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Setup Extensions and Modifications

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Bright-field illumination

Michael Redd from the Cell Imaging Core Facility at the University of Utah put together an Arduino-based solution to get proper bright-field illumination in an OpenSPIM setup: Brightfield.

Shutter

Instructions to make an Arduino-controlled shutter compatible with uManager: paper by Todd P. Meyrath and tutorial on the uManager page.

OpenSPIM for large samples

Field of view

The original OpenSPIM uses a 2X expander to enlarge the laser beam to 2 mm. For larger samples a larger field of view can be preferable. This means that you will need a larger beam to create a taller light sheet. Change the second beam expander lens to f=75 mm or 100 mm and increase the distance between lenses accordingly.

Objectives

Immersion objectives with long working distance are hard to find. However, at low NA and moderate magnification, a dry objective will also work; the higher NA, the more spherical aberrations will be present. Keep in mind that when using a dry objective in immersion, the WD will increase (approximately by a factor equal to the RI of the new medium, eg. 17 mm in air will increase to 22.6 mm in water).

Example parts list

The table below lists the parts used by the OpenSPIM for brains setup built by Monika Pawłowska (Nencki Institute of Experimental Biology, Warsaw).


Manufacturer Accessibility Description File or Link/Model # Image Quantity Price (EUR)
Nikon purchase 4X Nikon Plan Fluorite Imaging Objective, 0.13 NA, 17.2 mm WD, dry N4X-PF
Thorlabs N4X-PF objective.jpg
2 (or 3 for double illumination) 410
Thorlabs purchase Translating Lens Mount for Ø1" Optics (for the objectives LM1XY/M
Thorlabs LM1XY mount.jpg
2 (or 3) 125
Nikon purchase Infinity-Corrected Tube Lens for Plan Fluorite Objectives ITL200
Thorlabs ITL200 lens.jpg
1 405
Monika self made This chamber can be 3D printed. For windows use 24x24 mm microscopy cover glasses and glue (eg. two component epoxy) .stl on Github
Large samples chamber 3D.PNG
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Monika self made This holder includes two threaded holes so it's easiest to make from metal (eg aluminum). Holder is mounted on the 4D stage arm with M6 screw. Glass slide with 1 mm thickness can be held with nylon or nylon-tipped screw drawing on Github step file on Github
Holder.PNG
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