Imaging Technology Shows How RNA in Cells Can Affect Health

 

Insight into the cellular distribution of RNA, which is closely linked to cell functions, could help scientists better understand the relation between cellular processes and disease. Potentially, this could lead to more targeted treatments for neurodegenerative disorders and aging.

While many methods have been developed to study RNA distribution within cells, only a few have been applied on a transcriptome-wide scale.

To capture the transcriptome of target cell types at the tissue level and RNA content within subcellular compartments, a research team at the UT Southwestern Medical Center, led by professor Haiqi Chen, developed Photoselection of Transcriptome over Nanoscale (PHOTON).

PHOTON combines high-resolution imaging with high-throughput sequencing to achieve spatial transcriptome profiling of RNA at subcellular resolution. It identifies RNA molecules at their native locations within cells, showing where different RNA species are distributed spatially in response to cellular cues.

To build PHOTON, the researchers designed DNA-based molecular cages that bound to all the RNA in cells. The molecular cages open when they are exposed to light, allowing for further chemical labeling.

After observing microscopically that the cells bound to the molecular cages, the researchers shined a narrow, 200-300-nm, near-ultraviolet (NUV) laser beam on regions of interest, such as specific organelles. The light caused the molecular cages to open, allowing only the RNA molecules located in the illuminated regions to be labeled. The researchers then collected the labeled RNA molecules and sequenced them to learn their identities and functions.

The team used PHOTON to examine RNAs present in the nucleolus and mitochondria, showing that RNAs identified through PHOTON closely matched those in published databases that were produced by isolating the organelles from the cells.

The researchers applied PHOTON to stress granules — transient, membraneless structures formed by cells when the cells are under stress. Although most stress granule RNAs that were identified matched those in published databases, the researchers found some discrepancies using PHOTON.

At the tissue scale, PHOTON accurately captured the transcriptome of cells within their native tissue microenvironment. At the subcellular scale, it enabled selective sequencing of the RNA content in the nucleoli, the mitochondria, and the stress granules.

The researchers used PHOTON to investigate whether m6A, a chemical modification found on some RNA molecules, played a role in moving RNAs into stress granules. By analyzing RNA molecules identified through PHOTON, the researchers found that the RNAs in the stress granules carried significantly more m6A than those outside the granules, suggesting that m6A contributes to the movement of specific RNAs into stress granules.

The researchers showed that PHOTON could be flexibly applied across regions of interest that spanned different scales, from specific regions of mouse ovarian tissue to various subcellular compartments. In-line image segmentation enabled the researchers to generate regions of interest based on an extensive range of spatial features, and automated the targeted photocleavage process over large numbers of cells or features.

These results show that PHOTON has the potential to uncover connections between spatial and transcriptomic information at diverse length scales.

Existing techniques to spatially identify RNA species can be prohibitively expensive and typically require specialized technical expertise and sophisticated image processing and data analysis to complete.

Chen said that he and his colleagues plan to use PHOTON to study the distributions of RNA in various conditions, particularly in neurodegenerative disease and aging. By comparing distributions in diseased cells to those in healthy cells, Chen said, researchers may be able to identify new targets for therapies to treat these conditions.

“Aging and many neurodegenerative diseases impose significant stress on cells, causing a subset of cellular RNA to redistribute into various subcellular compartments such as the stress granules,” Chen said. “PHOTON allows us to detect the spatial redistribution of cellular RNA in diseases versus health, helping us understand how these diseases cause damage to cellular functions.”

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Quantum Sensor Generates Own Light Source to Detect Biomolecules

The development of a compact, self-illuminating plasmonic sensor could make high-performing optical biosensors more accessible for rapid diagnostics and environmental monitoring and in point-of-care settings. The plasmonic biosensor can focus light waves down to a scale small enough to detect proteins and amino acids, without needing a bulky, expensive external light source.

