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.

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Multifocus Microscope Pushes Limits of 3D Biological Imaging

A high-speed 3D imaging microscope developed by researchers at the University of California, Santa Cruz can capture detailed cell dynamics of an entire small whole organism at once. The ability to image 3D changes in real time over a large field of view could lead to new insights in developmental biology and neuroscience.

“Traditional microscopes are constrained by how quickly they can refocus or scan through different depths, which makes it difficult to capture fast, 3D biological processes without distortion or missing information,” said Eduardo Hirata Miyasaki, who performed the work while in Sara Abrahamsson’s lab at the University of California Santa Cruz (UCSC). “Our new system extends the multifocus microscopy (MFM) technique Abrahamsson developed by using a 25-camera array to push the limits of speed and volumetric imaging. This leap in efficiency opens the door to studying small living systems in motion without disrupting them.”

The researchers describe their new microscope, which combines diffractive optics with 25 tiny cameras to synchronously and simultaneously image at multiple depths. They demonstrate live imaging of 25-plane 3D volumes measuring up to 180 × 180 × 50 μm at acquisition speeds of more than 100 volumes per second.

“The new microscope, which we call the M25, is particularly useful for imaging swimming C. elegans worms, a model organism used to study development, neuroscience and locomotion,” said Hirata Miyasaki, now at the Chan Zuckerberg Biohub. “Traditionally, scientists could only see part of the organism clearly at any one time. With our new microscope, it is possible to watch the entire worm move naturally in 3D, allowing researchers to study how its nervous system controls movement and how behavior might change in response to a genetic mutation, disease or drug treatment.”

A key part of the new microscope is the diffractive optical elements used to distribute the various focal planes across an array of 25 cameras. Diffractive optics use microstructures to manipulate light, allowing more complex light control via a thinner, lighter component than traditional optical components such as prisms.

Building upon the original MFM technique, the researchers designed a multi-focus grating to split the incoming light so that each camera captures the same scene but with a focus at a different depth. They also made customized gratings to use in front of each camera lens to correct the chromatic dispersion introduced by the multi-focus grating. By replacing the traditional chromatic-correcting prism, which was difficult to scale beyond 3×3 arrays, these blazed gratings enabled high-resolution, high-speed bioimaging across more planes.

The gratings are made from nanometer-scale patterns that require specialized fabrication tools. After using simulations to determine the optimal designs, the researchers used the University of California Santa Barbara nanofabrication facility to etch the patterns into glass. With the fabrication process now established, these diffractive elements can be accurately reproduced at higher volumes.

“One of the key innovations of the M25 is its use of simplified chromatic correction architecture: By replacing bulky prism-based components with custom-designed blazed gratings, the system achieves efficient dispersion correction across all focal planes while remaining compact and scalable,” said Abrahamsson. “This streamlined optical design not only enables high-speed imaging but also supports compatibility with label-free modalities — a major advantage for applications like embryology, where minimally invasive imaging is essential.”

“When combined, the 25 images — all acquired simultaneously, with no mechanical scanning or moving parts — form a complete 3D snapshot,” said Hirata Miyasaki. “Because this happens at high speed, limited only by the camera’s acquisition speed and the sample’s brightness, we can record entire volumes over time, enabling studies of real biological dynamics.”

The M25 microscope can be used for both fluorescence and label-free modalities, such as brightfield and polarization microscopy, which are especially useful for imaging sensitive biological systems without introducing dyes or labels. This compatibility with minimally invasive techniques makes the M25 well-suited for applications like embryology, where preserving native physiology is critical.

To validate the instrument, the researchers built a prototype and confirmed that it could capture 25 distinct, evenly spaced focal planes simultaneously, without distortion or overlap, by imaging calibration targets. They also used the microscope to image live biological specimens, including common model organisms such as C. elegans, D. melanogaster and P. marinus, demonstrating real-time 3D imaging of moving organisms without the need for scanning or motion compensation.

The system mounts to the side port of a standard commercial microscope. Aside from the diffractive optics, it requires no specialized hardware, making it more straightforward to replicate than systems that rely on custom prisms or complex light path modifications.

Next, the researchers aim to further expand the system’s scale and applications. For example, they plan to use the system’s rich imaging data to train machine learning models that can identify cell states, track dynamic behaviors and detect disease-related changes directly from images.

