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EPFL scientists have published a guide for building an add-on that turns a standard light microscope into an instrument that can generate super-resolution 3D images of cells, organoids, and embryos.Credit: Ecole Polytechnique Fédérale de Lausanne
For hundreds of years, the only tool available to scientists who wanted to study the movement of cells, bacteria, and yeast was the light microscope. However, light diffraction makes it impossible to observe objects with resolutions below 100 nm, as the resulting images are too blurry to be useful.
This physical limitation, known as the diffraction barrier, was finally overcome about 15 years ago with the development of super-resolution microscopy, allowing scientists to peer deep inside living specimens, study the behavior of organelles, and It is now possible to observe how viruses and proteins interact with each other. and drug molecules.
One of these new methods, known as structured illumination microscopy (SIM), is popular among researchers because it can produce high-resolution and high-contrast images with fewer photon exposures.
Despite the advent of nanometer-resolution electron microscopy, optical imaging continues to play an important role in life science research. Optical imaging increases instrument flexibility and allows scientists to observe living samples under normal developmental conditions. However, cost and availability constraints still keep SIM imaging out of reach for many.
To get around this problem, scientists at EPFL's Bio-Nano Instrumentation Institute (LBNI), located within the Graduate School of Biotechnology (IBI) of the EPFL School of Engineering (STI), converted a standard light microscope into a light microscope. We have developed a method to do this. High resolution device using inexpensive off-the-shelf components.
The team has published a detailed how-to guide, a series of video tutorials, and a journal article in open access format. nature communications.
SIM overcomes the diffraction barrier by reconstructing regions of high spatial frequencies that normally appear blurry when viewed through a conventional optical microscope. This method increases resolution by a factor of two, allowing scientists to see parts as small as 100 nm in diameter.
SIM works by projecting a standard illumination pattern, such as a grid, onto the sample. Images captured with different illumination patterns are processed by an algorithm to generate high-resolution reconstructions using the moiré effect.
Obtained Ph.D. in 2019. Student Melanie Hannebell needed a microscope with just this feature for her research. That's when she got the idea to build it herself for her LBNI. Other labs had already produced similar devices, but they were complex, cumbersome, and difficult to reproduce. Hannebelle wanted to design a more compact alternative that could be built and used by non-experts and that did not require expensive upkeep and maintenance.
LBNI Professor Georg Fantner said: “We sourced electronic components of the type used to manufacture the video projectors we see in the classroom.'' “We modified and positioned them so that we could project a pattern of light onto the sample.”
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Diagram of optical sectioning effect when using openSIM. In wide-field images, out-of-focus light is noticeable in the image. The openSIM image shows only the part of the sample that is in direct focus. Sample: Fixed mouse intestinal organoids labeled for E-cadherin. Credit: LBNI
Tested and approved by life science researchers
The LBNI team wanted to find out if the new microscope was a viable and viable alternative. So they asked other labs to do the testing. They worked with the groups of Professors Andrew Oates, Matthias Rutolf, John McKinney and Aleksandra Radenovich to test the instrument on real-world research samples. “Our colleagues asked us questions, told us about their needs and shared samples with us,” says Professor Fantner. “We wanted to know if and how our equipment could help their research.”
Feedback was overwhelmingly positive, and the team was awarded an EPFL Open Science grant, allowing them to share their equipment in an open hardware format. Turning the device into something that other labs can reproduce, with instructions so detailed that colleagues don't give up midway through the process, has proven to be a painstaking and time-consuming process.
Esther Reis, another doctor. We have compiled detailed instructions, equipment lists, and video tutorials for lab students to share online. “The only prerequisite for our system is a high-quality light microscope, which most laboratories already have,” explains Professor Fantner.
OpenSIM is not intended to compete with more advanced equipment. For example, this approach has lower modulation contrast than commercially available counterparts, which limits the resolution gain to 1.7x compared to the theoretical 2x. But it serves its intended purpose. In other words, he wants to make SIM technology available to labs that only need it occasionally and who cannot afford to spend more than CHF 500,000 on a commercial-grade model.
The LBNI team is working to make its results available to a broader group of scientists and build a community of users to share their experiences. “Since this paper was shared on his BioRxiv.org, we have been contacted by several people who are interested in this idea and would like to learn more about how to build their own version of his OpenSIM.” says Professor Fantner.
For more information:
Mélanie TM Hannebelle et al., Open Source Microscope Add-on for Structured Illumination Microscopy, nature communications (2024). DOI: 10.1038/s41467-024-45567-7
Magazine information:
nature communications