eduSPIM - Light Sheet Fluorescence Microscopy in the Museum

  • Fig. 1: (a) LSFM principle and sample embedding: The sample is illuminated from the side with a thin sheet of laser light, while fluorescence signal from the whole plane is collected orthogonally and imaged onto a fast camera chip. The sample was supported by an FEP tube refractive index-matched to the surrounding PBS-filled cuvette. (b) Computer rendering of the setup. The sample chamber was positioned at the intersection of the illumination (top) and detection arms (bottom). The laser was fibre coupled into the system and expanded in a telescope (T1). A cylindrical lens (CL) was used to create the light sheet, which was imaged into the back-focal plane of the illumination objective (IO) using a second telescope (T2) with iris to crop the light sheet height. A red LED was used for transmission illumination. Transmission and fluorescence signal were collected with a long working distance air objective (20x 0.4), split using a dichroic mirror and bandpass filters and imaged onto two cameras. (c) eduSPIM setup at the museum: The optical components were on display beneath a glass cover, the electronic components were hidden beneath a wooden cover. Visitors interacted with the system using the control panel with seven push buttons.
  • Fig. 2: (a) When users moved the sample through the light sheet, the fluorescence signal of the acquired plane (green) was overlaid with the transmission image (gray). (b) When a stack was acquired, data were given a unique RGB value depending on position, and a transparency depending on intensity. These data were overlaid from back (blue) to front (red). (c) During the time in the museum, we logged the number of button presses. Overall usage increased during the winter months, and isolated spikes occurred during special events, such as the “Long night of the open museum”.
  • Fig. 3: After the end of the exhibition, we transferred eduSPIM into a dedicated transport- and display box. Again, the optical components are displayed beneath a glass cover and the electronic components hidden, but control panel and monitor are now integrated in the box. The photograph shows the eduSPIM demonstration during the 2016 EMBO practical course on LSFM in Dresden.

Light Sheet Fluorescence Microscopy (LSFM) combines the advantages of fast wide field detection and optical sectioning and is perfectly suited to image large and moving samples at cellular resolution. Its underlying principles are easily understood with basic knowledge in optics or photography. Still, little is known to the public about microscopy in general and LSFM in particular. To change this, we have built a robust and easy to operate light sheet microscope for a museum exhibition.

Science outreach programs communicate the benefits of scientific research. A successful outreach activity instills an understanding for the need of basic research in the public or sparks an interest in the sciences in students. Ideally, laymen explore the subject matter on their own terms in an interactive setting. To capture and hold their attention, surprising findings or attractive data visualization is needed.

We wanted to bring a fully functional, cutting-edge scientific instrument into an interactive museum exhibition. The design statutes for a museum exhibit differ strongly from the requirements in the lab. Appealing data visualization takes primacy to correct representation and simple, intuitive operation is more important than a large set of functions. A museum exhibit should be affordable and fit into the available space. At the same time access for service and debugging may be restricted. If the instrumentation was simplified sufficiently to meet these demands, imaging techniques promise to be a good candidate for museum exhibits, because they produce visual data that may need little post-processing to become “eye-catching”.

Selective Plane Illumination Microscopy (SPIM [1]), also called Light Sheet Fluorescence Microscopy (LSFM), is an established imaging technology in the life sciences. Its optical principles can be understood with basic knowledge in microscopy or photography: In both cases often only in-focus information is desired and the unwanted out-of-focus contribution can be eliminated by simply not illuminating. To achieve this, a SPIM consists of at least one illumination and one detection arm perpendicular to each other. A single plane in a fluorescent sample is illuminated from the side with a thin sheet of light (fig.

1a). The fluorescent signal is imaged onto a fast camera and a 3D dataset is acquired by imaging multiple planes consecutively. Because no out-of-focus fluorescence is excited, the photon budget is spent economically and light doses are low. LSFM produces attractive pictures with high dynamic range, which, combined with its easy-to-understand technology, makes it an ideal candidate for an interactive, unsupervised outreach project.

We have built an educational SPIM (eduSPIM [2]) that has been on display for a whole year in the Technische Sammlungen Dresden, Germany as part of a special exhibition on occasion of the UNESCO international year of light 2015. We continue to use eduSPIM for various scientific meetings and outreach projects after the end of the exhibition.

