Caltech Research Enables Coherent Spectral Broadening On-Chip

Broadband, coherent light sources are highly valued in R&D. But until now, they have been difficult to achieve without bulky, inefficient tabletop devices.

A Caltech team led by professor Alireza Marandi developed an efficient solution to integrating a broad spectrum of frequencies on a microchip. Using an optical parametric oscillator (OPO), the team demonstrated multi-octave frequency comb generation on a nanophotonic device with a threshold of only femtojoules (fJ) of pump energy.

The nanophotonic device has the potential to provide ultrabroadband (visible to MIR), on-chip light sources for applications in areas ranging from communications and imaging to spectroscopy.

To generate a frequency comb on a chip, the researchers engineered an OPO in lithium niobate (LiNbO3) and used dispersion engineering to shape the way that different wavelengths traveled through the device. An OPO is essentially a resonator that traps incoming laser light at one input frequency and uses a nonlinear crystal to generate light at different output frequencies. Typically, OPOs serve as laser-like light sources with tunable output frequencies. But. by using dispersion engineering in the work, the researchers ensured that the wavelengths remained together instead of spreading out.

The device demonstrated highly efficient, highly stable coherent spectral broadening with the OPO — a result that the team initially did not expect. “We turned it on and cranked up the power, and when we looked at the spectrum, we saw that it was extremely broad,” Marandi said. “We were particularly surprised that the super-broad spectrum was actually coherent. This was against the textbook descriptions of how OPOs work.”

In subsequent simulations, the researchers found that raising the incoming light energy above the threshold caused the spectrum to become incoherent — and therefore unable to generate a frequency comb. However, in the lab, the spectrum continued to remain coherent even when the device operated far above the threshold.

By leveraging an ultralow threshold and dispersion engineering, the researchers had accessed a previously unexplored OPO regime that enables coherent spectral broadening.

“It took us maybe six months to discover that there is this new regime of OPO operation in which the OPO is far above its threshold and the coherence is reestablished,” Marandi said. “Because the threshold of this OPO is orders of magnitude lower than previous OPOs, and the dispersion and the resonator are engineered unlike the previous realization of OPOs, we could observe this phenomenal spectral broadening, which is orders of magnitude more energy-efficient than other spectral broadening schemes.”

Creating a multi-octave frequency comb from an OPO could enable ultrabroadband, on-chip, nonlinear photonic capabilities for numerous applications.

One of the primary techniques used to make stable frequency combs requires significant broadening of the comb’s spectrum. The energy demands of this spectral broadening have, so far, created a bottleneck that has impeded the integration of frequency comb technologies on-chip. The team’s approach to building frequency combs could reshape how frequency comb-based technologies, currently found in table-top setups, could transition to integrated photonic devices.

Moreover, most of the advanced lasers and detectors used for measuring molecules operate in the NIR or visible range. OPOs that are launched from NIR lasers as the input frequency, and are then able to efficiently convert the light, outputting coherent light in the MIR range, could allow researchers, for example those working with spectroscopy, to access relevant information at lower frequencies.

“There have been two main challenges with frequency combs,” Marandi said. “One is that the sources are too big, and the second is that it’s challenging to make them in different desired spectral windows. Our work offers a path toward solving both of these problems.”

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Expansion Microscopy Technique Enables 20-nm Resolution

A new expansion microscopy (ExM) technique from MIT makes it possible to use a conventional light microscope to generate high-resolution images at the nanoscale, by expanding specimens 20-fold before imaging them.

Historically, nanoscale structures in cells and tissues have been imaged with high-powered, expensive, superresolution microscopes. The new ExM protocol, which achieves 20-fold expansion in just one step, provides a simple, inexpensive method that can be used by most biology labs to perform imaging at a resolution of about 20 nm.

“What this new technique allows you to do is see things that you couldn’t normally see with standard microscopes,” professor Laura Kiessling said. “It drives down the cost of imaging because you can see nanoscale things without the need for a specialized facility.”

The original version of the ExM technique, developed by professor Edward Boyden and his team in 2015, expanded tissue about 4-fold and provided images with a resolution of around 70 nm. In 2017, Boyden’s lab modified the process to include a second expansion step, achieving an overall 20-fold expansion.

“We’ve developed several 20-fold expansion technologies in the past, but they require multiple expansion steps,” Boyden said. “If you could do that amount of expansion in a single step, that could simplify things quite a bit.”

The new method reaches the same level of performance possible with iterative expansion methods, but with the simplicity of a single-shot protocol.

To implement ExM, the researchers embed the tissue specimen in an absorbent polymer and added water, creating a hydrogel that expands the polymer and pulls the biomolecules in the specimen apart. For one-step, 20-fold expansion, the researchers use gel that is extremely absorbent and mechanically stable to ensure that the gel does not fall apart when the specimen is expanded by 20x.

To further stabilize the gel and enhance its reproducibility, the researchers remove oxygen from the polymer solution prior to gelation, preventing side reactions that could interfere with crosslinking. Unlike previous expansion gels that require another molecule to be added to form crosslinks between the polymer strands, the gel used for the single-shot, 20-fold ExM technique forms crosslinks spontaneously.

Once the gel is formed, select bonds in the proteins that hold the tissue together are broken and water is added to make the gel expand. After the gel expands, target proteins in the tissue can be labeled and imaged. The new technique supports post-expansion staining for brain tissue to facilitate biomolecular labeling.

“This approach may require more sample preparation compared to other superresolution techniques, but it’s much simpler when it comes to the actual imaging process, especially for 3D imaging,” researcher Tay Won Shin said.

In one round of expansion, the new ExM technique, which the team calls 20ExM, enabled the researchers to image hollow microtubule structures in cultured cells and synaptic nanocolumns in the mouse somatosensory cortex on a conventional confocal microscope. The team could also visualize mitochondria and the organization of individual nuclear pore complexes in the cells.

The new ExM technique could be used for a variety of experiments where high resolution and single-step simplicity are desired. The researchers are currently using the technique to image glycans — carbohydrates, found on the surface of a cell that help control how the cell interacts with its environment.

20ExM could also be used to image tumor cells, providing insight into how proteins are organized within these cells. In principle, the new ExM technique could be used to simplify or enhance the resolution of other expansion-based technologies, such as in situ RNA detection and sequencing and genome imaging.

The single-shot, 20-fold expansion microscopy method provides a robust, simple, affordable solution to nanoscale-resolution imaging of preserved cells and tissues using conventional microscopes. The researchers believe that any biology lab could use the technique at a low cost, because it relies on standard, off-the-shelf chemicals and equipment that most labs already have or can easily access.

“Our hope is that with this new technology, any conventional biology lab can use this protocol with their existing microscopes, allowing them to approach resolution that can only be achieved with very specialized and costly state-of-the-art microscopes,” researcher Shiwei Wang said.

