A new retinal stimulation technique called Oz enabled volunteers to see colours that lie beyond the natural range of human vision. Developed by researchers at UC Berkeley, Oz works by stimulating individual cone cells in the retina with targeted microdoses of laser light, while compensating for the eye’s motion.
Colour vision is enabled by cone cells in the retina. Most humans have three types of cone cells, known as L, M and S (long, medium and short), which respond to different wavelengths of visible light. During natural human vision, the spectral distribution of light reaching these cone cells determines the colours that we see.
Spectral sensitivity curves The response function of M cone cells overlaps completely with those of L and S cones. (Courtesy: Ben Rudiak-Gould)
Some colours, however, simply cannot be seen. The spectral sensitivity curves of the three cone types overlap – in particular, there is no wavelength of light that stimulates only the M cone cells without stimulating nearby L (and sometimes also S) cones as well.
The Oz approach, however, is fundamentally different. Rather than being based on spectral distribution, colour perception is controlled by shaping the spatial distribution of light on the retina.
Describing the technique in Science Advances, Ren Ng and colleagues showed that targeting individual cone cells with a 543 nm laser enabled subjects to see a range of colours in both images and videos. Intriguingly, stimulating only the M cone cells sent a colour signal to the brain that never occurs in natural vision.
The Oz laser system uses a technique called adaptive optics scanning light ophthalmoscopy (AOSLO) to simultaneously image and stimulate the retina with a raster scan of laser light. The device images the retina with infrared light to track eye motion in real time and targets pulses of visible laser light at individual cone cells, at a rate of 105 per second.
In a proof-of-principle experiment, the researchers tested a prototype Oz system on five volunteers. In a preparatory step, they used adaptive optics-based optical coherence tomography (AO-OCT) to classify the LMS spectral type of 1000 to 2000 cone cells in a region of each subject’s retina.
When exclusively targeting M cone cells in these retinal regions, subjects reported seeing a new blue–green colour of unprecedented saturation – which the researchers named “olo”. They could also clearly perceive Oz hues in image and video form, reliably detecting the orientation of a red line and the motion direction of a rotating red dot on olo backgrounds. In colour matching experiments, subjects could only match olo with the closest monochromatic light by desaturating it with white light – demonstrating that olo lies beyond the range of natural vision.
The team also performed control experiments in which the Oz microdoses were intentionally “jittered” by a few microns. With the target locations no longer delivered accurately, the subjects instead perceived the natural colour of the stimulating laser. In the image and video recognition experiments, jittering the microdose target locations reduced the task accuracy to guessing rate.
Ng and colleagues conclude that “Oz represents a new class of experimental platform for vision science and neuroscience [that] will enable diverse new experiments”. They also suggest that the technique could one day help to elicit full colour vision in people with colour blindness.
Electron therapy has long played an important role in cancer treatments. Electrons with energies of up to 20 MeV can treat superficial tumours while minimizing delivered dose to underlying tissues; they are also ideal for performing total skin therapy and intraoperative radiotherapy. The limited penetration depth of such low-energy electrons, however, limits the range of tumour sites that they can treat. And as photon-based radiotherapy technology continues to progress, electron therapy has somewhat fallen out of fashion.
That could all be about to change with the introduction of radiation treatments based on very high-energy electrons (VHEEs). Once realised in the clinic, VHEEs – with energies from 50 up to 400 MeV – will deliver highly penetrating, easily steerable, conformal treatment beams with the potential to enable emerging techniques such as FLASH radiotherapy. French medical technology company THERYQ is working to make this opportunity a reality.
Therapeutic electron beams are produced using radio frequency (RF) energy to accelerate electrons within a vacuum cavity. An accelerator of a just over 1 m in length can boost electrons to energies of about 25 MeV – corresponding to a tissue penetration depth of a few centimetres. It’s possible to create higher energy beams by simply daisy chaining additional vacuum chambers. But such systems soon become too large and impractical for clinical use.
THERYQ is focusing on a totally different approach to generating VHEE beams. “In an ideal case, these accelerators allow you to reach energy transfers of around 100 MeV/m,” explains THERYQ’s Sébastien Curtoni. “The challenge is to create a system that’s as compact as possible, closer to the footprint and cost of current radiotherapy machines.”
Working in collaboration with CERN, THERYQ is aiming to modify CERN’s Compact Linear Collider technology for clinical applications. “We are adapting the CERN technology, which was initially produced for particle physics experiments, to radiotherapy,” says Curtoni. “There are definitely things in this design that are very useful for us and other things that are difficult. At the moment, this is still in the design and conception phase; we are not there yet.”
VHEE advantages
The higher energy of VHEE beams provides sufficient penetration to treat deep tumours, with the dose peak region extending up to 20–30 cm in depth for parallel (non-divergent) beams using energy levels of 100–150 MeV (for field sizes of 10 x 10 cm or above). And in contrast to low-energy electrons, which have significant lateral spread, VHEE beams have extremely narrow penumbra with sharp beam edges that help to create highly conformal dose distributions.
“Electrons are extremely light particles and propagate through matter in very straight lines at very high energies,” Curtoni explains. “If you control the initial direction of the beam, you know that the patient will receive a very steep and well defined dose distribution and that, even for depths above 20 cm, the beam will remain sharp and not spread laterally.”
Electrons are also relatively insensitive to tissue inhomogeneities, such as those encountered as the treatment beam passes through different layers of muscle, bone, fat or air. “VHEEs have greater robustness against density variations and anatomical changes,” adds THERYQ’s Costanza Panaino. “This is a big advantage for treatments in locations where there is movement, such as the lung and pelvic areas.”
