Dark-field microscopy gets a boost from Bragg mirrors

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A new simple chip powered by quantum dots allows standard microscopes to visualise difficult-to-image biological organisms.

MIT researchers have developed a multi-layered chip that could dramatically reduce the cost of dark-field microscopes

Dark-field microscopy can be used to reveal intricate details of objects such as translucent cells or aquatic organisms, as well as faceted diamonds and other precious stones, which would all otherwise appear very faint or even invisible under a typical bright-field microscope.

In order to generate dark-field images, standard microscopes can be fitted with a number of dedicated components that together enable the sample stage to be illuminated with a hollow, highly-angled cone of light. This cone can then scatter off the features of translucent samples in order to create an image on the microscope’s camera, in bright contrast to the dark background.

Such components include a mirrored casing that surrounds the microscope objective, which directs light onto the sample at a very high angle, as well as a dark-field filter cube that manipulates the incoming light in a way that it can be caught by the casing.

The issue with these components, however, is that in addition to being bulky – depending on the desired magnification – they can range from several hundred to several thousand dollars. This is far from ideal for those looking to perform dark-field imaging with lower-end microscopes, for example in high schools, as they can cost upwards of $1,000 already.

This could soon be an issue of the past, however, as a group of MIT researchers have recently developed a mirror-based solution that incorporates the illumination characteristics of these dedicated expensive components, at a fraction of the cost.

The solution takes the form of a single, three-layered chip that is slightly larger than a postage stamp while being as thin as a credit card. When placed on a microscope’s stage, the chip emits a hollow cone of light that can be used to generate detailed dark-field images of algae, bacteria, and similarly translucent tiny objects.

Inspired by butterfly wings, created with Bragg mirrors

Acting as the solution’s light source, the middle layer of the chip is made from a polymer infused with quantum dots: tiny nanoparticles that emit orange and red light when excited with blue fluorescent light.

Over this light-generating layer the researchers place a Bragg mirror, a structure made from alternating nanoscale layers of transparent materials with distinctly different refractive indices (the degrees at which they reflect incoming light).

Speaking with Electro Optics, Mathias Kolle, an associate professor of mechanical engineering at MIT, said that this Bragg mirror acts as a ‘gatekeeper’ for the photons that are emitted by the quantum dots. ‘The arrangement and thicknesses of the mirror’s layers is such that it lets photons escape up and out of the chip, but only if the light arrives at the mirror at high angles,’ he explained. ‘Light arriving at lower angles is bounced back down into the chip.’

Fluorescence observed from a SLED substrate with a variety of top Bragg mirror designs (small squares) that generate different angular emission profiles.

While Bragg mirrors are an old technology, according to Kolle they are often used in photonics applications for their ability to offer over 99 per cent reflectivity in a selected spectral band, as well as their ability to reject or transmit any part of the spectrum. ‘They are used in microscope filter cubes, for fluorescence microscopy, and for beam splitting optics,’ he said.

The third and final layer of the chip sits below the light-generating layer and offers the ability to recycle the photons initially rejected by the Bragg mirror. This layer is moulded out of solid, transparent epoxy coated with a highly reflective gold film. It resembles a miniature egg crate, pocked with small wells, each measuring about 4µm in diameter. This optical arrangement is intended to catch any light that reflects back down from the Bragg mirror, and ‘ping-pong’ that light back up at a new angle, at which it might pass the mirror.

According to the researchers, the design for this bottom layer was inspired by the microscopic scale structure in the wings of the Papilio butterfly, which feature intriguing egg crate-like structures with a lining similar to a Bragg mirror, giving them their iridescent colour.

A dramatic drop in price

The total cost of producing these chips is significantly less than having to purchase the standard dedicated components required to form a highly-angled cone of light. This could be brought down even further if certain alterations were made to their design, Kolle explained.

‘There is a simpler way of building this chip, using a Bragg mirror, a large-area LED to replace the quantum dots, and a rougher silver reflector to replace the gold film (a bit of aluminium foil could potentially even do the job here),’ he said.

With the extremely low cost of aluminium foil and LEDs, the highest costing component of this simplified set-up would by far be the Bragg mirror, which Kolle noted can be up to $200 depending on the rejection band quality. ‘That I think could come down, however, as the mirrors in our application don’t have to be quite as good, as long as they have a good rejection band in the lower angle of incidence ranges,’ he said.

In addition, while currently the Bragg mirrors being used are manufactured by the MIT scientists, if instead each design of mirror was mass-produced, the cost could come down further still. Kolle remarked that it could even come down to tens of dollars to produce each single mirrored chip.

In addition to being tailorable to fit simple, high school-grade microscopes, the chips could also be designed to fit within higher-end microscopes used in optical, physics, or materials science research labs. ‘Often high-end microscopes have darkfield components; however such microscopes can cost upwards of $50,000,’ said Kolle. ‘Our chip could be used to improve the cost of these microscopes dramatically should their manufacturers wish to work with us.’

With regards to the commercialisation of the chips, Kolle confirmed that a patent has been filed for the technology, which is currently waiting for approval.

Wide application potential

Desktop-based dark-field microscopes are not the only equipment being targeted by the researchers’ innovative mirrored chips. They could also be incorporated into miniaturised dark-field imaging devices, such as those used for bioanalytical applications in the field, or for point-of-care diagnostics. 

Such a device could be used, for example, by a marine biologist to prevent the need to bring samples all the way back to the lab before they can be analysed. The team has even tested the chips for such applications (albeit in a desktop format), collecting samples of seawater as well as non-pathogenic strains of the bacteria E. coli, and placing each sample on a chip that they set on the platform of a standard bright-field microscope. ‘With this simple setup, we were able to produce clear and detailed dark-field images of individual bacterial cells, as well as microorganisms in seawater, which were close to invisible under bright-field illumination,’ Kolle confirmed.

Published in the journal Nature Photonics, the MIT researchers’ work was supported by the National Science Foundation, the National Institutes of Health, and the US Army Research Office.

This has the potential for widespread impact in science and education through outfitting garden-variety microscopes with this technology,’ said James Burgess, programme manager for the Institute for Soldier Nanotechnologies at the Army Research Office. ‘Additionally, the ability to obtain superior contrast in imaging of biological and inorganic materials under optical magnification could be incorporated into systems for identification of new biological threats and toxins in Army Medical Center laboratories and on the battlefield.’

In summary, this new design of a mirrored chip can be added to any standard microscope with a sample stage as an affordable, downsized alternative to conventional dark-field components. The
chip may also be fitted into hand-held microscopes to produce images of microorganisms in the field.

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