A new high-speed multifocus microscope could facilitate discoveries in developmental biology and neuroscience thanks to its ability to image rapid biological processes over the entire volume of tiny living organisms in real time.

The pictures from many 3D microscopes are obtained sequentially by scanning through different depths, making them too slow for accurate live imaging of fast-moving natural functions in individual cells and microscopic animals. Even current multifocus microscopes that capture 3D images simultaneously have either relatively poor image resolution or can only image to shallow depths.

In contrast, the new 25-camera “M25” microscope – developed during his doctorate by Eduardo Hirata-Miyasaki and his supervisor Sara Abrahamsson, both then at the University of California Santa Cruz, together with collaborators at the Marine Biological Laboratory in Massachusetts and the New Jersey Institute of Technology – enables high-resolution 3D imaging over a large field-of-view, with each camera capturing 180 × 180 × 50 µm volumes at a rate of 100 per second.

“Because the M25 microscope is geared towards advancing biomedical imaging we wanted to push the boundaries for speed, high resolution and looking at large volumes with a high signal-to-noise ratio,” says Hirata-Miyasaki, who is now based in the Chan Zuckerberg Biohub in San Francisco.

The M25, detailed in Optica, builds on previous diffractive-based multifocus microscopy work by Abrahamsson, explains Hirata-Miyasaki. In order to capture multiple focal planes simultaneously, the researchers devised a multifocus grating (MFG) for the M25. This diffraction grating splits the image beam coming from the microscope into a 5 × 5 grid of evenly illuminated 2D focal planes, each of which is recorded on one of the 25 synchronized machine vision cameras, such that every camera in the array captures a 3D volume focused on a different depth. To avoid blurred images, a custom-designed blazed grating in front of each camera lens corrects for the chromatic dispersion (which spreads out light of different wavelengths) introduced by the MFG.

The team used computer simulations to reveal the optimal designs for the diffractive optics, before creating them at the University of California Santa Barbara nanofabrication facility by etching nanometre-scale patterns into glass. To encourage widespread use of the M25, the researchers have published the fabrication recipes for their diffraction gratings and made the bespoke software for acquiring the microscope images open source. In addition, the M25 mounts to the side port of a standard microscope, and uses off-the-shelf cameras and camera lenses.

The M25 can image a range of biological systems, since it can be used for fluorescence microscopy – in which fluorescent dyes or proteins are used to tag structures or processes within cells – and can also work in transmission mode, in which light is shone through transparent samples. The latter allows small organisms like C. elegans larvae, which are commonly used for biological research, to be studied without disrupting them.

The researchers performed various imaging tests using the prototype M25, including observations of the natural swimming motion of entire C. elegans larvae. This ability to study cellular-level behaviour in microscopic organisms over their whole volume may pave the way for more detailed investigations into how the nervous system of C. elegans controls its movement, and how genetic mutations, diseases or medicinal drugs affect that behaviour, Hirata-Miyasaki tells Physics World. He adds that such studies could further our understanding of human neurodegenerative and neuromuscular diseases.

“We live in a 3D world that is also very dynamic. So with this microscope I really hope that we can keep pushing the boundaries of acquiring live volumetric information from small biological organisms, so that we can capture interactions between them and also [see] what is happening inside cells to help us understand the biology,” he continues.

As part of his work at the Chan Zuckerberg Biohub, Hirata-Miyasaki is now developing deep-learning models for analysing dynamic cell and organism multichannel dynamic live datasets, like those acquired by the M25, “so that we can extract as much information as possible and learn from their dynamics”.

Meanwhile Abrahamsson, who is currently working in industry, hopes that other microscopy development labs will make their own M25 systems.  She is also considering commercializing the instrument to help ensure its widespread use.

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Optical fibres form the backbone of the Internet, carrying light signals across the globe. But some light is always lost as it travels, becoming attenuated by about 0.14 decibels per kilometre even in the best fibres. That means signals must be amplified every few dozen kilometres – a performance that hasn’t improved in nearly four decades.

