Photoacoustic Probes Enable Deep Brain Tissue Imaging

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Molecular engineering spearheaded by two groups at the European Molecular Biology Laboratory (EMBL) has developed an approach to create photoacoustic probes for neuroscience applications.

Scientists can learn more about biological processes by tracking certain chemicals, such as ions or biomolecules. Photoacoustic probes can act as ‘reporters’ for hard-to-detect chemicals by binding to them specifically. The probes can then absorb light when excited by lasers and emit sound waves that can be detected by specialized imaging equipment. For neuroscience applications, however, researchers have so far been unable to engineer targeted reporters that can visualize brain functions tailored for photoacoustics.

“Photoacoustics offer a way to capture imagery of an entire mouse brain, but we just lacked the right probes to visualize a neuron’s activity,” said Robert Prevedel, an EMBL group leader and a senior author on the paper.

To overcome this challenge, Prevedel enlisted the help of fellow EMBL group leader Claire Deo, also a senior author on the paper. She and her team specialize in chemical engineering.

“We have been able to show that we can actually label neurons in specific brain areas with probes bright enough to be detected by our customized photoacoustic microscope,” Prevedel said.

While researchers have experimented with using synthetic dyes as photoacoustic reporters of neuronal activity, controlling where the dye goes and what might be labelled has been challenging. Proteins have been particularly useful as probes for tagging specific molecules, but have not yet led to effective photoacoustic probes to monitor neural activity across the entire brain.

“In our case, we took the best of both of these sensors, combining a protein with a rationally designed synthetic dye, and we can now label and visualize neurons in specific regions of interest,” said Alexander Cook, first author of the study and a predoctoral fellow in the Deo group. In rational design approaches, researchers use existing knowledge and principles to build molecules with the desired properties, instead of blindly making and testing random compounds. The probe not only gave a static observation, Cook said, but showed a reversible, dynamic response to calcium, which is a marker of neuron activity.

According to Deo, an important challenge stood in the way of this technological development. Because photoacoustic probes have not been extensively studied, the researchers lacked a way to evaluate the probes they were building.

Consequently, the project began with Nikita Kaydanov, co-author of the study and predoctoral fellow in the Prevedel Group, who custom-made a spectroscopy setup.

“There is no commercial setup that can measure photoacoustic signals of a probe in test tubes or cuvettes, so we had to build one,” Kaydanov said. “We created our own photoacoustic spectrometer to assess and optimize the probes.”

“This allowed us to evaluate and characterize the different probes we made to assess a few things,” Deo said. “Did they produce a detectable photoacoustic signal? Are they sensitive enough? That’s how we inferred the next steps.”

Having proved that the probes could work in vial, the team took the work further by devising a way to deliver the probes into a mouse brain where they successfully detected photoacoustic signals from neurons inside the targeted brain regions.

“While we are excited about the progress, we need to be clear that this is just the first generation of these probes,” Deo said. “While they offer a very promising approach, we have a lot more work to do, but it’s a good first demonstration of what this system can enable and the potential it has in better understanding brain function.”

The researchers plan to continue development of the technology by improving the dye delivery system and confirming the ability to use them for dynamic imaging inside cells.

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Light Source Aims to Build on Nobel Prize-winning Technology

A team at Heriot-Watt University, led by professor Christian Brahms, is developing a light source for extremely fast laser pulses that will enable scientists to observe some of the fastest processes in the natural world as they occur. The new laser light source will capture natural processes like light absorption in photosynthesis in attoseconds.

The project, which is called FASTER — short for Flexible Attosecond Soliton Transients for Extreme Resolution — will build on the EUV attosecond technology that received the Nobel Prize in Physics in 2023.

Brahms and his team will design and build a laser light source that mimics natural sunlight, but in extremely short flashes. “My aim is to create laser pulses with similar extremely short duration to conventional attosecond science sources, but at the same ultraviolet and visible wavelengths as we get from the sun,” he said.

FASTER will bring attosecond time resolution to ultrafast spectroscopy experiments in the UV, visible, and IR regions of the electromagnetic spectrum. This will enable scientists to study ultrafast dynamics entirely with non-ionizing radiation and without the need for strong-field excitation or probing.

Ultrabroadband optical attosecond spectroscopy will be enabled by soliton self-compression. The researchers will create the optical attosecond pulses required for the new laser light source by building on the results of the recent High-energy soliton (HISOL) project.

