MesoSPIM: Big, Bold and Bright


Rebecca Pool

Thursday, November 14, 2019 - 15:30
Image: The mesoSPIM at Wyss Center Geneva.
In September this year, a pan-European team of researchers, led by Switzerland-based neuroscientists, launched the 'mesoSPIM initiative' to drive open source light-sheet microscopy forward.
The initiative aims to provide imaging communities with mesoscale selective plane illumination microscopes, a new generation of custom-built microscopes for large cleared samples. Incredibly, the instruments are compatible with any clearing method and can image an entire mouse central nervous system, a larger sample volume than any other available light sheet microscope.
As mesoSPIM developer, Dr Fabian Voigt from the group of Professor Fritjof Helmchen at the Brain Research Institute, University of Zurich, highlights: "We created the open-source mesoSPIM Initiative to share the latest developments in microscope instrumentation and software with the imaging community.”
“Anyone seeking high-quality anatomical data from large samples now has the information they need to build and operate their own mesoSPIM,” he adds.
XY view of mouse vasculature – cleared using the iDISCO clearing method. []
MesoSPIM started life as as a tiny side project for Voigt in 2015, when Adriano Aguzzi, University Hospital Zurich, asked Professor Helmchen for help in selecting a light-sheet microscope to swiftly screen hundreds of CLARITY-cleared mouse brains.
After unsatisfactory tests on existing commercial setups, Voigt and Helmchen decided to build their own instrument at the Helmchen Lab in Zurich. According to Voigt, existing light-sheet microscopes for cleared samples had, at the time, mostly been designed for a narrow subset of clearing techniques and did not allow multi-view imaging.
And while instruments designed for timelapse imaging in developing zebrafish and Drosophila combined straight-forward sample handling and allowed multi-view imaging, these were limited to small field-of-views of only around 1 mm.
“None of the existing instruments had the features we deemed essential for a cleared-tissue light-sheet microscope,” says Voigt. “Instruments designed to image pinhead-sized samples were incapable of imaging a mouse brain, which is the size of a fingertip, so this really motivated us to build our own microscope.”
Early days
The first mesoSPIM prototype emerged in October 2015, and was, as Voigt puts it: “built on a shoestring and assembled from left-over parts from single- and two-photon microscopes in our laboratory.”
Based on an Olympus MVX-10 microscope with a MVPLAPO2x objective, the instrument had a Hamamatsu Orca Flash V2 camera, taken from a widefield set-up, a cylindrical lens to form the light sheet and single-sided illumination. Voigt used spare electrophysiology stages for the sample XY movement, leftover stages from an old two-photon microscope for the focus stage, and lasers, borrowed from an old confocal microscope.
Given the need to rapidly screen many samples, Voigt's collaborator, Aguzzi, had asked for quick and simple sample handling and exchange. To this end, and inspired by the CLARITY-optimised light sheet microscope (COLM), Voigt started to mount samples in quartz cuvettes and designed a novel holder that used kinematic mounts from Thorlabs with magnets.
Magnetic sample holders []
As part of the set-up, the sample stages were designed to carry the mounts, allowing samples to be clicked into place and suspended below the stages within seconds. According to Voigt, the fragile CLARITY-cleared mouse brains could now be mounted and suspended in quartz cuvettes while more robust samples could be clamped into a holder. 
As Voigt points out: “Samples are sealed into the cuvettes but you can look at them from four directions.”
With the first prototype in hand, Voigt quickly realised that generating a light-sheet with a cylindrical lens caused extreme shadowing artifacts, so adopted the shadow reduction technique used in La Vision Biotec's light sheet microscope, the Ultramicroscope I. Here, a high-NA Gaussian beam is scanned to generate the light sheet and reduce shadows. 
Soon afterwards, the mesoSPIM prototype was moved onto a larger optical table, which also meant Voigt could add a second excitation path for dual-sided illumination. Other additions included replacing the 2X objective with a MVPLAPO 1X objective to increase the field of view and reduce distortion, and adding a Ludl filter wheel, bought from eBay, to motorise filter exchange.
Whole brain sample in a mesoSPIM. []
However, a further shortcoming of the microscope had also been preying on Voigt's mind; non-uniform axial resolution across the field of view was complicating image analysis and affecting data quality. As Voigt points out, the light sheet is shaped like an hour glass, with light being more confined at its waist zone. As a result, axial resolution is relatively high here, compared to the resolution away from this region and at the edge of the field of view.
So come 2017, the neuroscientist made the decision to convert the instrument to an axially-scanned-lightsheet (ASLM) microscope, translating the excitation beam waist through the sample using an electrically tunable lens from Optotune, Switzerland. By synchronising the rolling shutter of a Hamamatsu Orca Flash 4.0 with the moving waist, Voigt adapted the set-up so only this axially confined region of the light sheet was used to create an image, giving an optical section of uniform thickness across the FOV.
In this ASLM mode, the mesoSPIM could now achieve an axial resolution of around 6.5 micron across a 13.29 mm field of view; a 23.4-fold increase in usable field-of-view. And thanks to this modification, high resolution whole mouse brain imaging could now take place within 8 minutes, producing a relatively small, 15 GB dataset with no additional data processing required.
“A good axial resolution has a drastic impact on image quality so many researchers had tried to address this issue,” says Voigt. “So we opted for ASLM, a super simple approach of moving the waist region in synchrony with the image readout.”