Physicists at the University of Southampton, UK have now developed an alternative that could call time on that decades-long lull. Writing in Nature Photonics, they report hollow-core fibres that exhibit 35% less attenuation while transmitting signals 45% faster than standard glass fibres.

“A bit like a soap bubble”

The core of conventional fibres is made of pure glass and is surrounded by a cladding of slightly different glass. Because the core has a higher refractive index than the cladding, light entering the fibre reflects internally, bouncing back and forth in a process known as total internal reflection. This effect traps the light and guides it along the fibre’s length.

The Southampton team led by Francesco Poletti swapped the standard glass core for air. Because air is more transparent than glass, channelling light through it cuts down on scattering and speeds up signals. The problem is that air’s refractive index is lower, so the new fibre can’t use total internal reflection. Instead, Poletti and colleagues guided the light using a mechanism called anti-resonance, which requires the walls of the hollow core to be made from ultra-thin glass membranes.

“It’s a bit like a soap bubble,” Poletti says, explaining that such bubbles appear iridescent because their thin films reflect some wavelengths and lets others through. “We designed our fibre the same way, with glass membranes that reflect light at certain frequencies back into the core.” That anti-resonant reflection, he adds, keeps the light trapped and moving through the fibre’s hollow centre.

Greener telecommunications

To make the new air-core fibre, the researchers stacked thin glass capillaries in a precise pattern, forming a hollow channel in the middle. Heating and drawing the stack into a hair-thin filament preserved this pattern on a microscopic scale. The finished fibre has a nested design: an air core surrounded by ultra-thin layers that provide anti-resonant guidance and cut down on leakage.

To test their design, the team measured transmission through a full spool of fibre, then cut the fibre shorter and compared the results. They also fired in light pulses and tracked the echoes. Their results show that the hollow fibres reduce attenuation to just 0.091 decibels per kilometre. This lower loss implies that fewer amplifiers would be needed in long cables, lowering costs and energy use. “There’s big potential for greener telecommunications when using our fibres,” says Poletti.

Poletti adds that reduced attenuation (and thus lower energy use) is only one of the new fibre’s advantages. At the 0.14 dB/km attenuation benchmark, the new hollow fibre supports a bandwidth of 54 THz compared to 10 THz for a normal fibre. At the reduced 0.1 dB/km attenuation, the bandwidth is still 18 THz, which is close to twice that of a normal cable. This means that a single strand can carry far more channels at once.

Perhaps the most impressive advantage is that because the speed of light is faster in air than in glass, data could travel the same distance up to 45% faster. “It’s almost the same speed light takes when we look at a distant star,” Poletti says. The resulting drop in latency, he adds, could be crucial for real-time services like online gaming or remote surgery, and could also speed up computing tasks such as training large language models.

Field testing

As well as the team’s laboratory tests, Microsoft has begun testing the fibres in real systems, installing segments in its network and sending live traffic through them. These trials prove the hollow-core design works with existing telecom equipment, opening the door to gradual rollout. In the longer run, adapting amplifiers and other gear that are currently tuned for solid glass fibres could unlock even better performance.

Poletti believes the team’s new fibres could one day replace existing undersea cables. “I’ve been working on this technology for more than 20 years,” he says, adding that over that time, scepticism has given way to momentum, especially now with Microsoft as an industry partner. But scaling up remains a real hurdle. Making short, flawless samples is one thing; mass-producing thousands of kilometres at low cost is another. The Southampton team is now refining the design and pushing toward large-scale manufacturing. They’re hopeful that improvements could slash losses by another order of magnitude and that the anti-resonant design can be tuned to different frequency bands, including those suited to new, more efficient amplifiers.

Other experts agree the advance marks a turning point. “The work builds on decades of effort to understand and perfect hollow-core fibres,” says John Ballato, whose group at Clemson University in the US develops fibres with specialty cores for high-energy laser and biomedical applications. While Ballato notes that such fibres have been used commercially in shorter-distance communications “for some years now”, he believes this work will open them up to long-haul networks.