It’s also possible to manipulate VHEEs via electromagnetic scanning. Electrons have a charge-to-mass ratio roughly 1800 times higher than that of protons, meaning that they can be steered with a much weaker magnetic field than required for protons. “As a result, the technology that you are building has a smaller footprint and the possibility costing less,” Panaino explains. “This is extremely important because the cost of building a proton therapy facility is prohibitive for some countries.”
Enabling FLASH
In addition to expanding the range of clinical indications that can be treated with electrons, VHEE beams can also provide a tool to enable the emerging – and potentially game changing – technique known as FLASH radiotherapy. By delivering therapeutic radiation at ultrahigh dose rates (higher than 100 Gy/s), FLASH vastly reduces normal tissue toxicity while maintaining anti-tumour activity, potentially minimizing harmful side-effects.
The recent interest in the FLASH effect began back in 2014 with the report of a differential response between normal and tumour tissue in mice exposed to high dose-rate, low-energy electrons. Since then, most preclinical FLASH studies have used electron beams, as did the first patient treatment in 2019 – a skin cancer treatment at Lausanne University Hospital (CHUV) in Switzerland, performed with the Oriatron eRT6 prototype from PMB-Alcen, the French company from which THERYQ originated.
FLASH radiotherapy is currently being used in clinical trials with proton beams, as well as with low-energy electrons, where it remains intrinsically limited to superficial treatments. Treating deep-seated tumours with FLASH requires more highly penetrating beams. And while the most obvious option would be to use photons, it’s extremely difficult to produce an X-ray beam with a high enough dose rate to induce the FLASH effect without excessive heat generation destroying the conversion target.
“It’s easier to produce a high dose-rate electron beam for FLASH than trying to [perform FLASH] with X-rays, as you use the electron beam directly to treat the patient,” Curtoni explains. “The possibility to treat deep-seated tumours with high-energy electron beams compensates for the fact that you can’t use X-rays.”
Panaino points out that in addition to high dose rates, FLASH radiotherapy also relies on various interdependent parameters. “Ideally, to induce the FLASH effect, the beam should be pulsed at a frequency of about 100 Hz, the dose-per-pulse should be 1 Gy or above, and the dose rate within the pulse should be higher than 106 Gy/s,” she explains.
Into the clinic
THERYQ is using its VHEE expertise to develop a clinical FLASH radiotherapy system called FLASHDEEP, which will use electrons at energies of 100 to 200 MeV to treat tumours at depths of up to 20 cm. The first FLASHDEEP systems will be installed at CHUV (which is part of a consortium with CERN and THERYQ) and at the Gustave Roussy cancer centre in France.
“We are trying to introduce FLASH into the clinic, so we have a prototype FLASHKNiFE machine that allows us to perform low-energy, 6 and 9 MeV, electron therapy,” says Charlotte Robert, head of the medical physics department research group at Gustave Roussy. “The first clinical trials using low-energy electrons are all on skin tumours, aiming to show that we can safely decrease the number of treatment sessions.”
While these initial studies are limited to skin lesions, clinical implementation of the FLASHDEEP system will extend the benefits of FLASH to many more tumour sites. Robert predicts that VHEE-based FLASH will prove most valuable for treating radioresistant cancers that cannot currently be cured. The rationale is that FLASH’s ability to spare normal tissue will allow delivery of higher target doses without increasing toxicity.
“You will not use this technology for diseases that can already be cured, at least initially,” she explains. “The first clinical trial, I’m quite sure, will be either glioblastoma or pancreatic cancers that are not effectively controlled today. If we can show that VHEE FLASH can spare normal tissue more than conventional radiotherapy can, we hope this will have a positive impact on lesion response.”
“There are a lot of technological challenges around this technology and we are trying to tackle them all,” Curtoni concludes. “The ultimate goal is to produce a VHEE accelerator with a very compact beamline that makes this technology and FLASH a reality for a clinical environment.”
Brain–computer interfaces (BCIs) enable the flow of information between the brain and an external device such as a computer, smartphone or robotic limb. Applications range from use in augmented and virtual reality (AR and VR), to restoring function to people with neurological disorders or injuries.
Electroencephalography (EEG)-based BCIs use sensors on the scalp to noninvasively record electrical signals from the brain and decode them to determine the user’s intent. Currently, however, such BCIs require bulky, rigid sensors that prevent use during movement and don’t work well with hair on the scalp, which affects the skin–electrode impedance. A team headed up at Georgia Tech’s WISH Center has overcome these limitations by creating a brain sensor that’s small enough to fit between strands of hair and is stable even while the user is moving.
“This BCI system can find wide applications. For example, we can realize a text spelling interface for people who can’t speak,” says W Hong Yeo, Harris Saunders Jr Professor at Georgia Tech and director of the WISH Center, who co-led the project with Tae June Kang from Inha University in Korea. “For people who have movement issues, this BCI system can offer connectivity with human augmentation devices, a wearable exoskeleton, for example. Then, using their brain signals, we can detect the user’s intentions to control the wearable system.”
A tiny device
The microscale brain sensor comprises a cross-shaped structure of five microneedle electrodes, with sharp tips (less than 30°) that penetrate the skin easily with nearly pain-free insertion. The researchers used UV replica moulding to create the array, followed by femtosecond laser cutting to shape it to the required dimensions – just 850 x 1000 µm – to fit into the space between hair follicles. They then coated the microsensor with a highly conductive polymer (PEDOT:Tos) to enhance its electrical conductivity.
Between the hairs The size and lightweight design of the sensor significantly reduces motion artefacts. (Courtesy: W Hong Yeo)
The microneedles capture electrical signals from the brain and transmit them along ultrathin serpentine wires that connect to a miniaturized electronics system on the back of the neck. The serpentine interconnector stretches as the skin moves, isolating the microsensor from external vibrations and preventing motion artefacts. The miniaturized circuits then wirelessly transmit the recorded signals to an external system (AR glasses, for example) for processing and classification.