Robust Optics Design

To ensure that our eduSPIM was both easy to use and simple to understand, we stripped the design for a scientific light sheet microscope of all non-essential parts and hid all components not vital for optical function beneath a wooden cover. Visitors only saw the optical components, consisting of a single illumination and a single detection arm placed beneath a glass cover (fig. 1b,c).

The fixed sample was placed at their intersection of both arms. To ensure longevity of the sample, the laser was only turned on when the sample was moved and a still of the last acquired image was displayed instead of a live view. To prevent evaporation, the sample was embedded in a closed chamber and the whole chamber was translated to acquire a 3D stack of images. As a consequence of the refractive index mismatch between the imaging medium and air, the optical path length in the detection changed when the chamber was moved relative to the objective. Therefore, we also moved the detection objective to obtain sharp images regardless of the imaging position.

Fast Data Visualization

We chose zebrafish embryos with fluorescently stained vasculature as the sample, because the labelled structure is meaningful also for laymen and illustrates the working principle of LSFM as the optical sectioning becomes very apparent in the maze of vessels. While users moved the sample using push-buttons labelled with pictograms, fluorescence and transmission data were acquired simultaneously and overlaid (fig. 2a). When a user acquired a stack, the 3D data were visualized using a colormap encoding for depth that was calculated on-the-fly using only 2D drawing tools that did not need high-end graphics cards (fig. 2b).

Fail-Proof Software Framework and Usage Logging

To facilitate maintenance of the remotely-located eduSPIM, we incorporated a number of functions to handle errors efficiently [3]. When an error occurred, eduSPIM entered a “fall-back mode” simulating microscope function to decrease down-time. Only if “fall-back mode” failed, the microscope entered a “fatal mode” displaying an error message.

Interested visitors find more information about eduSPIM, LSFM and zebrafish online at eduspim.org. We also log each button press and show an “eduSPIM live” view with the latest acquired dataset. With this information, we estimated the reach of eduSPIM (fig. 2c) and assessed the current sample quality to determine when the sample needed to be exchanged.

Conclusions

eduSPIM has been continuously running in a one-year-long exhibition where its buttons have been pressed 170 000 times. We have such quantitative statistics, but lack feedback whether visitors actually understood the concepts and if any question remained unanswered. After the end of the exhibition, we have shown eduSPIM at the 2016 EMBO practical course on LSFM in Dresden, Germany and the 2016 LSFM meeting in Sheffield, UK (fig. 3). We want to continue showing eduSPIM, with experts available, for example when labs are opened to the public or scientists visit schools. eduSPIM was designed for display in the museum, but it is still a fully functional light sheet microscope and may be a good choice for research labs in need of an affordable, easy-to-use SPIM.

Acknowledgements

We thank Thorlabs, Physik Instrumente, Toptica, Zeiss and AHF for their generous support of the eduSPIM project and the Technische Sammlungen Dresden for hosting eduSPIM.

References
[1] Jan Huisken, Jim Swoger, Filippo Del Bene, Joachim Wittbrodt, and Ernst H. K. Stelzer: Optical Sectioning Deep Inside Live Embryos by Selective Plane Illumination Microscopy, Science 305, 1007-1009 (2004) DOI 10.1126/science.1100035
[2] Wiebke Jahr, Benjamin Schmid, Michael Weber, and Jan Huisken: eduSPIM: Light Sheet Microscopy in the Museum, PLoS ONE 11(8), e0161402 (2016) DOI 10.1371/journal.pone.0161402
[3] Benjamin Schmid, Wiebke Jahr, Michael Weber, and Jan Huisken: Software Framework for Controlling Unsupervised Scientific Instruments, PLoS ONE 11(8), e0161671 (2016) DOI 10.1371/journal.pone.0161671

Authors
Wiebke Jahr1, Benjamin Schmid2, Jan Huisken3       

Affiliations
1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany
2 Optical Imaging Centre Erlangen, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany
3 Morgridge Institute for Research, Madison, Wisconsin, USA

Contact
Wiebke Jahr

Max Planck Institute of Molecular Cell Biology and Genetics
Dresden, Germany
http://mpi-cbg.de/research-groups/current-groups/jan-huisken/home/

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