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Traumatic brain injury (TBI), caused by a sudden impact to the head, is a leading cause of death and disability. After such an injury, the most important indicator of how severe the injury is intracranial pressure – the pressure inside the skull. But currently, the only way to assess this is by inserting a pressure sensor into the patient’s brain. UK-based startup Crainio aims to change this by developing a non-invasive method to measure intracranial pressure using a simple optical probe attached to the patient’s forehead.

Can you explain why diagnosing TBI is such an important clinical challenge?

Every three minutes in the UK, someone is admitted to hospital with a head injury, it’s a very common problem. But when someone has a blow to the head, nobody knows how bad it is until they actually reach the hospital. TBI is something that, at the moment, cannot be assessed at the point of injury.

From the time of impact to the time that the patient receives an assessment by a neurosurgical expert is known as the golden hour. And nobody knows what’s happening to the brain during this time – you don’t know how best to manage the patient, whether they have a severe TBI with intracranial pressure rising in the head, or just a concussion or a medium TBI.

Once at the hospital, the neurosurgeons have to assess the patient’s intracranial pressure, to determine whether it is above the threshold that classifies the injury as severe. And to do that, they have to drill a hole in the head – literally – and place an electrical probe into the brain. This really is one of the most invasive non-therapeutic procedures, and you obviously can’t do this to every patient that comes with a blow in the head. It has its risks, there is a risk of haemorrhage or of infection.

Therefore, there’s a need to develop technologies that can measure intracranial pressure more effectively, earlier and in a non-invasive manner. For many years, this was almost like a dream: “How can you access the brain and see if the pressure is rising in the brain, just by placing an optical sensor on the forehead?”

Crainio has now created such a non-invasive sensor; what led to this breakthrough?

The research goes back to 2016, at the Research Centre for Biomedical Engineering at City, University of London (now City St George’s, University of London), when the National Institute for Health Research (NIHR) gave us our first grant to investigate the feasibility of a non-invasive intracranial sensor based on light technologies. We developed a prototype, secured the intellectual property and conducted a feasibility study on TBI patients at the Royal London Hospital, the biggest trauma hospital in the UK.

It was back in 2021, before Crainio was established, that we first discovered that after we shone certain frequencies of light, like near-infrared, into the brain through the forehead, the optical signals coming back – known as the photoplethysmogram, or PPG – contained information about the physiology or the haemodynamics of the brain.

When the pressure in the brain rises, the brain swells up, but it cannot go anywhere because the skull is like concrete. Therefore, the arteries and vessels in the brain are compressed by that pressure. PPG measures changes in blood volume as it pulses through the arteries during the cardiac cycle. If you have a viscoelastic artery that is opening and closing, the volume of blood changes and this is captured by the PPG. Now, if you have an artery that is compromised, pushed down because of pressure in the brain, that viscoelastic property is impacted and that will impact the PPG.

Changes in the PPG signal due to changes arising from compression of the vessels in the brain, can give us information about the intracranial pressure. And we developed algorithms to interrogate this optical signal and machine learning models to estimate intracranial pressure.

How did the establishment of Crainio help to progress the sensor technology?

Following our research within the university, Crainio was set up in 2022. It brought together a team of experts in medical devices and optical sensors to lead the further development and commercialization of this device. And this small team worked tirelessly over the last few years to generate funding to progress the development of the optical sensor technology and bring it to a level that is ready for further clinical trials.

In 2023, Crainio was successful with an Innovate UK biomedical catalyst grant, which will enable the company to engage in a clinical feasibility study, optimize the probe technology and further develop the algorithms. The company was later awarded another NIHR grant to move into a validation study.

The interest in this project has been overwhelming. We’ve had a very positive feedback from the neurocritical care community. But we also see a lot of interest from communities where injury to the brain is significant, such as rugby associations, for example.

Could the device be used in the field, at the site of an accident?

While Crainio’s primary focus is to deliver a technology for use in critical care, the system could also be used in ambulances, in helicopters, in transfer patients and beyond. The device is non-invasive, the sensor is just like a sticking plaster on the forehead and the backend is a small box containing all the electronics. In the past few years, working in a research environment, the technology was connected into a laptop computer. But we are now transferring everything into a graphical interface, with a monitor to be able to see the signals and the intracranial pressure values in a portable device.