Yeo and colleagues tested the performance of the BCI using three microsensors inserted into the scalp of the occipital lobe (the brain’s visual processing centre). The BCI exhibited excellent stability, offering high-quality measurement of neural signals – steady-state visual evoked potentials (SSVEPs) – for up to 12 h, while maintaining low contact impedance density (0.03 kΩ/cm2).
The team also compared the quality of EEG signals measured using the microsensor-based BCI with those obtained from conventional gold-cup electrodes. Participants wearing both sensor types closed and opened their eyes while standing, walking or running.
With the participant stood still, both electrode types recorded stable EEG signals, with an increased amplitude upon closing the eyes, due to the rise in alpha wave power. During motion, however, the EEG time series recorded with the conventional electrodes showed noticeable fluctuations. The microsensor measurements, on the other hand, exhibited minimal fluctuations while walking and significantly fewer fluctuations than the gold-cup electrodes while running.
Overall, the alpha wave power recorded by the microsensors during eye-closing was higher than that of the conventional electrode, which could not accurately capture EEG signals while the user was running. The microsensors only exhibited minor motion artefacts, with little to no impact on the EEG signals in the alpha band, allowing reliable data extraction even during excessive motion.
Real-world scenario
Next, the team showed how the BCI could be used within everyday activities – such as making calls or controlling external devices – that require a series of decisions. The BCI enables a user to make these decisions using their thoughts, without needing physical input such as a keyboard, mouse or touchscreen. And the new microsensors free the user from environmental and movement constraints.
The researchers demonstrated this approach in six subjects wearing AR glasses and a microsensor-based EEG monitoring system. They performed experiments with the subjects standing, walking or running on a treadmill, with two distinct visual stimuli from the AR system used to induce SSVEP responses. Using a train-free SSVEP classification algorithm, the BCI determined which stimulus the subject was looking at with a classification accuracy of 99.2%, 97.5% and 92.5%, while standing, walking and running, respectively.
The team also developed an AR-based video call system controlled by EEG, which allows users to manage video calls (rejecting, answering and ending) with their thoughts, demonstrating its use during scenarios such as ascending and descending stairs and navigating hallways.
“By combining BCI and AR, this system advances communication technology, offering a preview of the future of digital interactions,” the researchers write. “Additionally, this system could greatly benefit individuals with mobility or dexterity challenges, allowing them to utilize video calling features without physical manipulation.”
Reversible switching Schematic illustrating the hard/soft transition of the hydrogel/NAAC composite. (Courtesy: CC BY 4.0/Int. J. Extrem. Manuf. 10.1088/2631-7990/adbd97)
Complex hydrogel structures created using 3D printing are increasingly employed in fields including flexible electronics, soft robotics and regenerative medicine. Currently, however, such hydrogels are often soft and fragile, limiting their practical utility. Researchers at Zhejiang University in China have now fabricated 3D-printed hydrogels that can be easily, and repeatably, switched between soft and hard states, enabling novel applications such as smart medical bandages or information encryption.
“Our primary motivation was to overcome the inherent limitations of 3D-printed hydrogels, particularly their soft, weak and fragile mechanical properties, to broaden their application potential,” says co-senior author Yong He.
The research team created the hard/soft switchable composite by infusing supersaturated salt solution (sodium acetate, NAAC) into 3D-printed polyacrylamide (PAAM)-based hydrogel structures. The hardness switching is enabled by the liquid/solid transition of the salt solution within the hydrogel.
Initially, the salt molecules are arranged randomly within the hydrogel and the PAAM/NAAC composite is soft and flexible. The energy barrier separating the soft and hard states prevents spontaneous crystallization, but can be overcome by artificially seeding a crystal nucleus (via exposure to a salt crystal or contact with a sharp object). This seed promotes a phase transition to a hard state, with numerous rigid, rod-like nanoscale crystals forming within the hydrogel matrix.
Superior mechanical parameters
The researchers created a series of PAAM/NAAC structures, using projection-based 3D printing to print hydrogel shapes and then soaking them in NAAC solution. Upon seeding, the structures rapidly transformed from transparent to opaque as the crystallization spread through the sample at speeds of up to 4.5 mm/s.
The crystallization dramatically changed the material’s mechanical performance. For example, a soft cylinder of PAAM/1.5NAAC (containing 150 wt% salt) could be easily compressed by hand, returning to its original shape after release. After crystallization, four 9x9x12 mm cylinders could support an adult’s weight without deforming.
For this composite, just 1 min of crystallization dramatically increased the compression Young’s modulus compared with the soft state. And after 24 h, the Young’s modulus grew from 110 kPa to 871.88 MPa. Importantly, the hydrogel could be easily returned to its soft state by heating and then cooling, a process that could be repeated many times.
The team also performed Shore hardness testing on various composites, observing that hardness values increased with increasing NAAC concentration. In PAAM/1.7NAAC composites (170 wt% salt), the Shore D value reached 86.5, comparable to that of hard plastic materials.
The hydrogel’s crosslinking density also impacted its mechanical performance. For PAAM/1.5NAAC composites, increasing the mass percentage of polymer crosslinker from 0.02 to 0.16 wt% increased the compression Young’s modulus to 1.2 GPa and the compression strength to 81.7 MPa. The team note that these parameters far exceed those of any existing 3D-printed hydrogels.
Smart plaster cast
He and colleagues demonstrated how the hard/soft switching and robust mechanical properties of PAAM/NAAC can create medical fixation devices, such as a smart plaster cast. The idea here is that the soft hydrogel can be moulded around the injured bone, and then rapidly frozen in shape by crystallization to support the injury and promote healing.
