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Center for Microscopy and Image Analysis

Lightsheet - MesoSPIMs (Irchel)

Benchtop and V6 mesoSPIMs
The two mesoSPIMs available for booking: Benchtop (installed in 2024) and V6 "Revolver" (upgraded in 2023)

The mesoSPIM (mesoscale selective plane illumination microscopy) is the ideal instrument to quickly bridge scales from the µm- to the cm-level, which enables it to serve as an excellent tool for detailed three-dimensional anatomical investigations in many biological and medical fields (Voigt et al., 2019, Vladimirov et al, 2024).

Main features of both setups (Benchtop and V6):

  • Compatibility with all clearing techniques: Clarity, iDISCO, CUBIC, PEGASOS, ECI, etc.
  • Large field-of-view, ranging from 1.5 to 15 mm using a set of Mitutoyo Plan Apo objectives.
  • Large-sensor low-noise sCMOS cameras
  • Dual-sided excitation. Optimized for fast screening of whole mouse brain samples, yielding relatively small datasets (10-14 GB/brain/color) near-isotropic sampling (2-4 µm in X,Y & Z) due to an axially swept lightsheet which leads to uniform z-resolution across the entire field-of-view.
  • Modular sample holders and quick sample exchange.
  • Large travel range (45 x 45 x 100 mm) to allow imaging of very large samples.

Further information on the UZH mesoSPIM open-source project: MesoSPIM.org

Location

University Zurich, Irchel Campus, Room Y44-J-33.

Training Request

Pleaseregister to apply for an introduction to the microscopes.

Introduction session duration: 2 hours

If you have used mesoSPIM V6 before and would like to use the Benchtop, please request a training session as well, the systems have some differences in the startup sequence and interface.

Technical Specifications

Excitation path

Both systems have excitation based on axially scanned light-sheet microscopy (ASLM). The waist of the light-sheet is translated through the sample in synchrony with the rolling shutter of the camera, which enables high axial resolution across very large fields of view.

Light Sources and Lasers

mesoSPIM V6: iChrome MLE

Benchtop: Oxxius L4Cc

Laser lines available on both systems

  • 405nm (100mW)
  • 488nm (100mW)
  • 561nm (100mW)
  • 640nm (100mW)

Detection path

Both systems have long-working distance air plan apochromat objectives:

  1. Mitutoyo M Plan Apo 2x/0.055
  2. Mitutoyo M Plan Apo 5x/0.14
  3. Mitutoyo M Plan Apo 7.5x/0.21
  4. Mitutoyo M Plan Apo 10x/0.28
  5. Mitutoyo M Plan Apo 20x/0.28(t3.5) (corrected for 3.5 mm thick glass)

The microscopes offers lateral (XY) resolution of 1.5-2.6 µm (depending on the objective) and axial (Z) resolution  of 3.5 µm for all objectives, across a large field of view thanks to new-generation sCMOS cameras.

 

Emission (detection) filters

  • Quadband filter 405/488/561/640 (both systems)
  • 520/35 (mesoSPIM V6)
  • 590/36 (mesoSPIM V6)

If you need other filters, please talk to us.

Cameras

System mesoSPIM V6 Benchtop
Camera Hamamatsu Orca Lightning Photometrics Iris 15
image size 4608 x 2592 px (12 MP) 5056 x 2960 px (15 MP)
pixel size 5.5 µm 4.25 µm
sensor dimensions (h,w) 25.34 x 14.25 mm (diagonal 29 mm) 21.49 x 12.61 mm (diagonal 24.9 mm)

Pixel size of your images

The effective pixel size in acquired image depends on objective magnification you choose:

Pixel_size(image) = Pixel_size(sensor)/Magnification

Magnification mesoSPIM V6 pixel size, µm Benchtop pixel size, µm
1x 5.5 4.25
2x 2.75 2.125
5x 1.1 0.85
7.5x 0.73 0.567
10x 0.55 0.425
20x 0.275 0.212

Of course, the effective pixel size values are available in the meta-information files that accompany each dataset you acquire.