Following preliminary tests on patients, Crainio is now starting a new clinical trial. What do you hope to achieve with the next measurements?

The first study, a feasibility study on the sensor technology, was done during the time when the project was within the university. The second round is led by Crainio using a more optimized probe. Learning from the technical challenges we had in the first study, we tried to mitigate them with a new probe design. We’ve also learned more about the challenges associated with the acquisition of signals, the type of patients, how long we should monitor.

We are now at the stage where Crainio has redeveloped the sensor and it looks amazing. The technology has received approval by MHRA, the UK regulator, for clinical studies and ethical approvals have been secured. This will be an opportunity to work with the new probe, which has more advanced electronics that enable more detailed acquisition of signals from TBI patients.

We are again partnering with the Royal London Hospital, as well as collaborators from the traumatic brain injury team at Cambridge and we’re expecting to enter clinical trials soon. These are patients admitted into neurocritical trauma units and they all have an invasive intracranial pressure bolt. This will allow us to compare the physiological signal coming from our intracranial pressure sensor with the gold standard.

The signals will be analysed by Crainio’s data science team, with machine learning algorithms used to look at changes in the PPG signal, extract morphological features and build models to develop the technology further. So we’re enriching the study with a more advanced technology, and this should lead to more accurate machine learning models for correctly capturing dynamic changes in intracranial pressure.

The primary motivation of Crainio is to create solutions for healthcare, developing a technology that can help clinicians to diagnose traumatic brain injury effectively, faster, accurately and earlier

This time around, we will also record more information from the patients. We will look at CT scans to see whether scalp density and thickness have an impact. We will also collect data from commercial medical monitors within neurocritical care to see the relation between intracranial pressure and other physiological data acquired in the patients. We aim to expand our knowledge of what happens when a patient’s intracranial pressure rises – what happens to their blood pressures? What happens to other physiological measurements?

How far away is the system from being used as a standard clinical tool?

Crainio is very ambitious. We’re hoping that within the next couple of years we will progress adequately in order to achieve CE marking and all meet the standards that are necessary to launch a medical device.

The primary motivation of Crainio is to create solutions for healthcare, developing a technology that can help clinicians to diagnose TBI effectively, faster, accurately and earlier. This can only yield better outcomes and improve patients’ quality-of-life.

Of course, as a company we’re interested in being successful commercially. But the ambition here is, first of all, to keep the cost affordable. We live in a world where medical technologies need to be affordable, not only for Western nations, but for nations that cannot afford state-of-the-art technologies. So this is another of Crainio’s primary aims, to create a technology that could be used widely, because there is a massive need, but also because it’s affordable.

• To hear the full interview with Panicos Kyriacou listen to our Physics World Weekly podcast

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Optical coherence tomography (OCT), a low-cost imaging technology used to diagnose and plan treatment for eye diseases, also shows potential as a diagnostic tool for assessing rapid hearing loss.

Researchers at the Keck School of Medicine of USC have developed an OCT device that can acquire diagnostic quality images of the inner ear during surgery. These images enable accurate measurement of fluids in the inner ear compartments. The team’s proof-of-concept study, described in Science Translational Medicine, revealed that the fluid levels correlated with the severity of a patient’s hearing loss.

An imbalance between the two inner ear fluids, endolymph and perilymph, is associated with sudden, unexplainable hearing loss and acute vertigo, symptoms of ear conditions such as Ménière’s disease, cochlear hydrops and vestibular schwannomas. This altered fluid balance – known as endolymphatic hydrops (ELH) – occurs when the volume of endolymph increases in one compartment and the volume of perilymph decreases in the other.

Because the fluid chambers of the inner ear are so small, there has previously been no effective way to assess endolymph-to-perilymph fluid balance in a living patient. Now, the Keck OCT device enables imaging of inner ear structures in real time during mastoidectomy – a procedure performed during many ear and skull base surgeries, and which provides optical access to the lateral and posterior semicircular canals (SCCs) of the inner ear.