The researchers tested the smart plaster cast on an injured forearm. After applying a layer of soft cotton padding, they carefully wrapped around layers of the smart plaster bandage (packed within a polyethylene film to prevent accidental seeding). The flexible hydrogel could be conformed to the curved surface of limbs and then induced to crystallize.
Medical fixation device Application of the PAAM/NAAC composite to create a smart plaster cast. (Courtesy: CC BY 4.0/Int. J. Extrem. Manuf. 10.1088/2631-7990/adbd97)
After just 10 min of crystallization, the smart plaster cast reached a yield strength of 8.7 MPa, rapidly providing support for the injured arm. In comparison, a traditional plaster cast (as currently used to treat bone fractures) took about 24 h to fully harden, reaching a maximum yield strength of 3.9 MPa
To determine the safety of the exothermic crystallization process, the team monitored temperature changes in the plaster cast nearest to the skin. The temperature peaked at 41.5 °C after 25 min of crystallization, below the ISO-recommended maximum safe temperature of 50 °C.
The researchers suggest that the ease of use, portability and fast response of the smart plaster cast could provide a simple and effective solution for emergency and first aid situations. Another benefit is that, in contrast to traditional plaster casts that obstruct X-rays and hinder imaging, X-rays easily penetrate through the smart plaster cast to enable high-quality imaging during the healing process.
While the composites exhibit high strength and Young’s modulus, they are not as tough as ideally desired. “For example, the elongation at break was less than 10% in tensile testing for the PAAM/1.5NAAC and PAAM/1.7NAAC samples, highlighting the challenge of balancing toughness with strength and modulus,” He tells Physics World. “Therefore, our current research focuses on enhancing the toughness of these composite materials without compromising their modulus, with the goal of developing strong, tough and mechanically switchable materials.”
Multiphoton microscopy is a nonlinear optical imaging technique that enables label-free, damage-free biological imaging. Performed using femtosecond laser pulses to generate two- and three-photon processes, multiphoton imaging techniques could prove invaluable for rapid cancer diagnosis or personalized medicine.
Imaging biological samples with traditional confocal microscopy requires sample slicing and staining to create contrast in the tissue. The nonlinear mechanisms generated by femtosecond laser pulses, however, eliminate the need for labelling or sample preparation, revealing molecular and structural details within tissue and cells while leaving the sample intact.
Looking to bring these benefits to cancer diagnostics, Netherlands-based start-up Flash Pathology is developing a compact, portable multiphoton microscope that creates pathology-quality images in real time, without the need for sample fixation or staining.
Fast and accurate cancer diagnosis with higher harmonic imaging
The inspiration for Flash Pathology came from Marloes Groot of Vrije Universiteit (VU) Amsterdam. While studying multiphoton microscopy of brain tumours, Groot recognized the need for a portable microscope for clinical settings. “This is a really powerful technique,” she says. “I was working on my large laboratory setup and I thought if we start a company, we could transform this into a mobile device.”
Groot teamed up with Frank van Mourik, now Flash Pathology’s CTO, to shrink the imaging device into a compact 60 x 80 x 115 cm system. “Frank made a device that can be transported, in a truck, wheeled through corridors, and still when you plug it in, it’s on and it produces images – for a nonlinear microscope, this is pretty special,” she explains.
Van Mourik has now built several multiphoton microscopes for Flash Pathology, with one of the main achievements the ability to measure samples with extremely low power levels. “When I started in my lab, I used 200 mW of power, but we’ve been able to reduce that to 5 mW,” Groot notes. “We have performed extensive studies to show that our imaging does not affect the tissue.”
Flash Pathology’s multiphoton microscope is designed to provide rapid on-site histologic feedback on excised tissue, such as diagnostic biopsies or tissue from surgical resections. One key application is lung cancer diagnosis, where there is a clinical need for rapid intraoperative feedback on biopsies. The standard histopathological analysis requires extensive sample preparation and can take several days to provide results.
Rapid tissue analysis Multiphoton microscopy images of (left to right) adipose tissue, cartilage and lymphoid tissue (each image is 400 x 400 µm). The adipose tissue image shows large adipocytes (fat cells); the cartilage image shows a hyaline (glass-like) background with chondrocytes (cells); the image of lymphoid tissue shows many small lymphocytes (a type of immune cell). (Courtesy: VU Amsterdam)
“With lung biopsies, it’s challenging to obtain good diagnostic material,” explains Sylvia Spies, a PhD student at VU Amsterdam. “The lesions can be quite small and it can be difficult to get to the right position and take a good sample, so they use several techniques (fluoroscopy/CT or ultrasound) to find the right position and take multiple biopsies from the lesion. Despite these techniques, the diagnostic yield is still around 70%, so 30% of cases still don’t get a diagnosis and patients might have to come back for a repeat biopsy procedure.”
Multiphoton imaging, on the other hand, can rapidly visualize unprocessed tissue samples, enabling diagnosis in situ. A recent study using Flash Pathology’s microscope to analyse lung biopsies demonstrated that it could image a biopsy sample and provide feedback just 6 min after excision, with an accuracy of 87% – enabling immediate decisions as to whether a further biopsy is required.
“Also, many clinical fields are now focusing on a one-stop-shop with diagnosis and treatment in one procedure,” adds Spies. “Here, you really need a technique that can rapidly determine whether a lesion is benign or malignant.”
The microscope’s impressive diagnostic performance is partly due to its ability to generate four nonlinear signals simultaneously using a single ultrafast femtosecond laser: second- and third-harmonic generation plus two- and three-photon fluorescence. The system then uses filters to spectrally separate these signals, which provide complementary diagnostic information. Second-harmonic generation, for example, is sensitive to non-centrosymmetric structures such as collagen, while third-harmonic generation only occurs at interfaces with differing refractive indices, such as cell membranes or boundaries between the nucleus and cytoplasm.