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Optogenetic Light Source Transmits Information Between Neurons

Researchers at The Institute of Photonic Sciences (ICFO) demonstrated that photons, acting as neurotransmitters, can enable communication between neurons. The researchers developed an all-optogenetic, synaptic transmission system that enabled synthetic signaling between unconnected neurons and the generation of synaptic circuits.

The team’s findings could lead to therapies that use light instead of chemicals or drugs to restore communication between nerve cells in the treatment of diseases such as Alzheimer’s and Parkinson’s. In addition to treating neurological disorders, the team’s approach could potentially be used to rewire damaged neural circuits and improve learning.

The Photons as Synaptic Transmitters (PhAST) system connects two neurons by using light-emitting enzymes and light-sensitive ion channels. The researchers tested the PhAST system on the roundworm model C. elegans and showed that photon-based synaptic transmission can facilitate the modification of animal behavior.

After genetically modifying the roundworms to have faulty neurotransmitters, which made the worms insensitive to mechanical stimuli, the researchers engineered a luciferase enzyme to generate light inside the worms and a specially designed microscope for viewing the light being emitted by the worms. They also selected ion channels for the postsynaptic cells. The channels are highly sensitive, so that very little light is needed to open them.

The genetically engineered enzyme introduced into the worms as a light source requires calcium and the binding of a co-factor to emit light. The calcium increase occurs when the presynaptic cell activates, so the light is only on when the presynaptic neuron is on as well. When the light source and the presynaptic cell are activated, blue light is emitted from the presynaptic cell. The ion channel in the postsynaptic cell senses the blue light and activates the postsynaptic cell, which then transmits the information to the downstream pathway.

To follow the information flow, the researchers developed a device that delivered mechanical stresses to the animals’ noses, while at the same time measuring the calcium activity in the sensory neurons. To acquire the dim light signals coming from the neurons, they built a microscope with a sensor that is strong enough to detect a faint signal from just a few photons. The researchers then took a default model of an existing microscope and removed the optical elements that were not needed for bioluminescence and that could interfere with imaging. The researchers also used artificial intelligence to enhance the bioluminescence imaging capabilities of the microscope.

In experiments, the researchers established a new transmission between two unconnected cells, restoring neuronal communication in a defective circuit. They also suppressed the worms’ response to a painful stimulus, and they switched the worms’ response to an olfactory stimulus from attractive to aversive behavior. The researchers also used PhAST to study the calcium dynamics of the temporal pattern generator in a motor circuit for ovipositioning. The experimental results showed that the PhAST system can facilitate the modification of animal behavior.

Further, the PhAST system could help researchers better understand the underlying mechanisms of brain function and complex behaviors, and how different brain regions communicate with each other. It could lead to new ways to image and map brain activity with higher spatiotemporal resolution.

Limitations to the widespread use of the technology remain. Further improvements in the engineering of the bioluminescent enzymes and the ion channels and in the targeting of molecules would allow greater optical control of the neuronal function, with higher specificity and precision. However, the ICFO study demonstrated that chemical neurotransmitters can be replaced with light to overcome malfunctioning in neural circuits and help neurons communicate again. Now that the technology has been shown to work in vivo in worms, a potential next step could be use of the PhAST system for the study of more complex neural circuits.


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Light-Activated Tool Controls Protein Bonds and Tracks Cell Adhesion

Optical tools can be used to activate biological functions, but with current methods the effects are slow to appear, and sustained effects require continuous light activation. As a result, these light-activation tools provide limited control of fast biological processes and can lead to toxicity in cells and organisms.

Although light is a well-established tool for control of bond breakage, it is less firmly established for the control of specific bond formation in complex environments.

A team at Tampere University worked with researchers at the University of Cambridge and the University of Pittsburgh to develop a way to use visible light to control irreversible protein binding. The new optical technique for fast, irreversible protein conjugation could be especially valuable in processes where a short initial signal leads to long-term changes in cell or tissue function. Examples include the regulation of gene expression during stem cell differentiation and the activation of immune cells in viral infections.

The researchers built on their previous work with proteins to develop a system for the rapid, light-activated control of protein bond formation. Their “protein superglue” is a peptide/protein pair called SpyTag003/SpyCatcher003 that exhibits fast, irreversible binding. Based on an engineered protein, the SpyTag003/SpyCatcher003 peptide/protein pair allows the modular assembly of complex protein structures.

To achieve optical control of the protein superglue, the researchers looked beyond the 20 amino acids constituting human proteins. Using modified protein synthesis machinery from archaebacteria, they incorporated a light-reactive, unnatural amino acid into the SpyCatcher003 protein to make the protein photoreactive. The amino acid was strategically placed to block the peptide/protein pairing until it was activated by light.

In experiments, the researchers showed a uniform, specific reaction in cell lysate upon light activation.

“A short pulse of light was enough to trigger the rapid and efficient formation of the irreversible peptide/protein complex, both in the test tube and in living cells,” said Mark Howarth, a professor at the University of Cambridge. “Importantly, the activation only took place with specific wavelengths of light, making it possible to combine protein control with live-cell fluorescence microscopy.”

After validating their approach to optically controlling irreversible protein coupling, the researchers applied the technique to the covalent reconstitution of a talin protein that was split in half. The researchers used light to activate the talin — a central adhesion protein — inside living cells.

Optical control of talin reconstitution allowed the researchers to probe the timescale of the initial adhesion complex formation. By tracking the timing of protein recruitment into the adhesion complex, the team could determine a timeline of the events leading to the formation of the adhesion complex, and the hierarchy of the recruitment of key components for cell adhesion.

Cell-matrix adhesions — large protein complexes consisting of hundreds of different proteins — are highly dynamic. “Their dynamic structure and vast complexity make cell adhesions difficult to study,” Tampere University professor Vesa Hytönen said. “The details of how cell-matrix adhesions initially form and how they react to different stimuli have remained largely unknown.”

Researcher Rolle Rahikainen said that the team observed an immediate cell response after activating the talin protein with a short pulse of light. “We got very excited when we first realized how well the system worked in controlling complex cellular processes, such as the formation of adhesion and cell spreading.”

The findings demonstrate the potential of the light-activated protein superglue for investigating complex cellular processes. The results could also lead scientists to a more comprehensive understanding of the complex structure and function of adhesion.

The modular, Lego brick-like structure of the system makes it applicable to the study and control of diverse cellular functions. The precise, irreversible assembly of biological building blocks has many applications, from biomaterials to vaccines.

Beyond adhesion, SpyCatcher003 could be used for the photocontrol of biomolecules. The robust cellular response, initiated in seconds, opens possibilities for spatiotemporal control of highly dynamic intracellular and extracellular processes.