OCT offers a quicker, more accurate and less expensive way to see inner ear fluids, hair cells and other structures compared with the “gold standard” MRI scans. The researchers hope that ultimately, the device will evolve into an outpatient assessment tool for personalized treatments for hearing loss and vertigo. If it can be used outside a surgical suite, OCT technology could also support the development and testing of new treatments, such as gene therapies to regenerate lost hair cells in the inner ear.

Intraoperative OCT

The intraoperative OCT system, developed by senior author John Oghalai and colleagues, comprises an OCT adaptor containing the entire interferometer, which attaches to the surgical microscope, plus a medical cart containing electronic devices including the laser, detector and computer.

The OCT system uses a swept-source laser with a central wavelength of 1307 nm and a bandwidth of 89.84 nm. The scanning beam spot size is 28.8 µm and has a depth-of-focus of 3.32 mm. The system’s axial resolution of 14.0 µm and lateral resolution of 28.8 µm provide an in-plane resolution of 403 µm².

The laser output is directed into a 90:10 optical fibre fused coupler, with the 10% portion illuminating the interferometer’s reference arm. The other 90% illuminates the sample arm, passes through a fibre-optic circulator, and is combined with a red aiming beam that’s used to visually position the scanning beam on the region-of-interest.

After the OCT and aiming beams are guided onto the sample for scanning, and the interferometric signal needed for OCT imaging is generated, two output ports of the 50:50 fibre optic coupler direct the light signal into a balanced photodetector for conversion into an electronic signal. A low-pass dichroic mirror allows back-reflected visible light to pass through into an eyepiece and a camera. The surgeon can then use the eyepiece and real-time video to ensure correct positioning for the OCT imaging.

Feasibility study

The team performed a feasibility study on 19 patients undergoing surgery at USC to treat Ménière’s disease (an inner-ear disorder), vestibular schwannoma (a benign tumour) or middle-ear infection with normal hearing (the control group). All surgical procedures required a mastoidectomy.

Immediately after performing the mastoidectomy, the surgeon positioned the OCT microscope with the red aiming beam targeted at the SCCs of the inner ear. After acquiring a 3D volume image of the fluid compartments in the inner ear, which took about 2 min, the OCT microscope was removed from the surgical suite and the surgical procedure continued.

The OCT system could clearly distinguish the two fluid chambers within the SCCs. The researchers determined that higher endolymph levels correlated with patients having greater hearing loss. In addition to accurately measuring fluid levels, the system revealed that patients with vestibular schwannoma had higher endolymph-to-perilymph ratios than patients with Ménière’s disease, and that compared with the controls, both groups had increased endolymph and reduced perilymph, indicating ELH.

The success of this feasibility study may help improve current microsurgery techniques, by guiding complex temporal bone surgery that requires drilling close to the inner ear. OCT technology could help reduce surgical damage to delicate ear structures and better distinguish brain tumours from healthy tissue. The OCT system could also be used to monitor the endolymph-to-perilymph ratio in patients with Ménière’s disease undergoing endolymphatic shunting, to verify that the procedure adequately decompresses the endolymphatic space. Efforts to make a smaller, less expensive system for these types of surgical use are underway.

The researchers are currently working to improve the software and image processing techniques in order to obtain images from patients without having to remove the mastoid bone, which would enable use of the OCT system for outpatient diagnosis.

The team also plans to adapt a handheld version of an OCT device currently used to image the tympanic membrane and middle ear to enable imaging of the human cochlea in the clinic. Imaging down the ear canal non-invasively offers many potential benefits when diagnosing and treating patients who do not require surgery. For example, patients determined to have ELH could be diagnosed and treated rapidly, a process that currently takes 30 days or more.

Oghalai and colleagues are optimistic about improvements being made in OCT technology, particularly in penetration depth and tissue contrast. “This will enhance the utility of this imaging modality for the ear, complementing its potential to be completely non-invasive and expanding its indication to a wider range of diseases,” they write.

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