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Chip-scale microscope specified for high-throughput fluorescence imaging

25 Nov 2021

imec receives ERC grant to develop novel fluorescence microscope-on-chip; and new EC-funded scope project targets “superbugs”.

Conventional light microscopy has been instrumental for the study of cells and microorganisms; fluorescence microscopy has enabled visualization of even smaller cell features by selectively adding fluorescent labels to molecules.

But fluoresence microscopes are often bulky and expensive systems that require regular maintenance to keep the lenses aligned. For scientists to see more detail, larger optical components are required, but this causes the field of view to decrease.

Chip technology offers a different view: chips are compact and can integrate multiple functionalities. The scaling possibilities could allow chip-microscopes to be produced at a fraction of the cost of standard devices, says a research group at imec, the Belgium-based nanotechnology and photonics research center.

Niels Verellen, Principal Scientist at imec, has received an ERC Starting Grant to design a high-resolution microscope on-chip with a scalable field of view. At the halfway point of the five-year project, he has just described on the imec website the early successes and remaining challenges.

Lens-free fluorescence microscopy

To downsize the microscope, Verellen’s team removed the key part of standard optical microscopes: the lens. Lens-free options exist for light microscopy, where the viewer looks directly at the scattered light.

imec’s lens-free microscope, for example, uses the interference pattern of the excitation light to reconstruct the image holographically. These solutions do not work for fluorescence microscopy because fluorescence light is not coherent, meaning that there is no time-distance relationship between the excitation light and the fluorescence emission.

Verellen commented, “The goal of the ERC-project is to achieve the same advantages as the existing lens-free optical microscope, with its small size, scalability, large field of view and high resolution, for fluorescence microscopy. The operating principle of our microscope is similar to that of a traditional confocal laser scanning fluorescence microscope.

“The lens-free microscope contains an image sensor – a pixel array, topped with an integrated photonic circuit consisting of waveguides and phase modulators that form focused illumination spots. Unlike in a confocal microscope that operates traditionally with one focus point, we can generate and scan many spots simultaneously.”

The chip presents a high-throughput alternative to conventional microscopy, especially for sequencing-related applications. “We can only measure at the surface of a sample, within the evanescent field of total internally reflected light in a waveguide approximately 100nm deep. Aside from imaging membrane proteins, we see DNA sequencing as the most relevant application for our concept,” said Verellen.

Another major advantage of the chip based approach over confocal microscopy is its relative low cost. “A confocal microscope costs over €100,000 and can scan a well plate with a limited number of DNA fragments or other molecules,” said Verellen. “Imec’s microscope on-chip achieves the same resolution, but you can place ten chips side by side on a table at a fraction of the cost and floor space, and there is no need for expensive alignment maintenance.”

Light spot patterns

The essential components in the photonic chip are waveguides that guide and shape the light for sample illumination. Interference patterns can be generated using wave characteristics of the laser light. So a spot of light appears in regions where the combined waves reinforce each other.

“To generate the desired pattern, precise control over the interfering waves is crucial. We successfully built a mathematical model that achieves the pattern with a limited number of components on the photonic chip,” said Verellen.

One of the main innovations was translating this theoretical model into a chip architecture that allows imaging. For that, the team had to optimize, redesign and de-risk all optical components in the circuit. The initial results on test chips (without imagers) showed that the interference patterns could be nicely controlled and modulated.

“Until now, the components were designed and tested separately. However, we’re expecting the first photonic circuits on the imagers out of the cleanroom,” said Verellen. “The chips would be the first proof of concept device that can demonstrate the whole imaging concept. “In parallel, we’re already looking at ways to scale up to larger fields of view in the order of 1cm2. We are working on several custom passive and active optical circuit components to shape and modulate the light in an efficient and fast way.”

New microscope to focus on ‘superbugs’

Scientists working together in a new European project are developing a new super-resolution microscope that will use laser light to study the inner workings and behaviours of “superbugs” like Streptococcus Pneumoniae to gain new insights into how they cause disease. A leading cause of bacterial pneumonia, meningitis, and sepsis, Streptococcus Pneumoniae is estimated to have caused around 335,000 deaths worldwide in children aged under five in 2015 alone.

Current technologies do not allow a resolution that enables thorough studies of bacterial properties that affect disease development. But now, this super-resolution microscope will use laser light to illuminate proteins at incredibly high resolutions, allowing scientists to gain new insights.

Called the NANO-scale Visualisation to understand Bacterial virulence and invasiveness (NanoVIB for short), the project is expected to shed new light on how superbugs can cause disease, thereby providing the basis for the development of new antimicrobials to treat bacterial infections.

The European Commission has granted this health consortium €5.6 million via the Photonics Public Private Partnership to build this super-resolution microscope. The project, set to run through 2024, includes six partners from three countries: Kungliga Tekniska Hoegskolan (KTH), the coordinator, and Karolinska Institutet (Sweden); the Institut für Nanophotonik , Abberior Instruments, and Angewandte Physik und Elektronik (Germany); and Pi Imaging Technology (Switzerland).

Ten-fold resolution

Project coordinator, Professor Jerker Widengren, commented, “We expect our new microscope prototype to be a next-generation super-resolution system, making it possible to image cellular proteins marked with fluorescence emitters (fluorophores) with a ten-fold higher resolution than with any other fluorescence microscopy technique.

He added, “Using laser light, this new microscope will show how bacterial proteins localise on the surface of bacteria, allowing scientists to study the interaction of the pathogen with immune and host cells. It works based on the Miniflux concept, in which infrared laser light excites fluorophore-labelled molecules in a triangulated manner – leading to an increased resolution.”

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