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Microscope Design Uses Resonator to "Store" Light, Avoid Sample Degradation

In photography, taking a detailed image requires a lot of light. But in microscopy, too much light is often harmful to the sample, such as when imaging sensitive biological structures or investigating quantum particles. The aim is therefore to gather as much information as possible about the object under observation with a given amount of light.

In collaboration with the University of Vienna and the University of Siegen, researchers at TU Wien have developed a novel trick to achieve this: storing the light in a resonator in which the sample is also located. This allows them to obtain a clearer signal than with other methods.

“In a normal microscope, the light hits the sample once and then enters a lens,” said Maximilian Prüfer, who led the study as part of his fellowship at the Atomic Research Institute of TU Wien. “In our microscope, we place the sample in an optical resonator — between two mirrors.”

To turn this resonator into a microscope, the team developed an unusual experimental setup with additional lenses: After the light beam has passed through the sample, it is guided in a circle and hits the sample again. “Now the sample is illuminated again, but not with a normal, uniform beam of light as in the beginning, but with a beam of light that already contains the image of the sample, so to speak,” said Oliver Lueghamer of TU Wien, who built the microscope as part of his master's thesis.

Similar to a stamp that is pressed several times on the same spot, producing a clearly visible image even with faint ink, the image of the sample becomes clearer and clearer as it completes several rounds in the microscope.

Both theoretical calculations, which were developed in collaboration with Thomas Juffmann of the University of Vienna and Stefan Nimmrichter of the University of Siegen, and experiments show that this method provides more information than other microscopy techniques at a given light intensity. “The key figure is the signal-to-noise ratio,” said Maximilian Prüfer. “This ratio is better here than with other methods due to multiple scattering with the same disturbance of the sample.”

However, the practical suitability of the developed instrument and method also depends on how susceptible it is to disturbances. “When using optical resonators, as we do, it is often important to keep their length extremely constant,” Prüfer said.

“Normally, you have to go make a great effort to ensure that the distance between the two mirrors varies only minimally, otherwise the desired effect is lost. With our method, however, this is not the case.”

The distance between the mirrors can also show a certain instability without the enhancement disappearing. “This is important because it means that the method not only works in theory, but can also be used in practice with manageable effort,” Prüfer said.

One of the goals of the new microscopy technique is to image ultra-cold Bose-Einstein condensates and thereby study their quantum physical behavior.

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OLED Contact Lenses Expand Options for Diagnostics and Treatment

A wireless contact lens that integrates OLED technology into ophthalmic diagnostics could transform the way in which ocular health is monitored, benefiting both patients and practitioners.

The lens is the result of a collaboration among the Korea Advanced Institute of Science and Technology (KAIST), the Electronics and Telecommunications Research Institute (ETRI), and the Seoul National University Bundang Hospital. To create the wearable light source, the team led by professor Seunghyup Yoo designed a configuration and process flow that integrated an ultrathin OLED, an antenna, and a controller chip for wireless power reception with a contact lens.

Using a 433 megahertz (MHz) resonant frequency for power transmission, the researchers implemented a robust wireless power system for the lens. They embedded a wireless microcontroller into a sleep mask to optimize the lens and allow it to be synchronized with a smartphone.

The team demonstrated the efficacy of the OLED contact lens as a robust lighting solution for electroretinography (ERG) examinations in clinical settings.

It showed that the wearable light source could generate an ERG signal comparable to that of conventional, full-field light stimulation, and that the OLED lens system could be used for ERG measurement with minimal signal interference.

In an in vivo animal experiment, the researchers observed that the animal’s eye temperature remained well within safe limits while it was wearing the lens, mitigating the risk of corneal heat damage from the OLED. The researchers also found that the lens continued to maintain a robust optical performance during exposure to humid environments — an indicator of its potential for practical use in real-world settings.

In earlier lens designs, the light diffusion provided by inorganic LEDs was often inadequate, leading to excessive heat buildup. OLEDs offer a superior solution to LEDs due to their ability to emit light more uniformly across the surface area of the lens. This feature helps reduce heat generation and ensures retinal responses can be achieved even under low-light conditions.

During the study, the researchers observed retinal response results at a luminance level considerably lower than that of standard devices, further demonstrating the viability of the OLED technology as a diagnostic tool.