Field of view

Field of view can be calculated from the sensor size and objective magnifications by the formula:
FOV(h,w) = Sensor(h,w)/Magnification

Software (both systems)

The mesoSPIM-control software GUI.

The microscope software, mesoSPIM-control, is developed by us. It is open-source and written in Python. It allows users to specify sequences of z-stacks using a table-based acquisition manager. The software can also be used to acquire large-scale tiling acquisitions.

CAD models of custom 3D printed sample holders that accommodate samples from 3 mm to 75 mm.
CAD models of custom 3D printed sample holders that accommodate samples from 3 mm to 75 mm.

Depending on your sample, we have a variety of holders and imaging cuvettes.

 

Examples of samples imaged with Benchtop mesoSPIM

Examples of mesoSPIM sample types
Examples of mesoSPIM samples: a, Two pyramidal cells in the prefrontal cortex imaged at 20x (Thy1-GFPM Atto647N - yellow, propidium iodide - blue, cleared with vDISCO), with axons and basal dendrites resolved. b, Whole mouse body imaging at 0.9x magnification (P14 mouse, stained with propidium iodide PI, cleared with vDISCO). c, Peripheral nervous system of a chicken embryo at E9 imaged at 1.2x (neurofilament staining with mouse anti-RMO270, goat anti-Mouse Cy3; autofluorescence, cleared with BABB). d, Mouse brain at 1.2x (APPPS-1 line, amyloid plaques, arterial vessels, cleared with iDISCO). e, Mouse brain at 4x (Vglut2-Cre line, sparse retrograde AAV injection, iDISCO). f, Human brain tissue at 5x (area V2, stained with neutral red, cleared with MASH). g, CAD model of the SPIM-tower sample holder and the imaging results from high-throughput imaging session. g1, a molded agarose block depicting the sample as a cartoon drawing. g2, CAD model of a single layer of the SPIM-tower sample holder g3, Standardized imaging of 16 X. tropicalis tadpoles (stages 34 and 37) treated with BMS-453 (left) or control (right). Samples were stained with DAPI (cyan) and for Atp1a1/Col2a1/Tnnt2 (red), embedded in individual agarose blocks using the SPIM-mold; g4, pronephros at stage 42 (top: control, bottom: treated), g5 retina (top: control, bottom: treated). BMS-453 treatment affects both kidney and retinal development. i, Irradiated CaF2 crystal imaged at 20x magnification (color centers induced by gamma irradiation at 5 MRad, polished crystal, no clearing); i1, raw image (color-coded blue) showing SPIM-illuminated region vs background fluorescence in the irradiated CaF2 crystal; i2, Candidate track of color centers observed in repeated scans of the irradiated CaF2 crystal.

Differences between Benchtop and V6 mesoSPIMs

1. The way detection objectives can be exchanged

mesoSPIM V6: motorized objective turret ("revolver")

Benchtop: manual exchange (can be done without removing the sample).

2. Camera pixel size and total image size:

mesoSPIM V6: 5.5 µm pixel and 12 MP image.

Benchtop: 4.25 µm pixel and 15 MP image.

Other properties, such as lateral and axial resolution, maximum and minimum sample size, travel range of sample stages are nearly identical.

Custom accessories and unusual samples

For initial tests we provide standard accessories such as cuvettes, sample holders and immersion solutions. If you have unusual sample that requires customized accessories or supplies, talk us. We can suggest where to order custom accessories (such as ultra-long sample cuvettes), or even design and make them for you (such as specialized sample holders that are 3D printed form solvent-resistant polymers).

Further information (internal UZH use only)

Follow this link for further background information, documents and links.

Responsible Persons

If you have questions about the device please contact the responsible person.

Acknowledgements

Make sure to acknowledge the Center for Microscopy in your publication

How to acknowledge contributions of the Center for Microscopy

The major upgrade of existing mesoSPIM to V6 "Revolver" in 2023 and the introduction of Benchtop mesoSPIM in 2024 was made possible with the generous support of URPP AdaBD Program.

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