Overview of the mesoSPIM (Version 5)
The latest (and recommended) mesoSPIM version features a horizontal detection path, dual-sided excitation, modular sample holders, and sample stages with large travel range (52 x 52 x 102 mm). The sample stages are mounted to a gantry built from Qioptiq X-95 profiles. The footprint of the microscope is 1.1 x 0.75 x 0.65 m (length x width x height).
Simplified optical design of the excitation path
The mesoSPIM uses a multi-line laser combiner feeding the excitation light into a dedicated single-mode fiber for each of the two excitation paths. Light from the fiber outputs is collimated and sent to an electrically tunable lens (ETL) which allows for axially scanned light-sheet microscopy (ASLM). The focusing action of the tunable lens is relayed to the galvo scanner by a 4f system. The galvo scanner then creates the light-sheet by scanning a Gaussian beam through a scan lens.
Optical design of the excitation path
The optical path is folded several times. This allows to make the excitation path more compact, but is also required to be able to put the tunable lenses (ETLs) in a vertical section of the path to avoid gravity-induced sag of the lens. An additional fold mirror (M5) can be translated to adjust the tilt of the light-sheet.
Mechanical design of the excitation path
The excitation path is based on a Thorlabs TB1515/M large-area translation stage. This stage allows changing the distance between detection axis and excitation scan lenses - for example to accomodate immersion cuvettes of different sizes. The detection path also allows straightforward co-alignment of both light-sheets by a linear stage which controls the tilt of the light-sheet.
Mechanical design of the detection path
The detection path is based on the central Qioptiq FLS-95 rail and contains a focusing stage (PI M-406.4PD). This stage is necessary as the mesoSPIM uses air objectives which will show a focus drift when looking into immersion media of varying thickness and refractive indices. The detection path also contains a Ludl 10-position filter wheel for multicolor imaging.
Modular detection path
The detection path is based on a FLS-95 rail mounted on a focusing stage. In the standard configuration the rail carries an Olympus MVX-10 macroscope, filterwheel, and the camera mount. However, the detection rail offers ample space for custom detection paths, for example to accomodate other zoom macroscopes such as the Zeiss Axiozoom V.16 or the Nikon AZ 100.
The zoom axis of the MVX-10 macroscope is attached to a Robotis Dynamixel MX-28R servo. This allows computer control of the zoom (ranging from 0.63x to 6.3x) using mesoSPIM-control.
The mesoSPIM Version 5 features stages from Physik Instrumente with 52 x 52 x 102 mm travel range (PI L-509). In addition, a rotation stage (PI M-060.DG) provides 360° sample rotation. Ideally, the travel range should be sufficiently larger than your sample.
Sample mounting in cuvettes
A key advantage of the mesoSPIM is that it allows mounting samples in quartz or glass cuvettes which renders sample handling and exchange highly ergonomic. Cuvettes can be attached to the sample stages by magnetic holders. Especially for CLARITY-cleared samples which are index-matched to an index of 1.45, quartz cuvettes are the preferred way of imaging. These cuvettes are then immersed in a larger “immersion cuvette” which contains a medium of the same index to allow for sample translation and rotation without refocusing.
How to mount samples
Given that cleared samples come in all sizes and shapes, the mesoSPIM allows for a wide variety of sample mounting techniques. The most common are to put the specimen in a sample cuvette or to use clamping holders. Cuvette mounting is the preferred way for imaging CLARITY and CUBIC samples whereas clamping works well with dehydrated, hard samples generated using clearing methods based on organic solvents (such as BABB, iDISCO).
Open software: mesoSPIM-control
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.