“What I like about this technique is that you can see similar features as in conventional histology,” says Spies. “You can see structures such as collagen fibres, elastin fibres and cellular patterns, but also cellular details such as the cytoplasm, the nucleus (and its size), nucleoli and cilia. All these tiny details are the same features that the pathologists look at in conventional histology.”
Applying femtosecond lasers for 3D-in-depth visualization
The femtosecond laser plays a key role in enabling multiphoton microscopy. To excite two- and three-photon processes, you need to have two or more photons in the same place at exactly the same time. And the likelihood of this happening increases rapidly when using ultrashort laser pulses.
“The shorter the pulses are in the time domain, the higher the probability that you have an overlap of two pulses in a focal point,” explains Oliver Prochnow, CEO of VALO Innovations, a part of HÜBNER Photonics. “Therefore you need to have a very high-intensity, extremely short laser pulse. The shorter the better.”
The VALO Femtosecond Series of ultrafast fibre lasers can deliver pulses as short as 30 fs, which is achieved by exploiting nonlinear mechanisms to broaden the spectral bandwidth to more than 100 nm. As the optical spectrum and pulse duration are inherently related by Fourier transformation, a broadband spectrum will result in a very short pulse. And the shorter the pulse, at the same average power, the higher its peak power – and the higher the probability of producing multiphoton processes.
Laser parameters Left: typical temporal pulse profile highlighting the sub 50 fs pulse duration with very low pulse pedestal; the inset shows the typical beam profile. Right: typical optical spectrum of HÜBNER Photonics’ VALO Femtosecond Series lasers. (Courtesy: HÜBNER Photonics)
“If you decrease the pulse duration by a factor of five, this gives roughly a five times higher signal from two-photon absorption,” says Prochnow. “In contrast, a three-photon process scales with the third power of the intensity and with the inverse of the pulse duration squared. So you have a roughly 25 times higher signal, if you decrease the pulse duration by a factor of five at the same average power.” Crucially, the shorter pulses deliver this high peak power while maintaining a low average power, reducing sample heating and minimizing photobleaching.
The broadband optical spectrum is particularly important for enabling practical three-photon microscopy. The challenge here is that traditional ytterbium-based lasers with a wavelength of around 1030 nm produce a three-photon signal in the UV range, which is too short to be transmitted through standard optics.
Broadband spectrum Fundamental and third-harmonic generation (THG) spectra of a 30 fs broadband fibre laser (red) compared with standard 150 fs lasers. The solid black line shows the typical transmission characteristics of a standard microscopy objective. Only a THG spectrum generated from wavelengths of above 1080 nm will be transmitted. (Courtesy: HÜBNER Photonics)
The VALO Femtosecond Series overcomes this problem by having a broadband spectrum that extends up to 1140 nm. Frequency tripling then generates a signal with a long enough wavelength to pass through a standard microscope objective, enabling the VALO lasers to excite both two-photon and three-photon processes. “Our lasers provide the opportunity to perform simultaneous three-photon microscopy and two-photon microscopy using a simple fibre laser solution,” says Prochnow.
The lasers include an integrated dispersion pre-compensation unit to compensate for the dispersion of a microscope objective and provide the shortest pulses at the sample. Additionally, the lasers do not require water cooling, making them easy to use or integrate.
Towards future clinical applications
Flash Pathology is currently testing its microscope in several hospitals in the Netherlands, including Amsterdam UMC, as well as the Princess Maxima Center for paediatric oncology. “Sylvia performed a study in their pathology department and for a year measured all kinds of tissue samples that came through,” says Groot. “We also recently installed a device at the Queen Elizabeth Hospital in Glasgow, for a study on mesothelioma.”
With prototypes now available for research use, the company also plans to develop a fully certified multiphoton microscopy system. “Our ultimate goal is to sell a certified medical diagnostic device that will take a biopsy and produce images, but also contain artificial intelligence to help to interpret the images and give diagnostic conclusions about the nature of the illness,” says van Mourik.
Once fully realised in the clinic, the multiphoton microscopy system will provide an invaluable tool for rapid, in situ tissue analysis during bronchoscopy procedures or other operations. The unique combination of four nonlinear imaging modalities, made possible with a single compact femtosecond laser, delivers complementary diagnostic information. “This will be the big gain, to be able to provide a diagnosis bedside during a procedure,” van Mourik concludes.
Neutron-activated gold Novel activation imaging technique enables real-time visualization of gold nanoparticles in the body without the use of external tracers. (Courtesy: Nanase Koshikawa from Waseda University)
Gold nanoparticles are promising vehicles for targeted delivery of cancer drugs, offering biocompatibility plus a tendency to accumulate in tumours. To fully exploit their potential, it’s essential to be able to track the movement of these nanoparticles in the body. To date, however, methods for directly visualizing their pharmacokinetics have not yet been established. Aiming to address this shortfall, researchers in Japan are using neutron-activated gold radioisotopes to image nanoparticle distribution in vivo.
The team, headed up by Nanase Koshikawa and Jun Kataoka from Waseda University, are investigating the use of radioactive gold nanoparticles based on 198Au, which they create by irradiating stable gold (197Au) with low-energy neutrons. The radioisotope 198Au has a half-life of 2.7 days and emits 412 keV gamma rays, enabling a technique known as activation imaging.
“Our motivation was to visualize gold nanoparticles without labelling them with tracers,” explains Koshikawa. “Radioactivation allows gold nanoparticles themselves to become detectable from outside the body. We used neutron activation because it does not change the atomic number, ensuring the chemical properties of gold nanoparticles remain unchanged.”
In vivo studies
The researchers – also from Osaka University and Kyoto University – synthesized 198Au-based nanoparticles and injected them into tumours in four mice. They used a hybrid Compton camera (HCC) to detect the emitted 412 keV gamma rays and determine the in vivo nanoparticle distribution, on the day of injection and three and five days later.