By exploiting a quantum phenomenon called inelastic electron tunneling, researchers at the École Polytechnique Fédérale de Lausanne (EPFL), aided by colleagues at ETH Zurich, ICFO, and Yonsei University, created a biosensor that requires only a steady flow of electrons, in the form of an applied electrical voltage, to illuminate and detect molecules.

As an electron passes through a multilayer (metal-insulator-metal) film in the sensor structure, it transfers some of its energy to a plasmon, which then emits a photon. The intensity and spectrum of the light changes in response to contact with a biomolecule.

“If you think of an electron as a wave, rather than a particle, that wave has a certain low probability of ‘tunneling’ to the other side of an extremely thin insulating barrier while emitting a photon of light,” researcher Mikhail Masharin said. “What we have done is create a nanostructure that both forms part of this insulating barrier and increases the probability that light emission will take place.”

The multilayer structure has an aluminum electrode as the bottom layer, with a thin isolating layer of alumina, formed by thermal oxidation of the film, acting as a tunneling barrier. The upper electrode consists of a doubly periodic metasurface made of resonant gold nanowire antennas.

The plasmonic metasurface serves a dual purpose. It both creates the conditions for quantum tunneling and controls the resulting light emission, simultaneously providing enhanced electron-to-light conversion and far-field light emission. This dual capability is due to the arrangement of the gold nanowires, which act as nanoantennas to concentrate the light at the nm volumes required to detect biomolecules efficiently.

The optically resonant, doubly periodic nanowire metasurface provides uniform emission over large areas, amplified by the nanoantennas that simultaneously enhance the spectral and refractive index sensitivity.

“Inelastic electron tunneling is a very low-probability process, but if you have a low-probability process occurring uniformly over a very large area, you can still collect enough photons,” researcher Jihye Lee said. “This is where we have focused our optimization, and it turns out to be a very promising new strategy for biosensing.”

The researchers tested the biosensor with various analytes including thin layers of polymer and biomolecules. They observed that both the intensity and the spectral profile of the emitted light were modulated by the local refractive index changes produced by the presence of the analyte.

“Tests showed that our self-illuminating biosensor can detect amino acids and polymers at picogram concentrations — that’s one-trillionth of a gram — rivaling the most advanced sensors available today,” researcher Hatice Altug said.

The biosensor provides an integrated, nanoscale light source without requiring any labels. With plasmonic antennas serving both as a sensing element and a light source, the sensor has a considerably smaller device footprint compared with designs involving the integration of plasmonic structures on top of LEDs or photodetectors.

In addition to being compact and sensitive, the quantum platform is scalable and compatible with sensor manufacturing methods. Less than one square millimeter of active area is required for sensing, demonstrating the potential for its use in handheld biosensors. Because it removes the need for an external light source, the on-chip, optical biosensor could be appropriate for various point-of-care applications.

“Our work delivers a fully integrated sensor that combines light generation and detection on a single chip,” researcher Ivan Sinev said. “With potential applications ranging from point-of-care diagnostics to detecting environmental contaminants, this technology represents a new frontier in high-performance sensing systems.”

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Deep Learning-Trained Imager Magnifies Subwavelength Objects

An optical imaging system from UCLA goes beyond the traditional diffraction limit to enable imaging at subwavelength resolution. The new imager will make direct imaging of phase objects with subwavelength resolution less challenging for bioimaging, sensing, material characterization, and other applications that frequently use phase imaging.

The imager, developed in the lab of UCLA professor Aydogan Ozcan, enables subwavelength imaging of phase and amplitude objects. To enable the imager to recover high-frequency information corresponding to the subwavelength features of an object, the research team uses all-optical diffractive encoding and decoding with a solid-immersion layer.

The imager’s thin, high-index, solid-immersion layer transmits high-frequency information about the object to a spatially-optimized diffractive encoder. The encoder converts and encodes the high-frequency information into low-frequency spatial modes for transmission through air.

A diffractive decoder, which is jointly trained with the encoder surface, processes the encoded spatial information that is propagated through the air to create a magnified image of the input object. The magnified image reveals subwavelength features that would normally be washed out due to diffraction limitations.

