Multimodal Microscopy Imaging Method Charts Course for Monitoring Brain Metabolic Changes

A microscopy system developed by researchers at MIT addresses the challenges of using all-optical imaging techniques to visualize metabolic changes and neuronal activity deep within the brain. Using the system, which combines acoustic imaging and multiphoton excitation, the researchers achieved exceptional depth and sharp images by combining several advanced technologies into one microscope.

In the system, an ultrasound microphone located in the microscope detects the acoustic waves, and the recorded sound data is converted into high-resolution images. The solution additionally uses a near-infrared femtosecond (NIR-fs) laser for excitation, ensuring that the wavelength is long enough to penetrate deeply into tissue.

“We merged all these techniques — three-photon, label-free, photoacoustic detection,” researcher Tatsuya Osaki said. “We integrated all these cutting-edge techniques into one process to establish this ‘Multiphoton-In and Acoustic-Out’ platform.”

The label-free, multiphoton, photoacoustic microscope (LF-MP-PAM) could provide a way to monitor the metabolic changes in brain cells, both in vitro and in vivo, and measure brain activity without the need for external labels. Further, it could be used to investigate the normal and pathological mechanisms underlying neurodegenerative diseases and psychiatric disorders.

Using LF-MP-PAM with a 1300-nm fs laser, the researchers identified and imaged endogenous NAD(P)H at the single-cell level in living cultured cells, mouse brain slices, and human cerebral organoids. Nicotinamide adenine dinucleotide (NAD), a critical molecule in the cellular metabolic pathway, exists in two forms — oxidized (NAD(P)+ and reduced NAD(P)H. Real-time detection of NAD(P)H in the brain could serve as a biomarker for assessing the activity of neurons during normal functioning and disease progression.

The researchers confirmed the NADH photoacoustic signal with standard NADH imaging, validating the character of the photoacoustic energy, frequency, and acoustic transit time. They introduced NADH in cells and observed an increase in photoacoustic signals, which was confirmed through conventional, fluorescent-based NAD(P)H sensors. Although NAD(P)H emitted a weak fluorescent signal, the absorbed energy produced a localized thermal expansion of about 10 μm within the cell, generating sound waves that traveled with relative ease through the tissue compared to fluorescence emission.

Using photoacoustic detection, the researchers accessed depths of 700 μm in the mouse brain slices and 1100 μm in cerebral organoids from human stem cells. According to the team, LF-MP-PAM penetrated human organoid tissue at more than 5x the depth of other microscopy technologies. The researchers also achieved a strong photoacoustic NAD(P)H signal in the brain slices, at a depth 6x greater than the reported optical imaging depth for NAD(P)H.

“The major advance here is to enable us to image deeper at single-cell resolution,” professor Mriganka Sur said.

The researchers also developed an imaging subsystem and integrated it into the LF-MP-PAM platform to demonstrate a photoacoustic-generated spatial map of NAD(P)H in organoid and brain slice cells. They demonstrated simultaneous third-harmonic-generation imaging from three-photon stimulation, producing detailed renderings of cellular structures.

The team continues to refine the system’s signal processing capabilities, and is looking ahead to potential applications for LF-MP-PAM in neuroscience and clinical settings. It has already established that NAD(P)H imaging can inform wound care.

Levels of the NAD(P)H molecule in the brain are known to vary in patients who experience seizures and various neurological disorders like Alzheimer’s disease and Rett syndrome, making NAD(P)H a potentially valuable biomarker for these conditions. Because the new system is label-free, it could be used for deep tissue imaging during surgeries.

The next step for the researchers will be to demonstrate LF-MP-PAM in a live animal. To move beyond in vitro and ex-vivo tissue imaging, the team will first need to reposition the microphone to be on top of the sample, like the light source.


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Live Cell Superresolution Method Enables Full Range of Biological Mechanism Imaging

Researchers introduced a superresolution imaging technique that visualizes live, dynamic cellular structures at 60-100 nm while significantly reducing the risk of damaging the fragile cells. The microscopy advancement could inform research into DNA repair, chromosome activity, and other biological mechanisms.