“The Optotune lenses change the focus in a very controlled way and are cheap to buy,” he adds. “Also, many CMOS cameras used in microscopy will already have a programmable rolling shutter option.”
Rising interest
By now, the biology community was becoming more and more interested in mesoSPIM. As Voigt points out: “This was initially intended as a single instrument for our collaborators but when we showed it to other neurobiologists, they got really really excited.”
Come 2017, a second mesoSPIM was built at the Aguzzi Lab, Institute of Neuropathology, University Hospital Zurich, and then later that year, the Wyss Center Geneva started to build a third mesoSPIM to support its existing COLM instrument and image cleared samples at low magnifications. Voigt's side project was fast-becoming his full-time occupation.
Fabian Voigt
As the Wyss Center instrument was built, Voigt and Helmchen decided to unify mesoSPIM's mechanical stages, settling on the COLM stages from Physik Instrumente. They also streamlined its control software, which had been a combination of Labview, Micromanager and HCImage, to a Python-based light-sheet software which is available on Github.
Build and learn
The Wyss Center mesoSPIM was completed in 2018 with The Center for Microscopy and Image Analysis at the University of Zurich, and the Advanced Microscopy Facility at the Sainsbury Wellcome Centre for Neural Circuits and Behaviour, also installing mesoSPIMs that year. But as Voigt points out, these latest instruments, and updated versions, were suffering from mechanical artifacts due to a large rolling motion from the z-stage.
Given this, the very latest mesoSPIMs incorporate stages with tighter manufacturing tolerances, mounted in a more straightforward configuration. This Summer, The Aguzzi Lab, University Hospital Zurich, installed its second mesoSPIM while the Light Microscopy Platform at the Berlin Institute for Medical Systems Biology, Max Delbrück Center, is now also home to a mesoSPIM.
Rat cerebellum []
Within the last few weeks, an eighth instrument has come online at the Singapore Bioimaging Consortium, Agency for Science, Technology and Research (A*STAR), and Voigt is excited.
“From the project outset, I wouldn't have thought there was even a need for five instruments in Switzerland,” he says. “But this useful combination of easy sample handling, compatibility with all clearing methods, near-isotropic resolution and being able to image whole mouse brains in minutes, has seen mesoSPIM really take off.”
“The questions that people can ask and the answers that they get depend absolutely critically on the throughput of this method,” he adds. “Researchers are very willing to try out something new if, say, the sample preparation and imaging are fast. The combination of tissue clearing and mesoSPIM imaging allows that.”
Central nervous system of a mouse []
And applications abound. According to Voigt, researchers have been bringing a vast range of samples from lymph nodes to cleared teeth. Key projects include the study of Alzheimer's plaque distribution in mouse brains as well as whole central nervous system imaging. Here, researchers are tracing neurons from the surface of the mouse cortex all the way down to the spinal cord.
“Looking at data acquisition speed, mesoSPIM is not as fast as some other light-sheet methods, for example we cannot image a beating heart,” says Voigt. “But in terms of extracting anatomical information from a fixed a cleared mouse brain, the mesoSPIM is very fast.”
However, Voigt also cautions that the mesoSPIM model of installation may not be for everyone. As he points out, he and colleagues provide instructions and advice on how to set up the instrument through the website, workshops, and meetings, but they do not install the microscopes as a service.
“You need to invest your time here, and in the long-term these instruments also need dedicated support, so sometimes we do say that maybe a commercial instrument is better for you,” he says.
Different views of a nine day old chicken embryo, cleared with BABB neurofilament staining. []
Looking to the future, Voigt remains very enthusiastic about mesoSPIM. As he points out: “As a technology developer I have been a little embarrassed as the mesoSPIM does not push the limits of technology as much as we usually aspire to, all the ideas have been described before.”
But crucially, he and  Helmchen have combined existing techniques into a highly capable instrument for biologists.
“The embarrassment fades away when a biologist comes to you with a beautiful dataset and you can see how much they have enjoyed seeing their sample in a new light,” says Voigt. “And in the end that's what counts - making users happy.”
MesoSPIM design
The mesoSPIM modular light sheet microscope provides a large imaging volume – a whole mouse central nervous system can be imaged in its entirety - as well as excellent image quality over large fields of view with straightforward sample handling. The instrument layout is similar to the original selective plane-illumination microscope, with a horizontal detection path and vertical sample rotation axis, enabling multi-view acquisitions.
The actual instrument comprises two excitation arms and a detection arm. Each excitation arm contains the electrically tunable lens and galvo-scanner to reposition the waist region of the light sheet within a sample.
The galvo-scanner generates the light sheet by scanning a Gaussian beam up and down. A relay, placed between the tunable lens and galvo-scanner, ensures telecentric imaging conditions as the waist region moves.
Meanwhile, the detection arm comprises a Olympus MVX-10 zoom macroscope equipped with a Ludl filter-wheel and a Hamamatsu Orca Flash 4.0 sCMOS camera. The zoom macroscope delivers a field of view of 2 to 21 mm in combination with the sCMOS camera, enabling users to view large samples and also zoom in to study minute details, such as individual axons.
The latests mesoSPIMs achieve an axial resolution of around 6.5 micron across a 13.29 mm field of view.


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