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Simple Approach to Laser Color Conversion Uses SRS in Ionic Liquids

S

cientists from Brookhaven National Laboratory showed that ionic liquids provide an efficient means to convert one color of laser light into another. The discovery could lead to a way to create lasers with desired colors for a range of medical, scientific, and technological applications.

The method is based on the interaction between the laser and different types of ionic liquids (also known as liquid salts). The vibrational energy in the chemical bonds in the ionic liquid cause the laser’s energy to shift and change color.

“By adding a certain ion that has a particular vibrational frequency, we can design a liquid that shifts the laser light by that vibrational frequency,” chemist James Wishart said. “And if we want a different color, then we can switch out one ion and put in another that has a different vibrational frequency. The component ions can be mixed and matched to shift laser colors by different degrees as needed.”

The new approach to changing laser wavelengths has its roots in a project to boost the capabilities of a CO2 laser at Brookhaven’s Accelerator Test Facility. To improve the laser’s beam quality and repetition rate, the researchers wanted to pump the laser using optical excitation instead of electric discharge.

To create a laser with the appropriate wavelength for optical pumping, the researchers used stimulated Raman scattering (SRS) to shift the wavelength of an existing laser. SRS can be used to harness the vibrational frequencies of molecules in a solid, liquid, or gas form.

“Basically, the laser deposits energy into the molecular vibrations — the squishing and stretching of the chemical bonds that make up the material,” researcher Rotem Kupfer said. “Then the photons (particles of light) that come out have the original energy, minus the energy of those vibrations.” The lower-energy photons have a longer wavelength and, thus, a different color.

The researchers demonstrated that 1-ethyl-3-methylimidazolium dicyanamide (EMIM DCA), an ionic liquid, was an effective medium for converting 532-nm pulses from a Q-switched Nd:YAG laser to 603 nm. This corresponded to an approximate 2200 cm−1 shift, which could be used to generate mid-infrared radiation for optical pumping of CO2 lasers.

While choosing the best ionic liquid for pumping the CO2 laser, the researchers considered that their color-shifting approach could have a broader use.

In a proof-of-principle, single-pass conversion setup, the researchers obtained a threefold-higher Raman conversion efficiency in the ionic liquid compared with water under identical conditions, resulting in an efficient generation of high-quality orange laser pulses in a wavelength region that is difficult to access at high energies.

“There are a lot of hard ways to do Raman shifting. But for this one, we just filled a tube with a properly selected ionic liquid, shot a laser in from one end, and we got the color we wanted out — without any fine tuning,” Wishart said.

Kupfer said that other methods for achieving a shift in laser color require complex optical setups or the use of toxic materials. “Plus, those other processes ‘break’ the molecules; they wear out and have to be replaced,” he said. “In our case, it is a balance sheet. The molecules stay unharmed.”

The researchers determined that ionic liquids provide a framework to engineer liquids suitable for wavelength conversion over a broad spectral range. Careful selection of the molecular structures of the ionic liquid anions and cations lead to specific characteristics, such as a desirable Raman shift, low Brillouin scattering, and good optical transmission in the pump and Stokes wavelengths. An ionic liquid can interact with photons while offering a high density of energy-swapping sites.

Gas molecules have limited vibrational frequencies, and diffuse gaseous molecules mean scattering efficiency is low. Solids have more tightly packed molecules, making them more efficient, but their complex vibrational frequencies make them costly to produce.

“Liquids are somewhere in between,” Wishart said. “You’re still dealing with single molecules, but denser, meaning higher efficiency than gases. And with ionic liquids, you can engineer the molecules to give you the frequency you need.”

The large number of ionic liquids available makes it possible to precisely tune the energy loss caused by the ionic liquid-photon interaction, providing greater selective control over color. Optically transparent ionic liquids prevent background absorption of light. In addition, their viscosity prevents laser scattering from acoustic waves, which can diminish the color-shifting effect in low-viscosity liquids.

Although further improvements could optimize the process, the researchers said that overall, the made-to-order ionic liquids provide a suitable platform for efficient, simple, adjustment-free laser color shifting using SRS.

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Computational Technique Harnesses the Benefits of Spectral, Photographic Insights

Using an approach that combines computer vision, color science, and optical spectroscopy, researchers at Purdue University devised an approach that enables conventional photography to be used for optical spectroscopy and hyperspectral imaging. Using the mechanism, the researchers realized spectral resolution comparable to the resolution of scientific spectrometers with photos from a smartphone camera.

A range of industries, running the gamut from agriculture, environmental monitoring, and food quality analysis, to industrial quality control, defense and security, and medical diagnostics, could benefit from the technique, according to its developers.

The researchers hypothesized that the RGB values of reference colors, as captured by a traditional camera, could be used to design a spectral color chart that could then be used to decode spectral information. The fidelity of spectral recovery would be determined mainly by the spectral incoherence among the reference colors in the chart.

They developed a general computational framework, co-designed with spectrally incoherent color reference charts, to recover spectral information from a single-shot photograph. They optimized reference color selection and the computational algorithm to eliminate the need for training data or pretrained models.

The spectral color chart, together with the device-informed computation, can be used to recover spectral information from RGB values acquired using conventional cameras, such as smartphone cameras.

In transmission mode, data is acquired by photographing the spectral color chart through the sample of interest. Altered RGB values of reference colors are used to recover the spectral intensity of the sample. In reflection mode, the sample of interest is placed alongside the spectral color chart to recover the sample’s spectral hypercube without needing a hyperspectral imaging system. A spectral hypercube of the sample can be constructed from a single-shot photo, analogous to hyperspectral imaging.

The technique, which the researchers called computational photography spectrometry (CPS), has the potential to make optical spectroscopy and hyperspectral imaging accessible with off-the-shelf smartphones. Instead of limiting the user to multispectral data with only a few bands, the technique enables a high spectral resolution of 1-2 nm.

“Importantly, the spectral resolution — around 1.5 nm — is highly comparable to that of scientific spectrometers and hyperspectral imagers,” researcher Semin Kwon said. “Scientific-grade spectrometers have fine spectral resolution to distinguish narrow spectral features. This is critical in applications like biomedical optics, material analysis, and color science, where even small wavelength shifts can lead to different interpretations.”

Although methods exist to estimate and reconstruct spectral information from RGB values acquired using conventional cameras, they are limited in their ability to achieve a high degree of spectral resolution.

The researchers developed a generalizable method for extracting high-resolution spectral information from a single-shot photo of a sample without having to rely on task-specific training datasets or predetermined models. This provides an advantage over existing machine learning models for spectral reconstruction, which depend on task-specific training data or fixed models.

“From an algorithmic standpoint, to the best of our knowledge, our paper presents the first computational spectrometry method with 1.5-nm spectral resolution using a photograph of an arbitrary sample without relying on specific training data or predetermined algorithms,” professor Young Kim said.