To demonstrate the subwavelength diffractive imager, the researchers fabricated a multilayer, monolithic design that operates at the terahertz (THz) part of the spectrum. They tested this monolithic diffractive encoder-decoder pair with a customized, high-resolution THz imaging system. The experimental results confirmed that the 3D-fabricated, solid-immersion diffractive imager can resolve phase objects by directly performing transformations through the diffractive encoder-decoder pair.

At THz frequencies, the imager can resolve features as small as λ/3.4 (where λ is the illumination wavelength) by directly transforming them into magnified features at the output.

The trained subwavelength diffractive imager generalized to previously unseen objects from the same distribution as the objects used in training, demonstrating internal generalization. It also generalized to new types of objects from completely different datasets, demonstrating external generalization capability.

The user can operate the subwavelength imager at different parts of the electromagnetic spectrum by physically scaling — that is, by expanding or shrinking — the optimized diffractive features of the encoder and decoder surfaces in proportion to the illumination wavelength. This can be done without needing to redesign the diffractive features of the system.

The subwavelength imager offers the advantage of directly performing quantitative phase retrieval, eliminating the need for lengthy computer processing, which consumes a lot of power.

The researchers believe that the solid-immersion diffractive imager, with its compact size, cost-effectiveness, and ability to capture subwavelength features, could lead to significant advancements in bioimaging, sensing, and material inspection, among many other applications.

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Gamma Light, Sound Could Lessen Neurodegenerative Effects

An MIT study shows how 40 Hz sensory stimulation with light and sound helps sustain myelination, an essential process in the brain that insulates the signal-sending branches of neurons, called axons, with protective myelin sheaths.

Often called the brain’s “white matter,” myelin ensures electrical signal transmission in brain circuits. Demyelination, characterized by the loss of the myelin sheath and the oligodendrocyte cells that form it, leads to impaired axonal function, resulting in brain atrophy and neurodegeneration.

Early-stage trials in Alzheimer’s disease patients and studies in mouse models of the disease have suggested that exposure to light and sound at the gamma band frequency of 40 Hz can have a positive impact on the pathology and symptoms from neurodegenerative disorders.

“Gamma stimulation promotes a healthy environment,” said researcher Daniela Rodrigues Amorim. “There are several ways we are seeing different effects.”

The researchers used the cuprizone mouse model of demyelination to investigate the ways in which gamma sensory stimulation may promote myelination and reduce neuroinflammation. They divided the mice into four groups: mice that were fed a normal diet; mice that received no cuprizone but did receive gamma stimulation; mice that received cuprizone and constant, but not 40 Hz, stimulation; and mice that received cuprizone and 40 Hz stimulation.

The cuprizone-fed mice that received 40 Hz stimulation retained significantly more myelin, rivaling the myelin health of mice never fed cuprizone in some areas.

The team also investigated whether oligodendrocyte cells had higher survival rates in mice exposed to 40 Hz sensory stimulation. The number of oligodendrocyte cells was much closer to healthy levels in mice fed cuprizone and treated with gamma stimulation than in cuprizone-fed mice not exposed to gamma stimulation.

Electrophysiological testing of the neural axons showed that electrical performance improved in the cuprizone-fed mice that received gamma stimulation, compared to the cuprizone-fed mice not treated with 40 Hz stimulation.

To further explore how 40 Hz sensory stimulation might protect myelin, the researchers evaluated the protein expression from all four mouse groups. An analysis of the mice’s brain tissue identified distinct differences in protein expression between the cuprizone-fed mice exposed to control stimulation and the cuprizone-fed mice that received gamma stimulation.

The gamma-treated, cuprizone-fed mice showed an increase in microtubule-associated protein 2 (MAP2), a protein that helps preserve the functional integrity of myelin. Synaptic plasticity, also associated with the preservation of myelin, was better preserved in the mice exposed to 40 Hz stimulation. Exposure to gamma stimulation also helped to decrease oligodendrocyte cell death, which is linked to demyelination, by reducing ferroptosis.

