From your sample to data in less than 10 minutes
A mesoSPIM can provide you with isotropic data from a cleared and mounted whole mouse brain within 5-8 minutes. Typical single-color acquisitions in cleared mouse brains are done at 6.5 μm voxel size. And yes, that is a cleared mouse brain in the cuvette…
Isotropy across large FOVs
The mesoSPIM is an axially scanned light-sheet microscope (ASLM) for uniform z-resolution across the FOV. In ASLM, the waist of the light-sheet is scanned through the sample in synchrony with the rolling shutter readout of the camera. At the refractive index of a CLARITY-cleared sample (n=1.45) and using 488 nm illumination, the mesoSPIM achieves an axial resolution of 6.55 μm across a 13.3 mm FOV.
Capable of multiview imaging
The mesoSPIM has a vertical sample rotation axis similar to the original SPIM. This allows 360° rotation of the sample without changing the direction of gravity. This avoids any rotation-induced distortions of soft samples. Shown are maximum intensity projections of a multi-color acquisition (Alexa 594 & Autofluorescence excited at 405 nm) of a 7-day old chick embryo stained for neurofilament and cleared using BABB.
Image a whole mouse central nervous system.
The latest version of the mesoSPIM (V5) has 52 x 52 x 102 mm travel range. This is sufficient to image a whole CLARITY-cleared mouse CNS. Using the rotation stage, multi-view imaging can be performed in such large samples.
Mounting and exchanging samples is easy
The current generation of the mesoSPIM uses a 1x air objective (Olympus MVPLAPO1x). Samples are inserted from the top into an immersion cuvette (bottom) filled with a refractive index matching solution. The sample holders use kinematic mounts with magnets and can be exchanged within seconds. For CLARITY, samples are typically inserted into a sample cuvette (shown here), whereas hard samples (i.e. with iDISCO or BABB clearing) are clamped in a 3D-printed holder.
Compatible with all clearing techniques
The mesoSPIM has been tested with a variety of methods, ranging from active and passive CLARITY, X-CLARITY, CUBIC-X, BABB, iDISCO, to MASH (iDISCO/ECi) in a wide selection of samples from mouse brains, whole mouse CNS, a chicken embryo, Drosophila melanogaster, to human neocortex.
Quick screening of samples.
The combination of simple sample mounting and fast acquisitions allows quick screening of samples. For example, batches of CLARITY-cleared whole mouse brains can be prepared for screening by mounting them in imaging cuvettes. Thanks to the magnetic sample holders, mesoSPIM samples can be exchanged within seconds.
Reduced shadowing artifacts
The excitation path of the mesoSPIM is designed to reduce shadowing artifacts. In a standard SPIM, refraction, scattering, and absorption of the excitation light-sheet leads to shadowing artifacts (left side). The mesoSPIM uses a scanned Gaussian beam with NA 0.15 to create the light-sheet. This NA is higher than in a typical large-FOV digitally scanned light-sheet microscope (DSLM) which reduces shadows: The higher illumination NA shortens the shadow cone behind absorbing structures such as bubbles. This leads to a very uniform illumination profile in the sample and simplifies data analysis.
The mesoSPIM-control software is open-source and based on Python and PyQt5. It allows control of the ASLM mode and contains an acquisition manager, a table-based tool to create complex multichannel, multiview or tiling acquisitions.