OpenSPIM: an open access light sheet microscopy platform

Light sheet microscopy is an emerging technique that allows developmental and cell biologists to record entire living systems with minimal photo-damage over extended periods of time. The technology has thus far been applied to produce spectacular proof of principle recordings of model organism embryos which capture the developing specimen at cellular resolution throughout early development. True biological discoveries using this technology are, however, yet to be realized and will require that the microscopes move from the optical technology development labs to practicing cell and developmental biologists. To facilitate this transition the HFSP funded Tomancak and Huisken labs at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden developed in collaboration with the Laboratory for Optical and Computational Instrumentation (LOCI) in Madison an open access realization of the light sheet microscopy paradigm called OpenSPIM. The unique feature of the OpenSPIM platform is the publicly accessible wiki site (http://openspim.org) that documents in great detail how to assemble an affordable light sheet set-up.  The authors aim to establish an interdisciplinary community that will collaboratively adapt and improve the OpenSPIM design to address specific biological questions.

Pavel Tomancak received his education at the Masaryk University of Brno in the Czech Republic. After completing his PhD at EMBL on Drosophila developmental genetics, he started a post-doc at UC Berkeley where he became interested in using genomics approaches to study development. Since 2005 he has led an independent research group at the Max Planck Institute of Molecular Cell Biology and Genetics (MPI-CBG) in Dresden, Germany, working on the evolution of gene regulation in development using a diverse set of molecular, imaging and image analysis approaches. The lab is known for their contributions to the understanding of the hourglass model of morphological evolution, genome wide Drosophila resources and open access software and hardware projects - Fiji and OpenSPIM.

Jan Huisken received his PhD in physics in 2004 working on “Multi-view microscopy and multi-beam manipulation for high-resolution optical imaging” at EMBL with Prof. Stelzer. After an additional year as a postdoc at EMBL, having been awarded an HFSP Cross-Disciplinary Fellowship, he moved to the laboratory of Didier Stainier at UC San Francisco, expanding his optical work into a biological context. His latter work at UCSF was further supported by a Fellowship of the Cardiovascular Research Institute (CVRI). In 2010, he moved to the Max-Planck-Institute of Molecular Cell Biology and Genetics in Dresden where he started working with his own team, supported by an HFSP Career Development Award. His interest in noninvasive biomedical imaging remains at the center of his research, which is now fully devoted to organogenesis in zebrafish using advanced imaging technology.

Selective Plane Illumination Microscopy (SPIM) has resurrected an old light sheet illumination idea of the 1925 Nobel Prize winner Richard Adolf Zsigmondy and brought it into the 21st century. SPIM illuminates a single plane in a fluorescently labeled specimen with a laser light sheet and collects the fluorescence with a CCD camera oriented perpendicularly to the illuminated plane. Additionally, SPIM introduces a new sample mounting principle, where the living biological specimen is embedded in a rigid and transparent mounting medium (such as agarose) and suspended by gravity in a water filled chamber surrounded by the illumination and detection lenses. This arrangement allows sample rotation and imaging of relatively large specimens such as embryos from multiple angles.

An interesting feature of the SPIM technology is that it is relatively easy to build. All one needs is a laser, a CCD camera, off the shelf objectives, readily available optical components and a sample positioning system. We have exploited this feature to put together an affordable OpenSPIM system that realizes single sided light sheet illumination and detection using commercially available components centered on a custom made sample chamber (see figure).  The sample is mounted in a glass capillary inserted in a regular plastic syringe and positioned using a monolithic 4D positioning system developed specifically for OpenSPIM by Picard Industries (www.picard-industries.com). The system can be put together in 14 simple steps in about one hour.

The OpenSPIM hardware comes to life thanks to open source software that extends microscopy control plugin microManager to include SPIM specific functionalities such as sample rotation. All software is tightly incorporated into Fiji (http://fiji.sc) – an open source platform for biological image analysis, which uniquely offers advanced algorithms necessary to process the SPIM image data.

The wiki site documents the assembly and operation of the OpenSPIM in meticulous and even beginner-level detail. Rumor has it that IKEA instruction manuals seem opaque in comparison. There are exhaustive parts lists, countless pictures, CAD schematics for the production of custom parts, 3D renderings of all assembly steps as well as the corresponding real pictures of assembly intermediates and narrated videos of the assembly and alignment process. Description of sample mounting is very detailed, hopefully allowing even engineers with no prior experience in biology to put together a living sample for recording under the microscope. The software is documented for any user with minimal programming experience, but it also provides the nitty-gritty details necessary for experienced developers to extend it. We have spent a lot of time putting together cookbook style recipes describing the Fiji mediated image processing of the SPIM data (http://openspim.org/Operation#Data_processing). Together these resources provide a complete integrated solution for SPIM imaging, merging the ideas of open access software and hardware.

We have used OpenSPIM to image large specimens (zebra fish larvae), fast biological processes (beating fish heart); we recorded the developmental anatomy and gene expression dynamics in toto over time (using Drosophila embryo as a model) and imaged various marine organisms in 3D. Thus the OpenSPIM set-up offers a broad range of applications that span multiple disciplines and all that is just a beginning.

The OpenSPIM set-up is relatively affordable and therefore it is possible to build many such set-ups (OpenSPIM farms) allowing parallel long-term time-lapse imaging of multiple samples. OpenSPIM is an excellent teaching and outreach tool. It has thus far traveled (in a suitcase) to two EMBO courses in South Africa (Imaging Infection and Immunity) and Sweden (Marine Animal Models in Evolution and Development). OpenSPIM is kept simple on purpose, representing the very basic realization of light-sheet microscopy. It is meant to be a starting point for prototyping more advanced light sheet microscopy paradigms and for incorporating innovative engineering ideas such as open access electronics (Arduino) and 3D printing technology.

We are really hopeful that many researchers will build the set-up, tweak it to their needs, add cutting edge features or even completely redesign it. Everybody is welcome to do so and to contribute their experience and expertise back to the community by editing and further enhancing the wiki. Time will tell whether this idea will truly take off.

Reference

OpenSPIM - an open access platform for light sheet microscopy. Pitrone P., Schindelin J., Stuyvenberg L., Preibisch S., Weber M., Eliceiri K.W., Huisken J., Tomancak P. (2013). Nature Methods, 10, 598–599.

A longer pre-print version of the article has been deposited on arXiv: http://arxiv.org/abs/1302.1987