HISOL combines the high damage threshold and far UV (FUV) transparency of gas media, the long interaction lengths enabled by waveguides, the guidance of high-energy laser pulses in large-core hollow capillary fibers, and the nonlinear evolution of ultrafast laser pulses in the higher-order-soliton regime. This combination allows IR laser pulses to be converted to wavelength-tunable FUV pulses with a few-femtosecond duration and near-perfect beam properties.

Using tailored soliton dynamics in hollow-core waveguides, the FASTER team will convert femtosecond pulses to attosecond pulses. The resulting attosecond pulses will be used on various samples to perform ultrabroadband optical attosecond pump probe and 2D spectroscopy experiments, starting with condensed-matter targets.

While current attosecond technology, including the 2023 Nobel Prize-winning breakthrough, can create extremely short pulses of light at UV or X-ray wavelengths, it is limited when it comes to natural phenomena, because natural processes involve sunlight, not the wavelengths used in laboratory experiments.

FASTER will allow scientists to take “freeze-frame” images of exceptionally fast microscopic processes in molecules and materials. “This will fill in attosecond technology’s blind spots and directly relate our knowledge of ultrafast processes to other areas, like photochemistry or materials science,” Brahms said.

“Many of the most important breakthroughs in the history of science have been enabled by observing nature at scales far beyond the limits of human perception,” he said. “That’s exactly what we’ll be working on — pushing far beyond the limits of conventional laser sources to bring fundamental science into focus.”

The FASTER project to achieve a very fast laser light source for natural phenomena will take place over a five-year period and is scheduled to officially begin in the summer of 2025. It is one of 50 research projects in the UK to receive the European Research Council’s (ERC’s) Starting Grant in 2024. Brahms and his team will receive £2.5 million ($3.3 million) in ERC funding.

ERC funding supports research in a range of fields. “The new ERC Starting Grants winners aim to deepen our understanding of the world,” Iliana Ivanova, European Commissioner for Innovation, Research, Culture, Education, and Youth, said. “Their creativity is vital to finding solutions to some of the most pressing societal challenges.”

“Empowering researchers early on in their careers is at the heart of the mission of the ERC,” Maria Leptin, ERC president, said.

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Doublet Design Overcomes a Metalens Bottleneck in Microscopy

A research team from Nanjing University has developed a solution that improves metalens performance in and for microscopy applications. The researchers’ metalens-based microscope achieves both a wide field of view (FOV) and high-resolution imaging, addressing the inherent trade-off between these two critical parameters that, according to the researchers, has prevented metalenses to date from achieving performance comparable to conventional microscopes.

Metalenses face significant challenges in practical microscopy applications. Off-axis aberrations, which severely restrict metalens FOV and resolution capabilities, are the primary limitations to the use of metalenses in practical microscopy.

In their system architecture, the researchers used a doublet configuration of two metalenses on opposite sides of a transparent silica substrate combined with annular illumination. The two metalenses consist of silicon nitride nano-fins, crafted as high-aspect-ratio squares with precise dimensions and arranged at carefully calculated intervals. This design approach mitigates off-axis aberrations and increases resolution capabilities, thereby optimizing imaging performance.

The researchers’ prototype had a 1-mm FOV with a half-pitch resolution of 620 nm. Additionally, the prototype is compact, with a measurement of 4 cm × 4 cm × 5 cm. The researchers used their meta-microscope prototype to image cervical cancer cells. The system captured images of various stages of cancer development within the same FOV, revealing important cellular details such as nuclear enlargement, deformation, and division.

“Our experimental results demonstrate high-quality microscopic bioimages that are comparable to those obtained from traditional microscopes within a compact prototype, highlighting its potential applications in portable and convenient settings,” said Li.

According to the researchers, the technology could be used for research scenarios where traditional microscopes would be impractical. Further, it could be integrated into microelectronic devices and systems for clinical or biomedical automation.

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Raman Spectroscopy Undergoes Major Speed Upgrade

Researchers at the University of Tokyo have increased the measurement rate of Raman spectroscopy by 100-fold. Since the measurement rate of the technique has been a major limitation, the improvement is expected to aid advancements in multiple fields relying on the identification of molecules and cells, such as biomedical diagnostics and material analytics.