The spectral color chart and device-informed computation eliminate the need for complex hardware, simplifying the hardware requirements for the CPS technique. And, the CPS approach could potentially offer a simple, affordable, portable way to use smartphones for optical spectroscopy and hyperspectral imaging in day-to-day applications.

“Many mobile spectrometers require additional accessories and bulky components as mandatory attachments to smartphones,” Kwon said. “In contrast, our method leverages the built-in camera of the smartphone.”

The team is currently using the algorithm for digital and mobile health applications in both domestic and resource-limited settings.

“Photography is central to these applications, but color distortion has posed a persistent challenge, which is why we are focusing on these settings,” Kim said. “This algorithm provides a basis for quantifying and correcting colors, enhancing the reliability of medical diagnostics.”

The researchers believe that the generalized computational photography spectrometry technique could change how industry uses smartphones.

“A photograph is more than just an image. It contains abundant hyperspectral information,” Kim said. “We are one of the pioneering research groups to integrate computational spectrometry and spectroscopic analyses for biomedical and other applications.”

A patent for the algorithm is pending. Industry partners interested in developing or commercializing the algorithm should contact Patrick Finnerty, assistant director of business development and licensing-life sciences, at Purdue University.

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Spatial Light Modulation Gauges How Lenses Slow Progress of Myopia

Myopia, or nearsightedness, is one of the most common ocular disorders worldwide and a leading cause of visual impairment in children. Although specialized eyeglass lenses have been clinically tested to treat myopia progression, an in-depth optical characterization of the lenses has not yet been performed.

Researchers from the ZEISS Vision Science Lab at the University of Tübingen and the University of Murcia undertook a comprehensive characterization to investigate the properties of spectacle lenses designed to slow the progress of myopia. The results of their study could help increase the efficacy of future lens designs.

Myopia is typically caused when a person’s eyes become elongated, which affects how the eyes focus on faraway objects. The condition can progress in children and teens as their bodies grow.

To reproduce pupil shape and myopic ocular aberrations, researchers developed an instrument that reproduced the aberrations in myopic eyes and enabled physical simulation of the pupil. They based their instrument on spatial light modulation (SLM) technology.

“After exploring the state of the art, we didn’t find a method that could be used to characterize the optical properties of these eyeglass lenses under real viewing conditions,” said researcher Augusto Arias-Gallego. As a result, Arias-Gallego said, the researchers endeavored to build an instrument that can measure the lens’ optical response to different angles of illumination, while also reproducing the myopic eye’s pupil and refractive errors.

The team’s instrument uses an illumination source mounted on an arm that rotates around the lens. After the light passes through the lens, it is guided to an SLM by a rotating mirror. The SLM is composed of tiny liquid crystal cells that modify the propagating light, boosting its spatial resolution.

The SLM reproduces the refractive errors and pupil shape of myopic eyes, allowing the researchers to re-create myopic aberrations and to produce different aberrations depending on the angle of illumination. Using the SLM, the researchers programmed the aberrations as phase maps and induced programmed amounts of defocus to perform through-focus testing.

Tests helped the researchers determine the image quality within the proximity of a simulated retinal position, shedding light on how the special lens interacts with eye elongation signaled at the retina. “By combining the through-focus results with light-scattering measurements, we were able to accurately characterize several types of eyeglass lenses,” Arias-Gallego said. The researchers then compared measurements for each lens with their reported clinical efficacy for slowing myopia progression, he said.

The researchers quantified and compared the focusing and scattering properties of a single vision lens with two types of spectacle lenses for myopia progression management: defocus incorporated multiple segments (DIMS) and diffusion-optical technology (DOT). They calculated four optical metrics potentially related to myopia progress and quantified the scattered light from the peripheral lens zones. Scattering was quantified by implementing the optical integration method.

The characterization showed an increased contrast and sharpness of images through the DIMS lens at the peripheral retina when inducing myopic defocus, with respect to the single vision and DOT lenses. It further showed that contrast reduction by the DOT lens was dependent on the luminance at the pupil.

According to Arias-Gallego, the results both raised new questions and pointed to potential strategies that could increase the efficacy of future designs.

“Insights into the link between the optical properties of myopia progression management lenses and effectiveness in real-world scenarios will pave the way to more effective treatments,” Arias-Gallego said. “This could help millions of children and is fundamental in understanding the mechanisms by which these lenses work.”

The researchers are working to adapt the SLM instrument to include sources with varying wavelengths.

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DNA-Barcoded Fluorescence Imaging Illuminates Core Cell Components

Since many core components of cells — like DNA, RNA, proteins, and lipids — are just a few nanometers in size and substantially smaller than the resolution limit of traditional light microscopy, the exact composition and arrangement of these molecules and structures is thus often unknown. This results in a lack of mechanistic understanding of fundamental aspects of biology.

Drawing on recent improvements to superresolution imaging, including single-molecule localization microscopy, or SMLM, researchers from the Max Planck Institute of Biochemistry and Ludwig-Maximilians-Universität Munich have developed a technique that enhances the resolution of fluorescence microscopy down to the angstrom scale. The researchers’ technique enables the study of whole and intact cells over individual proteins — all the way down to the distance between two adjacent bases in DNA.

The researchers, from the group of Ralf Jungmann, called the technique resolution enhancement by sequential imaging, or RESI.

Current SMLM resolve structures on the order of 10 nm by temporally separating the structures’ individual fluorescence emission. As individual targets stochastically light up in an otherwise dark field of view, their location can be determined with subdiffraction precision. The SMLM technique of DNA points accumulation for imaging in nanoscale topography, or DNA-PAINT, uses the transient hybridization of dye-labeled DNA “imager” strands to their target-bound complements to achieve the light-up necessary to achieve superresolution.

To date, however, neither DNA-PAINT nor other superresolution methods have been able to resolve the smallest cellular structures.

RESI builds on DNA-PAINT and capitalizes on its ability to encode target identity via DNA sequences. By labeling adjacent targets, too close to each other to be resolved even by superresolution microscopy, with different DNA strands, an additional degree of differentiation — a barcode — is introduced into the sample. By sequentially imaging first one and then the other sequence to thereby capture the full target, the strands can now be unambiguously separated.

Critically, as they are imaged sequentially, the targets can be arbitrarily close to one other, which is a dynamic that existing techniques are unable to resolve. Further, RESI does not require specialized instrumentation and can be applied using any standard fluorescence microscope.

To demonstrate RESI’s leap in resolution compared to other methods, the researchers sought to resolve the separation between individual bases along a double helix of DNA, which is separated by less than 1 nm. They designed a DNA origami nanostructure that presented single-stranded DNA sequences that protrude from a double helix at one base pair distance.