The imaging approach, developed by a team at Queen Mary University of London in collaboration with industry partners, combines Fluorescence Recovery After Photobleaching (FRAP) with Lattice Structured Illumination Microscopy (diSIM/SIM2). The resulting application is named FRAP in the Superresolution regime (FRAP-SR).

“Our FRAP-SR approach enables us to visualize structures as small as 60 nanometers within living cells — a scale previously inaccessible for dynamic studies without causing significant cellular stress,” professor Viji Draviam, who led the research, said. “This resolution, 2000x smaller than the width of a human hair, allows us to probe the nanoscale organization and behavior of cellular components in real time.”

Using FRAP-SR, the researchers investigated the dynamics of a protein that is key to the repair of double-strand DNA breaks, called 53BP1. They analyzed the dynamics of the 53BP1 protein within nuclear structures at 60-nm resolution. FRAP-SR enabled them to correlate protein diffusion with subcellular structural changes in the superresolution regime without perturbing the live-cell samples.

The approach revealed sub-compartments within 53BP1 foci. These sub-compartments displayed faster 53BP1 protein mobility than other foci without sub-compartments.

The researchers characterized two distinct types of 53BP1 foci that differed in their activities. Some foci appeared as stable, compact structures, while others exhibited more fluid, dynamic shapes. The compact foci displayed uniform recovery after photobleaching, but showed greater heterogeneity in the recovery rates between different foci. The amorphous foci contained discrete sub-compartments with varying protein mobility, suggesting functional specialization within these DNA repair centers.

Using lattice light-sheet movies of aphidicolin-treated cells, the researchers confirmed faster recovery of 53BP1 in amorphous foci compared to compact foci. The study also revealed that the dynamics of the foci are influenced by cellular conditions such as recovery from DNA replication stress.

The team believes that, over the long term, the combination of FRAP and diSIM will enable scientists to overcome existing limitations to examining photosensitive subcellular structures with varied protein mobilities, activities, and roles. Long-range joining of DNA breaks are important, as defective cells can experience extensive degradation of the unrepaired coding ends, leading to genomic instability.

FRAP-SR could accelerate the development of drug targeting and drug screening methods based on live-cell dynamics. The global market for DNA repair drugs was valued at approximately $9.18 billion in 2024 and is projected to reach $13.97 billion by 2030. The use of FRAP-SR to study the DNA damage marker, 53BP1, in live cells could foster the development of DNA repair drugs and candidate drugs for personalized medicine.

“This will transform the field of optogenetics in the superresolution regime," Draviam said. "It will also enable the development of new anti-cancer drugs that target DNA damage repair pathways that are dynamic.”

The study utilized the ZEISS Elyra 7 system, enhanced with FRAP capabilities from Rapp OptoElectronics. The system provided the superresolution imaging necessary to resolve the sub-compartments of 53BP1 foci. The researchers worked with ZEISS and Rapp OptoElectronics to integrate FRAP and SIM, which allowed for precise quantification of protein dynamics.

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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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Photoacoustic Microscopy Images Stents Non-Invasively

 

Each year, around two million people in the U.S. are implanted with a stent to improve blood flow in narrowed or blocked arteries. Stents need to be monitored for problems such as fractures or improper positioning, but conventionally used techniques require invasive procedures or radiation exposure.

Researchers at Xi’an Jiaotong-Liverpool University found a way to safely monitor those stents using photoacoustic microscopy to image through the skin

“It is critical to monitor stents for problems such as fractures or improper positioning, but conventionally used techniques require invasive procedures or radiation exposure,” said co-lead researcher Myeongsu Seong from Xi’an Jiaotong-Liverpool University in China. “This inspired us to test the potential of using photoacoustic imaging for monitoring stents through the skin.”

The research work demonstrated that photoacoustic microscopy can be used to visualize stents covered with mouse skin under various clinically relevant conditions, including simulated damage and plaque buildup.

