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Center for Microscopy and Image Analysis

Lightsheet - MesoSPIM (Irchel)

MesoSPIM v6 "Revolver" (upgraded in 2023)
The mesoSPIM 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, 2023).

Main features (upgrade 2023):

  • 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 camera Hamamatsu Orcal Lighting (29 mm diagonal)
  • 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.

For further information: MesoSPIM.org

Location

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

Training Request

Follow this link to apply for an introduction to the microscope.

Technical Specifications

Excitation path

The mesoSPIM excitation path is 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. The waist is translated with electrically tunable lenses by Optotune.

Light Sources and Lasers

The iChrome MLE comprises 3 diode lasers and 1 DPSS laser fully integrated in one compact housing.

Lasers

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

Detection path

The mesoSPIM detection path consists of five 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 microscope 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 camera Hamamatsu Orca Lightning.

 

Emission (detection) filters

  • Quadband filter 405/488/561/640
  • 520/35
  • 590/36

Camera

Hamamatsu Orca Lightning sCMOS:

  1. image size: 4608 x 2592 px (12 MP)
  2. sensor pixel size: 5.5 µm,
  3. sensor dimensions (h,w): 25.34 x 14.25 mm (diagonal 29 mm).

Pixel size

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

Pixel_size(image) = Pixel_size(sensor)/Magnification

Magnification Image pixel size, µm
1x 5.5
2x 2.75
5x 1.1
7.5x 0.73
10x 0.55
20x 0.275

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

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 recently imaged with 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.
  • For initial tests and the first experiments, the ZMB will serve users with the needed experimental accessories such as cuvettes and immersion solutions. If they become regular user of the microscope, they have to organize the accessories by themselves.

Literature and Links

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.

 

Make sure to acknowledge the Center for Microscopy in your publication to support us.

How to acknowledge contributions of the Center for Microscopy

Weiterführende Informationen

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