The researchers then imaged these single strands sequentially and resolved a distance of 0.85 nm, or 8.5 Å between adjacent bases. They accomplished these measurements with a precision of 1 Å, or one ten-billionth of a meter.

According to the researchers, the technique is universal, with application beyond DNA nanostructures. In separate tests, Jungmann and his team investigated the molecular mode of action of rituximab, an anti-CD20 monoclonal antibody used for treatment of CD20-positive blood cancer. Investigating the effects of such drug molecules on molecular receptor patterns has been beyond the spatial resolution capabilities of traditional microscopy techniques. Understanding whether and how such patterns change in health and disease as well as upon treatment is important for mechanistic research and the design of targeted therapies.

The researchers used RESI to reveal the natural arrangement of CD20 receptors in untreated cells as dimers and uncovered how CD20 re-arranged to chains of dimers upon drug treatment.

Because RESI is performed in whole, intact cells, the technique closes the gap between purely structural techniques such as x-ray crystallography or cryogenic electron microscopy and traditional lower-resolution whole-cell imaging approaches.


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Agate Sensors Raises $6.6M for Everyday Spectroscopy Tech

Agate Sensors, a spinout of Aalto University developing smart sensors for material analysis, has raised €5.6 million (~$6.6 million) to commercialize a research breakthrough that shrinks spectroscopy from suitcase-sized lab equipment to a single pixel smaller than a grain of sand — integrated into a chip compact enough to sit on the tip of a finger.

The startup’s technology allows devices to analyze the spectral signatures of materials in real time, bringing high-precision material sensing out of the lab and into everyday devices, from smartphones and wearables to medical equipment and defense systems.

“We’ve taken a spectrometer once confined to specialized labs and made it small and affordable enough to live inside everyday devices,” said Tommi Leino, CEO of Agate Sensors. “One sensor can shift between functions entirely through software — from diagnosing a health condition to detecting, identifying, and classifying objects and materials — changing how we interact with the physical world.”

Manufacturing of initial chips is expected by year-end, enabling proof-of-concept demonstrators throughout 2026 and first commercial smart wearable products targeted for late 2027. According to the company, one of the earliest market ready applications lies in defense, as the sensor enables the distinction between real foliage and synthetic camouflage, or the identification of specific vehicle types via paint signatures.

The platform also has applications in machine vision, including multi-biomarker health monitoring in wearables, detection of counterfeit goods in supply chains, identifying environmental hazards in industry, and early intervention in smart agriculture and forestry, among other potential uses.

“This technology is the result of over a decade of research in semiconductor physics and nanotechnology at Aalto University,” said Andreas Liapis, CTO of Agate Sensors. “For the first time, we are able to bring laboratory-grade spectroscopy to an integrated form factor suitable for mass market use.”

The round includes €4 million in seed funding led by Voima Ventures and LIFTT, plus €1.6 million in grants from Business Finland.

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Two-Photon Microscopy Connects Hypoxia in Brain to Stalled Blood Flow

Using high-resolution imaging with two-photon phosphorescent lifetime microscopy (2PLM), researchers learned that even brief interruptions in blood flow to capillaries in the brain can cause rapid, localized drops in oxygen that probably extend into nearby brain tissue. These stalls in blood flow, in the smallest vessels in the brain, could play a role in brain diseases like stroke, Alzheimer’s disease, and traumatic brain injury, where such disruptions are common.

Using a two-photon phosphorescent probe, a team comprising researchers from Boston University and Massachusetts General Hospital monitored capillary flux and partial pressure of oxygen (pO2) in the mouse cortex. The researchers sought to quantify oxygen dynamics around capillary stalls as they occurred in vivo.

2PLM provided high-resolution measurement of the pO2 in the brain, enabling the researchers to investigate the distribution and consumption of oxygen. It offered the spatial and temporal resolution necessary to capture stalls using single point measurements.

The researchers excited phosphorescence at 950 nm with a pulse laser, and controlled excitation power with an electro-optic modulator. The excitation and emission light were split with a primary dichroic, and excess laser power was blocked by a filter in the detection path. Photons were detected by a photomultiplier tube after passing through a secondary dichroic and emission filter.

Imaging planes started at about 50 μm below the cortical surface and extended to an approximate depth of 250-300 μm. The researchers selected 10-20 points in each plane, based on capillaries with clear cross-sections parallel to the imaging plane.

At each point, the researchers performed 1000 cycles of 10 microsecond (10-μs) excitation, and 290 μs of collection and photon counting.

The researchers measured red blood cell passage and oxygen levels in more than 300 mice capillaries. Using 2PLM, they tracked the moments when red blood cells temporarily stopped moving through a blood vessel and monitored the resulting oxygen changes in real time.

To quantify the effect of stalling on oxygen, they monitored capillary oxygen, flux, and speed in 10 to 20 capillaries for about 10 minutes. They repeated these 10-minute recordings in several different regions of interest.

They found that every stall caused an immediate decline in oxygen within the capillary, which was likely to spread to surrounding tissue. About 40% of stalls dropped to levels considered hypoxic, and about 25% fell to levels where cells could not sustain normal energy production.

The severity of hypoxia differed depending on the animal’s state. Awake animals were far more vulnerable than mice under anesthesia, highlighting how dependent the brain is on uninterrupted microvascular flow under normal conditions.

The researchers also found that nearby capillaries sometimes showed small drops in oxygen when a neighboring vessel stopped flowing. This suggests that the impact of a stall may extend to the microvascular network surrounding the blocked vessel.

Because some capillaries tend to stall repeatedly, the tissue in their vicinity may experience repeated bouts of hypoxia over time, the team found. This could be one way that capillary dysfunction contributes to brain diseases where stalling is common. With the increased incidence of stalling that occurs with age or diseases such as Alzheimer’s and stroke, the potential for acute metabolic disruption is increased.

Future work could extend the measurements to deeper cortical layers and provide comparisons between healthy and diseased models. Flow and oxygen distributions are different across brain regions and could potentially be found to result in different dynamics around stalling events.

The brain depends on a constant supply of oxygen, and unlike other organs, it has minimal stores of energy. While it is known that blockages in larger vessels can have devastating consequences for the brain, less is understood about the effects of momentary stalls in the smallest vessels, the capillaries. These stalls have been observed more often in aging brains and in conditions such as Alzheimer’s disease, stroke, and traumatic brain injury.

The use of 2PLM to study when and where stalls in capillary flow occur, and result in pockets of low oxygen in the brain, could provide valuable information on a way that the conditions for various brain diseases may be exacerbated.

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FPGA-Based Data Compression Drives Brain Imaging Performance Gains

To help broaden the use of single-photon avalanche diode (SPAD) cameras for multispeckle diffuse correlation spectroscopy (DCS), researchers at the University of Edinburgh developed a data compression scheme for a large-pixel-count SPAD camera using a field-programmable gate array (FPGA).