The 12.5-μm-thick OLED contact lens is designed to optimize both comfort and function. It is engineered to be an effective, yet unobtrusive, device that will allow patients to engage actively during retinal diagnoses. Unlike conventional ERG, which requires a darkened space and restricts patient mobility for prolonged periods, the OLED contact lens allows patients to rest comfortably, even with their eyes closed, while they undergo retinal evaluations.

In addition to ensuring efficiency and patient comfort during retinal exams, the ultrathin OLED contact lens could be a valuable tool for personalized healthcare monitoring. By combining wearable technology and healthcare, the OLED contact lens could help shape the future of ocular care.

Further integration of smart contact lens technology into ocular diagnostics and therapies could expand the scope of digital healthcare applications. As digital healthcare technology evolves, the adaptability and functionality of the OLED contact lens could give it an important role in many future ocular innovations.

An interdisciplinary approach to OLED lens development could lead to solutions for myopia and other ocular issues. The fields of augmented reality and light-based neurostimulation could also benefit from the new OLED lens 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.

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SPAD Camera Characterizes Large Samples of Molecules at Same Time

 

A new widefield fluorescence lifetime imaging technique, achieved with a time-gated, single-photon avalanche diode (SPAD) camera, enables thousands of molecules to be characterized rapidly, accurately, and at the same time.

Developed by a team at the Swiss Federal Institute of Technology in Lausanne (EPFL), single-molecule fluorescence lifetime imaging microscopy (smFLIM) could be a significant advancement for multitarget, single-molecule localization microscopy.

Traditional FLIM typically relies on time-correlated single-photon counting (TCSPC), a precise but low-throughput method, to discriminate molecules or probe their nanoscale environment.

Unlike conventional imaging methods, smFLIM detects molecules at a specific point in time immediately after they are subjected to an excitation pulse. It captures an alternating series of images — one image immediately after excitation and another a few nanoseconds later — with picosecond-scale resolution.

The images are analyzed to determine the molecule’s fluorescence lifetime — that is, the short delay between the excitation laser pulse and the fluorescence emitted by the molecule — and the individual molecules in the sample are characterized.

Using smFLIM, the researchers obtained measurements with a precision only about 3x less than TCSPC — but with a system capable of imaging with multiple pixels (512 × 512) to enable the spatial multiplexing of more than 3000 molecules. The new technique can take precise measurements of a molecule’s unique light-emission signature at the scale of a billionth of a second.

SmFLIM can provide precise information on thousands of molecules in less than one minute, compared to the one hour required for existing techniques. “Our method is slightly less accurate than conventional ones, but it is faster and can detect an unprecedented number of molecules at once,” professor Aleksandra Radenovic said.

For the design of smFLIM, the researchers revisited a gated imaging scheme introduced 35 years ago. Although time-gated cameras have shown potential for high-throughput FLIM, until now their use in single-molecule microscopy has not been explored extensively. The time-gated SPAD camera used for smFLIM has almost one million sensors, each of which detect a photon.

The team used smFLIM to demonstrate parallelized lifetime measurements of many labeled, pore-forming proteins on supported lipid bilayers, and temporal, single-molecule Förster resonance energy transfer (FRET) measurements to detect the distance between molecules.

“Measuring the fluorescence lifetime of a pair of molecules provides information on the distance between them at a scale of just a few nanometers,” researcher Nathan Ronceray said. “The current approach can only be applied to small samples, but our system can expand it to allow for the rapid study of dynamic phenomena on thousands of molecules.”

The team said that, although it chose single-molecule FRET for its demonstration, smFLIM can be used with other lifetime-changing phenomena, such as non-radiative energy transfer to bulk metal or 2D materials.

Based on the results of the study, smFLIM is a promising tool for diverse areas of science and technology. “One promising direction is its potential to improve multiplexed analyses, that is, to measure several parameters simultaneously in a single sample,” Radenovic said. “It is likely to be useful in fields such as spatial transcriptomics, which aims to measure gene expression in a tissue while preserving spatial information about the exact location of cells or structures in the tissue.”

This approach could benefit lifetime-based assays of biomolecules for structural biology, diagnostic assays, biopolymer sequencing, and single-molecule superresolution microscopy.

By enabling the simultaneous reading of many molecular species throughout life, the method could serve as a powerful complement to high-resolution omics tools used to study the different biological layers of an organism in a comprehensive and systematic way, often on a cellular or molecular scale.

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