“While our photoacoustic microscopy results are preliminary, further development could enable frequent, noninvasive monitoring of stent status — without the need for surgical access or X-ray exposure,” said co-lead researcher Sung-Liang Chen from Shanghai Jiao Tong University in China. “This would make it easier and safer to monitor the condition of stents in patients.”

Photoacoustic imaging is a label-free technique that detects sound waves generated when materials absorb light and release energy. Because sound scatters less than light, this imaging method can be used to acquire higher-resolution images at greater depths than purely optical methods.

Although other studies have used photoacoustic imaging via an endoscope to image stents, this still requires that patients undergo a procedure. In the current study, the researchers examined whether photoacoustic microscopy could enable noninvasive stent monitoring through the skin.

To do this, they mimicked different stent scenarios, including fractures, compression and movement of overlapped stents. They also used butter to mimic deposition of plaque or blood clots after stenting. Using photoacoustic microscopy at various wavelengths, including 670 nm and 1210 nm, they were able to image these various stent conditions through excised mouse skin.

“One of the most interesting results is that we could easily differentiate between the butter we used to mimic a lipid plaque and the stent,” said Seong. “Because plaque and stents absorb light differently, using two wavelengths helped us distinguish them.”

The researchers said that photoacoustic microscopy could potentially be used to image stents placed in dialysis access sites, which are typically located just beneath the skin. For stents in deeper areas like the carotid artery, a related method called photoacoustic computed tomography may be more suitable.

The researchers pointed out that before photoacoustic imaging can be used for clinical noninvasive stent monitoring, in vivo animal experiments and preliminary clinical experiments would have to be performed. The system would also need to be optimized for use in various parts of the body.


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Evident's Pramana Acquisition Will Combine Clinical Microscopy, Digital Pathology Capabilities

Evident has agreed to acquire Pramana, a manufacturer of whole slide imaging technologies and digital pathology solutions. Terms of the deal have not been announced.

Founded in 2021 by nference, a leader in multimodal and agentic AI innovation, Pramana develops fully autonomous image scanning systems, which are designed for use in hospitals, research facilities, and educational institutions. The systems use volumetric imaging techniques to scan specimens at varying fields of view and combine the images into a single fully focused image. Real-time AI algorithms optimize efficiency, reduce costs, and enhance safety during the scanning process. According to Pramana, these built-in AI algorithms and automated quality control enable its solution to eliminate up to 70% of manual workflow steps while capturing previously undetectable tissue features, radically improving clinical diagnostics and research.

According to Evident, the acquisition complements and expands its portfolio of technologies to keep pace with daily caseloads, automate quality control, seamlessly integrate data and communication systems, and harness advances in AI-driven imaging and analysis.

Evident formed in April 2022 when Olympus Corporation finalized the separation of its Scientific Solutions business to a wholly-owned subsidiary. Evident completed the sale of it Inspection Technologies division, formerly part of Olympus' Scientific Solutions division, in a $1.78 billion dollar deal last month.

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OCT Technique Improves Accuracy of Deep Brain Stimulation Mapping

Deep brain stimulation (DBS), a surgical procedure that can be used to treat Parkinson’s, obsessive-compulsive disorder, and other neurological disorders, involves implanting electrodes in specific brain regions to regulate abnormal neural activity. The precise placement of these electrodes is crucial for a successful clinical outcome.

Magnetic resonance imaging (MRI), the tool commonly used for DBS mapping, lacks the resolution and contrast needed to accurately pinpoint the small, deep brain nuclei targeted for electrode placement. Consequently, researchers are exploring optical imaging techniques with better contrast, higher resolution, and lower costs than MRI to serve as supplementary tools in intraoperative DBS.

A study by Laval University and Harvard Medical School explores one such tool, polarization-sensitive optical coherence tomography (PS-OCT), and demonstrates its potential as a complementary imaging technique for guiding DBS surgery.

Unlike MRI, which provides mm-scale resolution, PS-OCT can visualize brain structures at the μm level. This enables it to provide detailed information essential to accurately target electrodes used in DBS surgery.