The camera system’s large sensor array enabled a substantial signal-to-noise ratio (SNR) gain over a single-pixel system: The researchers demonstrated an SNR gain of 110, with respect to single-pixel multispeckle DCS, using half of the 192 × 128 SPAD array. The pixel active fill factor was 13% — an order-of-magnitude-larger pixel count than in prior works, according to the team.

FPGA compression for large-array multispeckle DCS could democratize the use of SPAD cameras in the biomedical research community, the researchers believe, extending the benefits of multispeckle DCS across many areas of biomedical research.

DCS quantifies cerebral blood flow — an important indicator of brain health — by measuring the autocorrection function of diffused light introduced through the scalp. The light scatters through the deep tissue and returns a speckle pattern at the detector. The pattern fluctuates in intensity in response to the movement of the tissue and the blood circulating within it.

SPAD cameras have made it possible to capture many independent speckles at the same time, leading to multispeckle DCS instruments with high sensitivity. However, multispeckle DCS systems are hampered by the small number of pixels in a SPAD array and by a lack of camera-embedded processing capabilities. The extremely high data rates of SPAD cameras, which exceed the maximum data transfer rates of commonly used communication protocols, require large computing resources and limit the scalability of SPAD cameras to the higher pixel resolutions.

To enable practical use of SPAD cameras for multispeckle DCS, the researchers, led by professor Robert K. Henderson, connected a SPAD sensor array composed of 192 × 128 pixels to a commercial FPGA. They embedded an autocorrelation algorithm in the FPGA to make data compression scalable to large SPAD arrays. The algorithm can perform most of the calculations needed for DCS, and the system demonstrated the ability to calculate 12,288 autocorrelations in real time from the SPAD array output.

Shifting the computational burden from a host computing system to the hardware directly connected to the SPAD sensors alleviated the need for high-powered computing resources and extremely fast data transfer rates. The researchers ran the multispeckle DCS measurements in real time using a standard PC.

“Our proposed system achieved a significant gain in the signal-to-noise ratio, which is 110 times higher than that possible on a single-speckle DCS implementation and 3× higher than other state-of-the-art multispeckle DCS systems,” Henderson said.

Although the focus of the work is on FPGA-embedded processing for multispeckle DCS, the system can also operate in multispeckle time-domain DCS with a precision of 33-ps time-of-flight resolution, the researchers said.

In the future, the FPGA-based design for data compression could enable SPAD arrays for multispeckle DCS that provide high pixel resolution without requiring specialized, high-performance computing to calculate autocorrelators for real-time measurements.


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Skin-Like Microfiber Grating Gauges Cardiovascular Wellness

According to the World Health Organization, 17.9 million people die annually due to cardiovascular diseases. Soft wearable devices are well suited for monitoring physiological signals from electrocardiogram, phonocardiogram, and pulse wave. Advantages of these types of devices include real-time operation capability, skin-like mechanical properties, and high signal-to-noise sensing capability.

However, monolithic hemodynamic parameters achieved by current wearable devices cannot adequately and precisely reflect the health status of regional vasculature. Spatiotemporal hemodynamic monitoring techniques are needed to satisfy growing demand for clinical treatment and daily health management of the cardiovascular system.

Researchers from Nanjing University have developed a hemodynamics monitoring technique that relies on a configurable, skin-like microfiber Bragg grating group to deliver information on the real dynamics of the systemic cardiovascular system, such as heartbeat, angiectasis, and pulse wave propagation. The system overcomes common bottlenecks to biophotonic sensing mechanisms that use commercial fiber Bragg grating (FBG) devices.

Although the conventional DOF sensing technique represented by the fiber Bragg grating FBG is well suited for spatiotemporal hemodynamic monitoring, the researchers said, the traditional optical fiber commonly used in the method has a large distinct mechanical property with the skin and a low response on physiological signals. This makes it difficult to be worn on the body stably and comfortably. And, while flexible packaging technology has been used to address the mechanical mismatch, thick encapsulation and the low sensitivity of commercial FBG devices pose an obstacle in detecting subtle physiological signals, thereby limiting their potential applications in wearable devices.

Similarly, optical microfibers have been proven to have excellent flexibility, configurability, and large evanescent fields for high-sensitivity sensing. However, existing devices based on optical microfiber are difficult to achieve spatially distributed, time-synchronized, and multiparameter sensing capabilities without a wavelength encoding strategy.

The researchers’ skin-like microfiber grating group combines ultrathin flexible packaging technology with microfiber to produce skin-like fiber patches. The researchers used femtosecond laser direct writing technology to noninvasively inscribe the Bragg gratings into the interior of the microfiber. This provided different wavelength encodings for multiple microfiber patches, which in turn enabled synchronous multichannel sensing capabilities

By connecting microfiber grating patches in series, the researchers detected multiple physiological signals at different nodes of the human body simultaneously and distinguished them by different working wavelengths. Since the light-based physiological signals propagate at close to the speed of light in the microfiber grating group, the time synchronization is only limited by the FBG interrogator.

The researchers ultimately activated the monitoring technology by detecting the proximal ballistocardiograph signal and the distal pulse wave at each superficial artery in the human cardiovascular system and then calculating the pulse wave transmit time. By detecting mechanical signals at the proximal and distal ends of the cardiovascular system instead of electrophysiological activity signals, the monitoring technique can present the real dynamics of the systemic cardiovascular system, such as heartbeat, angiectasis, and pulse wave propagation.

In addition, the mechanism showed favorable repeatability and stability — under 10,000 stress circles.

In their development of the technique, the researchers presented three distinct hemodynamic monitoring modes. They said that the technology has the working capability of real-time and dynamic evaluation of local blood vessel health status in the whole cardiovascular system, demonstrating great potential in the diagnosis of cardiovascular diseases such as arrhythmia, angiosclerosis, hypertension, and thrombosis. Further, the advancement could serve to facilitate precise clinical diagnosis, the fast screening of lesions, and daily health management.

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More Efficient Microcombs on the Road to Commercialization

Researchers at Chalmers University have developed a method to make microcombs 10 times more efficient, opening pathways to discovery in space and health care, and paving the way for high-performance lasers in a range of technologies. The team has established a company to commercialize the new technology.

Laser frequency combs can measure frequency with extreme levels of precision, analogous to a ruler made of light. The principle is based on a laser sending photons that circulate within a small cavity — a so-called microresonator — where the light is divided into a wide range of frequencies. These frequencies are precisely positioned in relation to each other, like the markings on a ruler. Therefore, a new kind of light source can be created consisting of hundreds, or even thousands, of frequencies, like lasers beaming in unison.