The HCC, which incorporates two pixelated scintillators, a scatterer with a central pinhole, and an absorber, can detect radiation with energies from tens of keV to nearly 1 MeV. For X-rays and low-energy gamma rays, the scatterer enables pinhole-mode imaging. For gamma rays over 200 keV, the device functions as a Compton camera.
The researchers reconstructed the 412 keV gamma signals into images, using an energy window of 412±30 keV. With the HCC located 5 cm from the animals’ abdomens, the spatial resolution was 7.9 mm, roughly comparable to the tumour size on the day of injection (7.7 x 11 mm).
In vivo distribution Images of 198Au nanoparticles in the bodies of two mice obtained with the HCC on the day of administration. (Courtesy: CC BY 4.0/Appl. Phys. Lett. 10.1063/5.0251048)
Overlaying the images onto photographs of the mice revealed that the nanoparticles accumulated in both the tumour and liver. In mice 1 and 2, high pixel values were observed primarily in the tumour, while mice 3 and 4 also had high pixel values in the liver region.
After imaging, the mice were euthanized and the team used a gamma counter to measure the radioactivity of each organ. The measured activity concentrations were consistent with the imaging results: mice 1 and 2 had higher nanoparticle concentrations in the tumour than the liver, and mice 3 and 4 had higher concentrations in the liver.
Tracking drug distribution
Next, Koshikawa and colleagues used the 198Au nanoparticles to label astatine-211 (211At), a promising alpha-emitting drug. They note that although 211At emits 79 keV X-rays, allowing in vivo visualization, its short half-life of just 7.2 h precludes its use for long-term tracking of drug pharmacokinetics.
The researchers injected the 211At-labelled nanoparticles into three tumour-bearing mice and used the HCC to simultaneously image 211At and 198Au, on the day of injection and one or two days later. Comparing energy spectra recorded just after injection with those two days later showed that the 211At peak at 79 keV significantly decreased in height owing to its decay, while the 412 keV 198Au peak maintained its height.
The team reconstructed images using energy windows of 79±10 and 412±30 keV, for pinhole- and Compton-mode reconstruction, respectively. In these experiments, the HCC was placed 10 cm from the mouse, giving a spatial resolution of 16 mm – larger than the initial tumour size and insufficient to clearly distinguish tumours from small organs. Nevertheless, the researchers point out that the rough distribution of the drug was still observable.
On the day of injection, the drug distribution could be visualized using both the 211At and 198Au signals. Two days later, imaging using 211At was no longer possible. In contrast, the distribution of the drug could still be observed via the 412 keV gamma rays.
With further development, the technique may prove suitable for future clinical use. “We assume that the gamma ray exposure dose would be comparable to that of clinical imaging techniques using X-rays or gamma rays, such as SPECT and PET, and that activation imaging is not harmful to humans,” Koshikawa says.
Activation imaging could also be applied to more than just gold nanoparticles. “We are currently working on radioactivation of platinum-based anticancer drugs to enable their visualization from outside the body,” Koshikawa tells Physics World. “Additionally, we are developing new detectors to image radioactive drugs with higher spatial resolution.”
A team at the Trento Proton Therapy Centre in Italy has delivered the first clinical treatments using proton arc therapy, an emerging proton delivery technique. Following successful dosimetric comparisons with clinically delivered proton plans, the researchers confirmed the feasibility of PAT delivery and used PAT to treat nine cancer patients, reporting their findings inMedical Physics.
Currently, proton therapy is mostly delivered using pencil-beam scanning (PBS), which provides highly conformal dose distributions. But PBS delivery can be compromised by the small number of beam directions deliverable in an acceptable treatment time. PAT overcomes this limitation by moving to an arc trajectory.
“Proton arc treatments are different from any other pencil-beam proton delivery technique because of the large number of beam angles used and the possibility to optimize the number of energies used for each beam direction, which enables optimization of the delivery time,” explains first author Francesco Fracchiolla. “The ability to optimize both the number of energy layers and the spot weights makes these treatments superior to any previous delivery technique.”
Plan comparisons
The Trento researchers – working with colleagues from RaySearch Laboratories – compared the dosimetric parameters of PAT plans with those of state-of-the-art multiple-field optimized (MFO) PBS plans, for 10 patients with head-and-neck cancer. They focused on this site due to the high number of organs-at-risk (OARs) close to the target that may be spared using this new technique.
In future, PAT plans will be delivered with the beam on during gantry motion (dynamic mode). This requires dynamic arc plan delivery with all system settings automatically adjusted as a function of gantry angle – an approach with specific hardware and software requirements that have so far impeded clinical rollout.
Instead, Fracchiolla and colleagues employed an alternative version of static PAT, in which the static arc is converted into a series of PBS beams and delivered using conventional delivery workflows. Using the RayStation treatment planning system, they created MFO plans (using six noncoplanar beam directions) and PAT plans (with 30 beam directions), robustly optimized against setup and range uncertainties.
PAT plans dramatically improved dose conformality compared with MFO treatments. While target coverage was of equal quality for both treatment types, PAT decreased the mean doses to OARs for all patients. The biggest impact was in the brainstem, where PAT reduced maximum and mean doses by 19.6 and 9.5 Gy(RBE), respectively. Dose to other primary OARs did not differ significantly between plans, but PAT achieved an impressive reduction in mean dose to secondary OARs not directly adjacent to the target.
The team also evaluated how these dosimetric differences impact normal tissue complication probability (NTCP). PAT significantly reduced (by 8.5%) the risk of developing dry mouth and slightly lowered other NTCP endpoints (swallowing dysfunction, tube feeding and sticky saliva).