The team assessed gene expression in the mice using single-cell RNA sequencing technology and found that gamma stimulation had an anti-inflammatory effect in the brain. When exposed to 40 Hz light and sound, fewer cells became inflammatory. Direct observation of tissue showed that microglia became more proficient at clearing away myelin debris, a key step in repairing myelin, in the gamma-stimulated group.

The results of the study suggest that 40 Hz sensory stimulation with light and sound could be therapeutic for numerous disorders that exhibit myelin degeneration, including multiple sclerosis and Alzheimer’s disease. The course of these neurological conditions comprises severe neurodegenerative processes, including neuroinflammation, profound myelin damage, and brain atrophy.

Cognito Therapeutics, the spin-off company that licensed MIT’s sensory stimulation technology, published phase II human trial results in the Journal of Alzheimer’s Disease in early 2024, which indicated that 40 Hz light and sound stimulation significantly slowed the loss of myelin in volunteers with Alzheimer’s. In 2024, the lab of professor Li-Huei Tsai also published a study showing that gamma sensory stimulation helped mice withstand neurological effects of chemotherapy medicines, including by preserving myelin.

“Previous publications from our lab have mainly focused on neuronal protection,” Tsai said. “But this study shows that it’s not just the gray matter, but also the white matter that’s protected by this method.”


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Accurate Skin Tone Measurement with Smartphone Improves Pulse Oximetry

A smartphone-based imaging technique for measuring skin pigmentation could improve the accuracy of pulse oximetry readings, particularly for people with darker skin tones.

Pulse oximeters are used to measure oxygen saturation levels in the blood. These devices often rely on light absorption through the skin, which varies depending on pigmentation. Pulse oximeters can overestimate oxygen levels in people with darker skin, which can lead to missed diagnoses and delays in treatment. Yet, skin tone is seldom measured directly in clinical settings.

To objectively measure skin tone, researchers at Brown University and Morgan State University developed a method to extract individual typology angle (ITA) values from a patient’s skin using a smartphone camera. ITA values are the metric that is used to classify levels of skin pigmentation.

Using a cell phone camera, the researchers captured the backscattered light from the dorsal and palmar sides of the fingers (a common measurement site for pulse oximetry) of four volunteers. They acquired skin-color data across a diverse set of skin tones, under various camera exposure settings and ambient lighting conditions. They developed an algorithm to extract a standardized skin-tone (i.e., ITA) value from an ITA spatial mapping derived from the RGB images captured by the smartphone.

The smartphone measurements compared favorably to those obtained using an industry-standard tristimulus colorimeter. When taken under controlled lighting conditions, the smartphone-based readings closely matched those from the high-end instrument.

The best results were achieved when both the camera flash and room lights were turned off, and the phone was set to a specific exposure level. The researchers identified an exposure setting of 0.7 as a reliable configuration for achieving ITA alignment with industry-standard colorimeter values.

Under these optimal conditions, the smartphone-based method for measuring skin tone proved consistent across different skin tones and required no extra equipment beyond the phone itself. By validating the technique under various lighting conditions and across different levels of skin pigmentation, the researchers were able to identify settings and configurations that are resilient to skin-tone variations.

To facilitate the use of smartphone cameras for pulse oximetry in nonlaboratory settings, the researchers recommended ways to minimize errors caused by ambient light scattering, which can affect skin-tone readings. They provided simple guidelines for using the technique in hospitals, such as avoiding measurements over tattoos or scars, turning off automatic camera features, and keeping the camera at a consistent distance from the skin.

Although the research was limited to a small group of young adults and tested outside a clinical setting, it could lay the groundwork for similar studies in real-world environments. The findings demonstrate that smartphone-based imaging could provide an affordable, effective way to evaluate skin tone.