Because virtually all optical measurements are connected to light frequencies, the microcomb has myriad applications, from calibrating instruments that measure signals at light-year distances in space in the search for exoplanets, to identifying and keeping track of health via exhaled air.

A fundamental problem with microcombs has been that their efficiency has been too weak to reach their transformative potential. The conversion efficiency between the laser and the microcomb was too weak, meaning that only a fraction of the power contained in the laser beam was usable.

According to Victor Torres-Company, professor of photonics at Chalmers, the new method breaks what was believed to be a fundamental limit for optical conversion efficiency. The method increases the laser power of the soliton microcomb by 10 times and raises its efficiency from about 1% to more than 50%.

Rather than using just one microresonator, the new method uses two. They form a unique ensemble with properties greater than the sum of its parts. One of the resonators enables the light coming from the laser to couple with the other resonator; similar to impedance matching in electronics.

According to the researchers, the high conversion efficiency and uniform spectrum make the devices ideal for applications in optical communications and dual-comb spectroscopy. With further engineering of the coupling region and dispersion, the researchers believe the results pave the way for the realization of octave-spanning microcombs and self-referencing using only integrated components.

Additionally, a shifted resonance can be achieved in multiple different systems, and it is not limited to the coupled-cavity design that the researchers have presented. As such, they said, the work provides important insights for realizing high-efficiency solitons using other schemes, like photonic crystal resonators or linearly coupled transverse modes, and, potentially, when using a feedthrough pump cavity.

The technology was recently patented and the researchers founded Iloomina AB, a company that will launch the technology onto a wider market.

According to Torres-Company, the new microcombs enable high-performance laser technology in numerous markets. “For example, frequency combs could be used in lidar modules for autonomous driving, or in GPS satellites and environmental sensing drones, or in data centers to enable bandwidth-intensive AI apps,” he said.

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Lasers Help Mimic Biosystems as Team Prods Organisms’ Reproduction

Researchers at New York University (NYU) have devised a system of asynchronous, optically driven micro-rotors that could be used to study far-from-equilibrium phenomena such as turbulent weather and biosystems. The advancement could potentially be used to replicate natural phenomena in engineered systems.

In vortical flows, which are found in both meteorological and biological systems, particles move into orbital motion in the flow generated by their own rotation, resulting in a range of complex interactions. To better understand these dynamics, the researchers sought to replicate vortical flows at their most basic level. They created a system to move micro-particles using micro-rotors and a laser beam.

Direct observation of hydrodynamic coupling between artificial micro-rotors has been restricted by the details of the drive that is used, either through synchronization (using external magnetic fields) or confinement (using optical tweezers). The NYU system is enabled by a tweezing-free optical field.

The researchers designed a force-free torque field using a collimated beam of circularly polarized light and developed a synthetic route for birefringent, silica-coated colloids to show the spinning of hundreds of micro-particles using photonic angular momentum. They systematically quantified the micro-rotors’ optical and hydrodynamic properties. Unlike previous synthetic micro-rotor systems, the particles rotated asynchronously in the optical torque field while freely diffusing in the plane.

The researchers also found that the rotating particles affected each other’s orbital motion.

Analysis of the particles’ spinning rates revealed that pairs of rotating particles mutually advected one another, and that their translation and rotation were coupled hydrodynamically. The coupling was geometric, indicating that it could potentially have general application in active systems, from living organisms to robotic systems.

For example, the researchers found similarities in their system to the dynamics observed by other scientists in “dancing” algae, that is, in algae groupings that move in concert with each other.

“The spins of the synthetic particles reciprocate in the same fashion as that observed in algae — in contrast to previous work with artificial micro-rotors,” said Matan Yah Ben Zion, a doctoral student at the time of the work and now a researcher at Tel Aviv University. Synthetically, and on the micron scale, the researchers successfully reproduced an effect that is seen in living systems, he said.

The NYU system could be used to investigate isotropic rotating ensembles with broken time-reversal symmetry and parity in order to shed light on new material properties theoretically predicted in active matter. These include odd viscosity and quantum hall fluids. Free optical rotors using nonspherical particles could also be used to study the effect of morphology and steric interactions in tandem with hydrodynamic coupling.

Further, the NYU system of optical rotors, if combined with rotors driven by an external magnetic field, could enable the experimental study of ensembles of counter-rotating particles. In these mechanisms, optical rotors rotate independently from the magnetic rotors. Experimental investigation of an ensemble of counter-rotors could expand scientific understanding of far-from-equilibrium states of matter.

“Collectively, these findings suggest that the ‘dance of algae’ can be reproduced in a synthetic system, better establishing our understanding of living matter,” Ben Zion said. “Living organisms are made of materials that actively pump energy through their molecules, which produce a range of movements on a larger cellular scale.

“By engineering cellular-scale machines from the ground up, our work can offer new insights into the complexity of the natural world.”

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Breath-Activated Sensor for Diagnosing Diabetes

Currently, diagnosing diabetes and prediabetes means a visit to a doctor's office or lab work, both of which can be expensive and time-consuming. Research from Huanyu "Larry" Cheng at Penn State has yielded a sensor that can help diagnose diabetes and prediabetes on-site in a few minutes with just a breath sample.

Previous diagnostic methods have used glucose found in blood or sweat, but the current sensor however, this non-invasive test uses a sensor to detect acetone levels in breath. While acetone in breath is a normal byproduct from the burning of fat, an acetone level of 1.8 parts per million is a sign of diabetes.

“While we have sensors that can detect glucose in sweat, these require that we induce sweat through exercise, chemicals or a sauna, which are not always practical or convenient,” Cheng said. “This sensor only requires that you exhale into a bag, dip the sensor in, and wait a few minutes for results.”

While there have been other breath detection methods in the past, they have required lab analysis. Acetone can be detected and read on-site, making the new sensors cost-effective and convenient.

Beyond using acetone as the biomarker, Cheng said another novelty of the sensor came down to design and materials — primarily laser-induced graphene. To create this material, a CO2 laser is used to burn the carbon-containing materials, such as the polyimide film in this work, to create patterned porous graphene with large defects desirable for sensing.

The porous nature of the graphene helps to let the gas pass through, which means there is a higher likelihood of the acetone molecules being captured. By itself, laser graphene didn’t identify acetone as precisely as needed, which the team remedied by combining the graphene with zinc oxide.

“A junction formed between these two materials that allowed for greater selective detection of acetone as opposed to other molecules,” Cheng said.

Cheng said another challenge was that the sensor surface could also absorb water molecules, and because breath is humid, the water molecules could compete with the target acetone molecule. To address this, the researchers introduced a selective membrane, or moisture barrier layer, that could block water but allow the acetone to permeate the layer.