As a mode of identification for cells and molecules, Raman spectroscopy is widely used, but it’s limited in its ability to keep up with the speed of changes in certain chemical and physical reactions due to the low scattering cross section.

Over the last decade, various broadband-coherent Raman scattering spectroscopy techniques have been developed to address the limitation, achieving a measurement of 500 kSpectra/s (kilospectra per second).

In order to further improve the measurement rate, the team built a system from scratch, leveraging a mode-locked ytterbium laser system developed by Takuro Ideguchi and his team at the Institute for Photon Science and Technology at the University of Tokyo.

In building the system, the team combined coherent Raman spectroscopy — a version of Raman spectroscopy that produces stronger signals than the conventional, spontaneous Raman spectroscopy— with their previously developed specifically designed ultrashort pulse laser and time-stretch technology using optical fibers.

The developed system provides a 50 MSpectra/s (megaspectra per second) measurement rate, a 100-fold increase compared to the previous fastest rate of 500 kSpectra/s. The system enables highly efficient Raman scattering with an ultrashort femtosecond pulse and sensitive time-stretch detection with picosecond probe pulse at a high repetition of the laser.

As a proof-of-concept, the team measured broadband coherent Stokes Raman scattering spectra of organic compounds covering the molecular fingerprint region from 200 to 1200 cm-1.

“We aim to apply our spectrometer to microscopy, enabling the capture of 2D or 3D images with Raman scattering spectra,” Ideguchi said. “Additionally, we envision its use in flow cytometry by combining this technology with microfluidics. These systems will enable high-throughput, label-free chemical imaging and spectroscopy of biomolecules in cells or tissues.”

According to the researchers, the high-speed broadband vibrational spectroscopy technique holds promise for unprecedented measurements of sub-microsecond dynamics of irreversible phenomena and extremely high throughput measurements.

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Light-Based Control of Bacteria Aims to Quell Antibiotic Resistance

Bacterial resistance to antibiotics is a growing challenge for the healthcare and environmental sectors. Bacterial persistence is usually the first step leading to resistance, which involves a change in an organism’s genome.

In the future, it may be possible to address antibiotic persistence and resistance by using light to regulate bacterial response to antibiotics. A technique developed at the Polytechnic University of Milan (Politecnico di Milano), by a team participating in the Engineering of Bacteria to See Light (EOS) project, allows bacteria to sense light and convert light energy into electrical signals across the bacterial membrane, without the need for genetic modification.

To demonstrate photocontrol of bacterial membrane potential, the researchers attached phototransducing molecules to bacterial surfaces to make the bacteria light-responsive. When the researchers exposed the phototransducers to light, the electrical potential of the bacterial membrane changed.

The researchers used Ziapin2, a photoswitchable chemical compound, with two broad-spectrum antibiotics, Kanamycin and Ampicillin. These antibiotics have different modes of activation. Kanamycin requires cytosolic uptake to inhibit protein synthesis, while Ampicillin targets cell wall polymerization at the cell envelope.

When the researchers exposed B. subtilis to Kanamycin and Ziapin2 through photoactivation with 470-nm light, the previously lethal effects of Kanamycin were markedly reduced. In contrast, Ampicillin-treated samples remained largely unaffected by light-induced membrane modulation.

“This interplay between light and electrical signaling allows us to control key biological processes such as movement, biofilm formation, and antibiotic sensitivity,” professor Giuseppe Maria Paternò said. “By manipulating the membrane potential on demand, we can influence antibiotic uptake and restore or even enhance the effectiveness of treatments against resistant strains.”

Possible applications for the technique include developing next-generation antimicrobial platforms that use light to target resistant pathogens, and biocompatible, light-guided “bacterial robots” capable of delivering drugs to specific areas of the body — even the areas that are hard to reach, such as the gastrointestinal tract.

More broadly, the potential to modulate and control the interaction between bacterial cells and antibiotics using light and synthetic photoswitches, without genetic engineering, in real time, represents a first step in developing a new approach to combating antibiotic resistance.

In the future, photoswitchable chemical compounds could be membrane-targeted and tailored to work with different classes of antibiotics, advancing bacterial bioelectric regulation and its applications in antimicrobial therapies.


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Advanced OCT System Integral to Eye Transplantation

An adaptation of OCT put into use at Northwestern University, visible OCT (vis-OCT), will be a key component in an Advanced Research Projects Agency for Health (ARPA-H)-funded project that aims to conduct successful eye transplants in the next several years. The technology will be used to examine the structural integrity of eyes in donors, such as the health of retinal layers and the flow of blood as well as monitoring their continued viability throughout the process.