The smartphone approach is accessible to the clinical community and others interested in carrying out pulse oximetry across a diversity of skin tones in a manner that standardizes skin-tone assessment. With additional testing and refinement, it could potentially enhance healthcare equity by providing clinicians with a consistent method for quantifying skin tone in a variety of environments.

To develop a reliable, user-friendly tool for real-world applications, future studies could encompass a broader range of skin tones and use lighting conditions commonly encountered in clinical settings.

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Optical Redox Imaging Monitors Heart Cell Development

An optical imaging tool for monitoring the growth of human heart cells known as cardiomyocytes (CMs) could lead to a reproducible means to generate human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) for biomanufacturing. The imaging technique, along with various synthetic hydrogel substrates, was developed by a team at the University of Wisconsin-Madison.

CMs derived from iPSCs are a promising tool for combatting heart disease, which is a leading cause of death worldwide. IPSCs can be used to improve drug screening platforms, build accurate disease models, and develop personalized regenerative medical treatments. They can be reprogrammed to generate high-purity CM batches. However, in vitro maturation of iPSC-CMs remains difficult.

IPSC-CM differentiation and maturation studies typically rely on heterogeneous substrates and destructive verification methods. The technique that is traditionally used to grow iPSC-CMs is highly variable, prone to contamination, and degrades easily over time. The assays used to screen CM cells to determine their maturity often require harsh chemicals that damage the cells.

The University of Wisconsin-Madison team recognized that a touch-free monitoring approach and tunable substrates might provide a way to produce homogenous, functional CM cells and determine the ideal conditions for the cells’ successful growth.

The researchers developed a label-free, wide-field optical imaging technique to observe live CM cells throughout their differentiation and early-to-late maturation and identify key metabolic shifts in the cells. They grew the cells on synthetic, in-house developed hydrogels.

Using autofluorescence lifetime imaging (FLIM), the researchers verified increased oxidative metabolism and NAD(P)H protein binding activity throughout cell differentiation. To increase throughput and screen more hydrogel formulations, they used widefield NAD(P)H and FAD autofluorescence imaging to calculate the optical redox ratio at multiple timepoints throughout differentiation. The optical redox ratio represents the metabolic shifts in the cells’ energy production and consumption.

The researchers developed several different compositions of synthetic, modifiable hydrogels to induce stem cell differentiation and maturation. Their approach to formulating the gels led to uniform results.

“The goal is having something that will be tunable and reproducible,” researcher Danielle Desa said. “The dream application of using these synthetic materials would be for biomanufacturing, because you’d want something robust and repeatable.”

The researchers found that the optical redox ratio decreased in CMs grown in all the synthetic hydrogel formulations during differentiation, early maturation, and late maturation. These results indicate that the CMs’ metabolic pathways shifted from glycolysis (i.e., breakdown of glucose to access energy quickly) to oxidative phosphorylation (i.e., use of oxygen to produce energy).

The team screened CMs grown in the hydrogels out to 100 days post-differentiation and continued to see metabolic changes over that period. “It was useful to see that they didn’t degrade in that time,” Desa said. “We wanted to do repeated imaging on the same gels over time and see if the technique was sensitive to any metabolic changes.”

The researchers also observed metabolic differences in low- and high-efficiency differentiation batches of cells. The sensitivity of the optical redux imaging technique could serve as an early predictor of which batch of cells is most likely to mature into functional CMs. This feature could make optical redux imaging a useful screening tool for cell manufacturing.

The efficient production and widespread use of iPSC-CMs will require consistent manufacturing of functionally mature cell batches and touch-free, nondestructive monitoring. The optical redux imaging technique and synthetic hydrogels could provide an accessible, medium-throughput method to monitor iPSC metabolism in response to interventions that aim to increase batch yield, consistency, and maturity without manipulating or destroying the cells. The technique allows the same cells to be monitored repeatedly and used for complementary analyses.


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Photoacoustic Probes Enable Deep Brain Tissue Imaging

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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