Currently, the method requires that a person breathe directly into a bag to avoid interference from factors such as airflow in the ambient environment. The next step is to improve the sensor so that it can be used directly under the nose or attached to the inside of a mask, since the gas can be detected in the condensation of the exhaled breath. He said he also plans to investigate how an acetone-detecting breath sensor could be used to optimize health initiatives for individuals.

“If we could better understand how acetone levels in the breath change with diet and exercise, in the same way we see fluctuations in glucose levels depending on when and what a person eats, it would be a very exciting opportunity to use this for health applications beyond diagnosing diabetes,” Cheng said.

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Light-Activated Biomimetic Gel Offers Potential for Corneal Repair

Tens of millions of people around the world suffer from corneal diseases, with only a small fraction eligible for corneal transplantation. In a recent study, researchers at the University of Ottawa showed that biomimetic materials activated with low-energy blue light can reshape and thicken damaged corneal tissue to promote healing and recovery. The research results could provide a safe way to treat corneal thinning, as well as a practical alternative to corneal transplantation.

Further, the dosage of pulsed blue light needed to activate the biomaterial is minimal, which mitigates the possibility of cytotoxic effects from the light.

The biomaterial is injected within the corneal tissue after a tiny pocket is surgically created. The injectable biomaterial, which is in the form of a viscous liquid, is made from short peptides and glycosaminoglycans that assemble into a hydrogel when irradiated with low-energy blue light. The hydrogel hardens and forms a tissue-like 3D structure with properties similar to those found in pig corneas. The use of low-energy pulsed light irradiation allows the researchers to safely use photocuring to photo-crosslink the biomimetic materials designed for injection into thinning corneas.

Data showed that the materials used to obtain experimental test results could remain in an animal model for several weeks. As a result, professor Emilio Alarcon and the other researchers anticipate that the material will remain stable and be nontoxic in human corneas.

In their study, the researchers observed that the way the light was delivered affected the formation of the hydrogel. Pulsed irradiation allowed for better recovery of the oxygen levels within the hydrogel, compared to a continuous dosage of light. Pulsing the light for 2.5 s on, 2.5 s off produced optimal results.

To keep the light dosage under standard safety values, the researchers selected a low in vivo radiance dosage of 8.5 mW cm−2 for 10 min of pulsed light, which is equivalent to only 5 min of light exposure. This radiance level is below the category of low risk when direct blue light is exposed to the eye for up to 166 min. The researchers performed in vivo experiments for light toxicity on animal models to verify the safety of their light irradiation regime.

In vivo experiments using a rat model indicated that the light-activated hydrogel could thicken corneas without side effects. The researchers also successfully tested the technology in an ex vivo pig cornea model.

According to Alarcon, the researchers developed the technology to be clinically translatable; all components must be designed to be manufacturable following strict standards for sterility. In clinical conditions, reducing the amount of light delivered to the eye will translate into a faster and safer procedure. It will shorten the period during which eye movement must be minimized to ensure stability of the injected biomaterial volume and shape until it turns into a soft hydrogel. Good control of the corneal front surface curvature is of primary importance for adequate refraction of the light within the eye.

Although testing in large animal models will be necessary prior to clinical human trials, the researchers have begun the patent application process for the technology.

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Single-Cell Analysis Optofluidics Tool Delivers on Demand

An on-demand optical system for exporting target droplets from a static droplet array (SDA) provides a simple way to export specific cells or analytes for analysis without compromising efficiency or accuracy. Researchers at the Qingdao Institute of Bioenergy and Bioprocess Technology of the Chinese Academy of Sciences developed the system, called optical on-demand droplet release (OODR). The developers and their collaborators believe that OODR could promote SDAs as a valuable tool for use with high-capacity screening assays with applications in diverse fields. They said that the technique in its current stage of development has the potential to be used in single-molecule/cell analysis, drug screening, and phenotype-based cell sorting.

The OODR system incorporates a 1064-nm laser-responsive indium tin oxide (ITO) layer into a microchamber, array-based, droplet microfluidic chip. When the laser is focused onto the ITO layer of the chip, local heating causes microbubbles to form. The microbubbles push the droplets out of the chamber on a selective basis.

The researchers fabricated the chip using a low-cost, readily available ITO glass as a photoresponsive layer. The ITO layer was bonded to a PDMS layer with an array of microchambers to provide the capability to selectively release target droplets and enable the SDA to work rapidly.

According to the researchers, the size of the microbubble proved to be critical to the successful release of the droplet based on the chip design used by the team. They identified the optimal size for the microbubble as 40 μm, and they tuned the laser to the range necessary to generate the amount of heat needed to form a correctly sized microbubble without degrading the integrity of the cell. Under the appropriate conditions, OODR can release a droplet within three seconds.

OODR precisely heats the ITO layer, via laser, to create microbubbles that allow for the selective pushing of a target droplet out of the chamber on the microfluidics chip. This is based on the microdroplet single-cell sorting system, such as EasySort Compact, for example. The team used the EasySort Compact system to achieve automatic single-cell sorting, said professor Bo Ma.

Once the released droplet is pushed out of the chamber, it is carried by the flow to the outlet. The droplet can be easily exported in the one-droplet-one-tube (ODOT) manner by a pipette tip, via the inherent capillary force, which allows the movement of liquid without applying external force. The droplet is exported into a well or tube in a high-throughput manner for further analysis.

The released droplet is identified by using white or fluorescent imaging. These images can be used to sort the morphology of the target bacteria, which can be a challenge without a static image for reference.

The researchers used OODR to selectively release droplets containing fluorescein sodium from an SDA consisting of 6400 microchambers. OODR achieved a success rate of about 100% (nine out of 6400 droplets were successfully released). It also exhibited low residual, with only about 5% of the droplet volume remaining in the chamber.

The team demonstrated on-demand release of single-cell and multicell droplets for both E. coli and yeast, based on white or fluorescence imaging. The successful use of OODR with E. coli and yeast cells suggests that the system is applicable to other types of cells. According to Ma, the technique not only targets single cells, but enables the sorting of microdroplets that contain one cell, multiple cells, and/or reagents only.

Beyond avoiding causing any effect to the cell’s ability to be cultivated, or accuracy, the researchers said, the successful cultivation of the cell-containing droplets in an ODOT manner indicates that the isolation method has minimal impact on cell viability. This, they said, is essential when further live-cell analyses are needed. It also demonstrates the potential to seamlessly couple OODR with downstream ODOT-based assays, such as human or microbial single-cell sequencing. OODR also reduces the sample size and amount of reagent needed for analysis, making SDA more efficient and cost-effective. And the current system is user-friendly, the researchers said.

Professor Jian Xu said that the team is currently using artificial intelligence and machine learning to automate the system via EasySort. This will reduce human involvement in the system’s operation, making it easier for nonprofessionals to use, which could further expand the use of OODR and SDA.

Bio Photonics Research Award

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