, created

by scanning coaxial beam arrays that are synchronized with the detection camera are used in TLS-SPIM to image a large FOV, by which the imaging speed of TLS-SPIM can be increased, and the raw image data size can be decreased proportionally to the number of the coaxial beams contained in the coaxial beam array. The primary feature of discontinuous light sheets is that they have multiple foci, or multiple regions of ideal sheet thickness.