To verify the feasibility of clinical PAT, the researchers delivered MFO and PAT plans for one patient on a clinical gantry. Importantly, delivery times (from the start of the first beam to the end of the last) were similar for both techniques: 36 min for PAT with 30 beam directions and 31 min for MFO. Reducing the number of beam directions to 20 reduced the delivery time to 25 min, while maintaining near-identical dosimetric data.
First patient treatments
The successful findings of the plan comparison and feasibility test prompted the team to begin clinical treatments.
“The final trigger to go live was the fact that the discretized PAT plans maintained pretty much exactly the optimal dosimetric characteristics of the original dynamic (continuous rotation) arc plan from which they derived, so there was no need to wait for full arc to put the potential benefits to clinical use. Pretreatment verification showed excellent dosimetric accuracy and everything could be done in a fully CE-certified environment,” say Frank Lohr and Marco Cianchetti, director and deputy director, respectively, of the Trento Proton Therapy Center. “The only current drawback is that we are not at the treatment speed that we could be with full dynamic arc.”
To date, nine patients have received or are undergoing PAT treatment: five with head-and-neck tumours, three with brain tumours and one thorax cancer. For the first two head-and-neck patients, the team created PAT plans with a half arc (180° to 0°) with 10 beam directions and a mean treatment time of 12 min. The next two were treated with a complete arc (360°) with 20 beam directions. Here, the mean treatment time was 24 min. Patient-specific quality assurance revealed an average gamma passing rate (3%, 3 mm) of 99.6% and only one patient required replanning.
All PAT treatments were performed using the centre’s IBA ProteusPlus proton therapy unit and the existing clinical workflow. “Our treatment planning system can convert an arc plan into a PBS plan with multiple beams,” Fracchiolla explains. “With this workaround, the entire clinical chain doesn’t change and the plan can be delivered on the existing system. This ability to convert the arc plans into PBS plans means that basically every proton centre can deliver these treatments with the current hardware settings.”
The researchers are now analysing acute toxicity data from the patients, to determine whether PAT reduces toxicity. They are also looking to further reduce the delivery times.
“Hopefully, together with IBA, we will streamline the current workflow between the OIS [oncology information system] and the treatment control system to reduce treatment times, thus being competitive in comparison with conventional approaches, even before full dynamic arc treatments become a clinical reality,” adds Lohr.
Treatment with low-temperature plasma is emerging as a novel cancer therapy. Previous studies have shown that plasma can deactivate cancer cells in vitro, suppress tumour growth in vivo and potentially induce anti-tumour immunity. Researchers at the University of Tokyo are investigating another promising application – the use of plasma to inhibit tumour recurrence after surgery.
Lead author Ryo Ono and colleagues demonstrated that treating cancer resection sites with streamer discharge – a type of low-temperature atmospheric plasma – significantly reduced the recurrence rate of melanoma tumours in mice.
“We believe that plasma is more effective when used as an adjuvant therapy rather than as a standalone treatment, which led us to focus on post-surgical treatment in this study,” says Ono.
In vivo experiments
To create the streamer discharge, the team applied a high-voltage pulse (25 kV, 20 ns, 100 pulse/s) to a 3 mm-diameter rod electrode with a hemispherical tip. The rod was placed in a quartz tube with a 4 mm inner diameter, and the working gas – humid oxygen mixed with ambient air – was flowed through the tube. As electrons in the plasma collide with molecules in the gas, the mixture generates cytotoxic reactive oxygen and nitrogen species.
The researchers performed three experiments on mice with melanoma, a skin cancer with a local recurrence rate of up to 10%. In the first experiment, they injected 11 mice with mouse melanoma cells, resecting the resulting tumours eight days later. They then treated five of the mice with streamer discharge for 10 min, with the mouse placed on a grounded plate and the electrode tip 10 mm above the resection site.
Experimental setup Streamer discharge generation and treatment. (Courtesy: J. Phys. D: Appl. Phys. 10.1088/1361-6463/ada98c)
Tumour recurrence occurred in five of the six control mice (no plasma treatment) and two of the five plasma-treated mice, corresponding to recurrence rates of 83% and 40%, respectively. In a second experiment with the same parameters, recurrence rates were 44% in nine control mice and 25% in eight plasma-treated mice.
In a third experiment, the researchers delayed the surgery until 12 days after cell injection, increasing the size of the tumour before resection. This led to a 100% recurrence rate in the control group of five mice. Only one recurrence was seen in five plasma-treated mice, although one mouse that died of unknown causes was counted as a recurrence, resulting in a recurrence rate of 40%.
All of the experiments showed that plasma treatment reduced the recurrence rate by roughly 50%. The researchers note that the plasma treatment did not affect the animals’ overall health.
Cytotoxic mechanisms
To further confirm the cytotoxicity of streamer discharge, Ono and colleagues treated cultured melanoma cells for between 0 and 250 s, at an electrode–surface distance of 10 mm. The cells were then incubated for 3, 6 or 24 h. Following plasma treatments of up to 100 s, most cells were still viable 24 h later. But between 100 and 150 s of treatment, the cell survival rate decreased rapidly.
The experiment also revealed a rapid transition from apoptosis (natural programmed cell death) to late apoptosis/necrosis (cell death due to external toxins) between 3 and 24 h post-treatment. Indeed, 24 h after a 150 s plasma treatment, 95% of the dead cells were in the late stages of apoptosis/necrosis. This finding suggests that the observed cytotoxicity may arise from direct induction of apoptosis and necrosis, combined with inhibition of cell growth at extended time points.
In a previous experiment, the researchers used streamer discharge to treat tumours in mice before resection. This treatment delayed tumour regrowth by at least six days, but all mice still experienced local recurrence. In contrast, in the current study, plasma treatment reduced the recurrence rate.