The researchers tested PS-OCT on three primary DBS targets in a postmortem animal. To simulate a DBS procedure, they inserted a PS-OCT probe into the brain along predefined trajectories. As the probe was pulled through the tissue, it collected data and captured high-resolution images of the brain’s internal structure. The researchers matched these images with MRI scans and anatomical references to assess their accuracy.

The PS-OCT system used a rotating catheter with a tiny lens and prism to direct light into the tissue and measure how the light’s polarization changed as it passed through different structures. This change, or birefringence, reflects the alignment and density of fibers in the brain’s white matter. The use of polarized light to detect subtle structural differences in tissue and capture birefringence could enable more accurate identification of white matter fiber tracts — bundles of nerve fibers in the brain that are crucial landmarks for DBS targeting.

The researchers used a simplified segmentation approach to compare the performance of PS-OCT with MRI. They averaged data along the probe path and applied clustering to separate tissue types. This allowed them to create “tissue barcodes” showing transitions between white and gray matter.

The results showed that PS-OCT was able to distinguish between white and gray matter more clearly than MRI. PS-OCT also captured fine fiber structures that MRI missed, such as the internal capsule, a dense bundle of fibers important for DBS planning. In one case, PS-OCT identified highly organized fiber tracts near the external pallidum that were invisible in MRI scans.

Overall, PS-OCT’s polarization-sensitive reconstruction algorithms provided more detailed, accurate information than MRI, while remaining consistent with MRI findings.

PS-OCT could provide surgeons with supplementary intraoperative feedback during DBS procedures, improving accuracy and reducing the risk of electrode misplacement. Moreover, the system’s compact form factor and imaging paradigm could be integrated seamlessly into the surgical workflow.

“Catheter-based PS OCT shows strong promise as a tool complementary to MRI in DBS neurosurgery,” researcher Shadi Masoumi said. “By providing high-resolution structural information and visualizing critical fiber pathways, it could help surgeons target brain regions more precisely.”

Although PS-OCT offers superior resolution, future advancements could further broaden its applicability. It currently measures fiber orientation in 2D only, and the ability to capture fiber orientations in 3D would increase its value as a visualization tool. The PS-OCT probe used in the study was slightly larger than standard DBS electrodes, but smaller probes are now available and could be adapted for clinical use.

Next steps include live testing, integration into surgical workflows, and direct comparisons with diffusion MRI, another technique used to map brain fibers. If successful, PS-OCT could become a valuable addition to the neurosurgical toolkit, improving outcomes for patients undergoing DBS.

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Hypervision and imec Collaborate on Hyperspectral Imaging for Surgery

Hypervision, a spin-out company from King’s College London that aims to advance computer-assisted tissue analysis for improved surgical precision and patient safety, has signed a strategic development agreement with imec. The collaboration targets the co-development of scalable technologies tailored for surgical applications, as the company works to scale its on-chip hyperspectral imaging and real-time AI analytics.

Hypervision's technology delivers tissue-level insights, including on oxygenation, perfusion, and tissue differentiation. Its regulatory-cleared intraoperative imaging platform combines on-chip hyperspectral imaging with real-time AI analytics operating at over 60 fps. Additionally, the technology is designed to integrate into existing surgical vision platforms and workflows. The company's platform is currently under clinical evaluation in U.K. hospitals, with a primary focus on gastrointestinal surgery.

Though hyperspectral imaging is already used in medical applications, previous hyperspectral systems have struggled with integration due to hardware complexity, slow processing speeds, and poor compatibility with surgical workflows. These bottlenecks can restrict the use of these system to research settings or post-operative analysis.

As part of the collaboration, imec is leveraging its expertise in semiconductor fabrication, equipment, and process technology to develop on-chip spectral imaging and to design and manufacture interference-based optical filters at the wafer level. imec's CMOS infrastructure provides compact, clean, and high-yield optical filter integration with scalability to high-volume production at low cost.

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