This work will be supported by over $50 million in ARPA-H funds to multiple institutions, earmarked for viability, imaging, surgical, immunomodulation, ocular preservation, and neuroregeneration strategies within the Transplantation of Human Eye Allografts (THEA) project. The Northwestern team, led by Hao Zhang, a professor of biomedical engineering, and Cheng Sun, a professor of mechanical engineering, will be part of group directed by Jeffrey Goldberg, professor and chair of ophthalmology at the Byers Eye Institute at Stanford University.

Opticent Health, a spinoff company of Zhang’s lab at Northwestern, created the Halo 375 for animal research and the Aurora X4 for human research. These center on a vis-OCT system that includes a supercontinuum white light laser based on photonic crystals, fundus camera for fast imaging, an adjustable field of view, and software to analyze the generated data. It has not yet been approved by the FDA, but is in use for clinical research.

“At this point, we have created the system, it is about overcoming logistical issues associated with this project,” Zhang said. “We have to adapt it for a robotic arm and account for multiple instruments at the bedside. A traditional OCT instrument is used with a chin rest when the patient is sitting, but obviously that wouldn’t work for something like this.”

He said the ARPA-H funding includes expectations of meeting certain guidelines, such as a system ready for use in a medical setting within the next two years, and a clinical protocol within the next three years.

Eye transplants are not new, as more than 70,000 people in the U.S. donate their eyes each year when they die. But these historically have involved corneal transplants, whereas the most common forms of vision loss are caused by retinal degeneration. That is where the whole-eye transplants fit into global eye health. And Zhang acknowledged the questions that need to be answered go beyond the imaging itself.

“With a whole-eye transplant you would need to look at how the muscles attach and whether the blood will flow as it should,” he said. “Then there’s the question of attaching and regenerating the nerves. But I know there are lots of people working on that part of the problem, too.”


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Microscopy Method Doubles the Depth Limit for Live Tissue Imaging

Label-free imaging using two-photon autofluorescence of reduced form nicotinamide adenine dinucleotide phosphate, or NAD(P)H, provides nondestructive, high-resolution, 3D visualization of cellular activities in living systems. Due to light scattering, however, this imaging technique typically can only penetrate as far as 300 μm into living tissues.

To enable deep imaging of thick tissues, researchers at MIT implemented multimode fiber-based, three-photon excitation of NAD(P)H with a low repetition rate and high peak power. They used living, engineered, human multicellular microtissues as test samples.

With this approach, the researchers more than doubled the standard depth limit of NAD(P)H imaging, extending it beyond 700 μm. They achieved deep and dynamic simultaneous localization and mapping (dSLAM) microscopy for structural and metabolic imaging of intact, living biosystems.

The dSLAM microscopy technique attained a high peak power exceeding 0.5 megawatts (MW) at a band of 1100 nm, plus or minus 25 nm. This was achieved by adaptively modulating multimodal, nonlinear pulse propagation with a compact fiber shaper.

The new, noninvasive imaging technique could help biomedical researchers study the body’s immune responses in living tissue and develop new medicines.

The ability to capture the metabolic dynamics of living biosystems is essential for basic biomedical research and laboratory testing. The enhanced depth provided by dSLAM microscopy, combined with the improved imaging speed, could help fuel new investigations into complex cellular interactions.

The flexibility provided by the modular design — a step-index multimode fiber with a slip-on fiber shaper — makes this imaging methodology suitable for demanding in vivo and in vitro imaging applications, including cancer research, immune responses, and tissue engineering.

Advanced microscopy technologies have led to a better understanding of biology, with each technique offering unique advantages for specific applications. With its noninvasive, deep tissue imaging capabilities, dSLAM microscopy could serve as a complementary tool to other imaging techniques for studying cellular dynamics in living tissues. It requires minimal sample preparation and no exogenous labels.

Continued advancements in beam optimization, system design, and streamlined data analysis could make dSLAM microscopy increasingly accessible to biomedical researchers, as a valuable addition to the existing arsenal of microscopy techniques for investigating living tissues.

“It opens new avenues for studying and exploring metabolic dynamics deep in living biosystems,” professor Sixian You said.


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