The difference may be due to different mechanisms by which plasma inhibits tumour recurrence: cytotoxic reactive species killing residual cancer cells at the resection site; or reactive species triggering immunogenic cell death. The team note that either or both of these mechanisms may be occurring in the current study.
“Initially, we considered streamer discharge as the main contributor to the therapeutic effect, as it is the primary source of highly reactive short-lived species,” explains Ono. “However, recent experiments suggest that the discharge within the quartz tube also generates a significant amount of long-lived reactive species (with lifetimes typically exceeding 0.1 s), which may contribute to the therapeutic effect.”
One advantage of the streamer discharge device is that it uses only room air and oxygen, without requiring the noble gases employed in other cold atmospheric plasmas. “Additionally, since different plasma types generate different reactive species, we hypothesized that streamer discharge could produce a unique therapeutic effect,” says Ono. “Conducting in vivo experiments with different plasma sources will be an important direction for future research.”
Looking ahead to use in the clinic, Ono believes that the low cost of the device and its operation should make it feasible to use plasma treatment immediately after tumour resection to reduce recurrence risk. “Currently, we have only obtained preliminary results in mice,” he tells Physics World. “Clinical application remains a long-term goal.”
The COVID-19 pandemic provided a driving force for researchers to seek out new disinfection methods that could tackle future viral outbreaks. One promising approach relies on the use of nanoparticles, with several metal and metal oxide nanoparticles showing anti-viral activity against SARS-CoV-2, the virus that causes COVID-19. With this in mind, researchers from Sweden and Estonia investigated the effect of such nanoparticles on two different virus types.
Aiming to elucidate the nanoparticles’ mode of action, they discovered a previously unknown antiviral mechanism, reporting their findings in Nanoscale.
The researchers – from the Swedish University of Agricultural Sciences (SLU) and the University of Tartu – examined triethanolamine terminated titania (TATT) nanoparticles, spherical 3.5-nm diameter titanium dioxide (titania) particles that are expected to interact strongly with viral surface proteins.
They tested the antiviral activity of the TATT nanoparticles against two types of virus: swine transmissible gastroenteritis virus (TGEV) – an enveloped coronavirus that’s surrounded by a phospholipid membrane and transmembrane proteins; and the non-enveloped encephalomyocarditis virus (EMCV), which does not have a phospholipid membrane. SARS-CoV-2 has a similar structure to TGEV: an enveloped virus with an outer lipid membrane and three proteins forming the surface.
In this latest investigation, the team aimed to determine whether one of these potential mechanisms – blocking of surface proteins, or membrane disruption via oxidation by nanoparticle-generated reactive oxygen species – is the likely cause of TATT’s antiviral activity. The first of these effects usually occurs at low (nanomolar to micromolar) nanoparticle concentrations, the latter at higher (millimolar) concentrations.
Mode of action
To assess the nanoparticle’s antiviral activity, the researchers exposed viral suspensions to colloidal TATT solutions for 1 h, at room temperature and in the dark (without UV illumination). For comparison, they repeated the process with silicotungstate polyoxometalate (POM) nanoparticles, which are not able to bind strongly to cell membranes.
The nanoparticle-exposed viruses were then used to infect cells and the resulting cell viability served as a measure of the virus infectivity. The team note that the nanoparticles alone showed no cytotoxicity against the host cells.
Measuring viral infectivity after nanoparticle exposure revealed that POM nanoparticles did not exhibit antiviral effects on either virus, even at relatively high concentrations of 1.25 mM. TATT nanoparticles, on the other hand, showed significant antiviral activity against the enveloped TGEV virus at concentrations starting from 0.125 mM, but did not affect the non-enveloped EMCV virus.
Based on previous evidence that TATT nanoparticles interact strongly with proteins in darkness, the researchers expected to see antiviral activity at a nanomolar level. But the finding that TATT activity only occurred at millimolar concentrations, and only affected the enveloped virus, suggests that the antiviral effect is not due to blocking of surface proteins. And as titania is not oxidative in darkness, the team propose that the antiviral effect is actually due to direct complexation of nanoparticles with membrane phospholipids – a mode of antiviral action not previously considered.
“Typical nanoparticle concentrations required for effects on membrane proteins correspond to the protein content on the virus surface. With a 1:1 complex, we would need maximum nanomolar concentrations,” Kessler explains. “We saw an effect at about 1 mM/l, which is far higher. This was the indication for us that the effect was on the whole of membrane.”
Verifying the membrane effect
To corroborate their hypothesis, the researchers examined the leakage of dye-labelled RNA from the TGEV coronavirus after 1 h exposure to nanoparticles. The fluorescence signal from the dye showed that TATT-treated TGEV released significantly more RNA than non-exposed virus, attributed to the nanoparticles disrupting the virus’s phospholipid membrane.
Finally, the team studied the interactions between TATT nanoparticles and two model phospholipid compounds. Both molecules formed strong complexes with TATT nanoparticles, while their interaction with POM nanoparticles was weak. This additional verification led the researchers to conclude that the antiviral effect of TATT in dark conditions is due to direct membrane disruption via complexation of titania nanoparticles with phospholipids.
“To the best of our knowledge, [this] proves a new pathway for metal oxide nanoparticles antiviral action,” they write.
Importantly, the nanoparticles are non-toxic, and work at room temperature without requiring UV illumination – enabling simple and low-cost disinfection methods. “While it was known that disinfection with titania could work in UV light, we showed that no special technical measures are necessary,” says Kessler.
Kessler suggests that the nanoparticles could be used to coat surfaces to destroy enveloped viruses, or in cost-effective filters to decontaminate air or water. “[It should be] possible to easily create antiviral surfaces that don’t require any UV activation just by spraying them with a solution of TATT, or possibly other oxide nanoparticles with an affinity to phosphate, including iron and aluminium oxides in particular,” he tells Physics World.
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