Coffee as a staining agent substitute in electron microscopy

Researchers at TU Graz have proven that espresso is a favourable alternative to the highly toxic and radioactive uranyl acetate in the analysis of biological samples

Graz University of Technology

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image: 

Electron microscope image of algae pre-treated with espresso to create stronger contrasts.

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Credit: Claudia Mayrhofer - FELMI-ZFE

To ensure that the tissue structures of biological samples are easily recognisable under the electron microscope, they are treated with a staining agent. The standard staining agent for this is uranyl acetate. However, some laboratories are not allowed to use this highly toxic and radioactive substance for safety reasons. A research team at the Institute of Electron Microscopy and Nanoanalysis (FELMI-ZFE) at Graz University of Technology (TU Graz) has now found an environmentally friendly alternative: ordinary espresso. Images of the samples treated with it were of equally good quality as images of comparative samples, which were prepared with uranyl acetate. The researchers have published their findings in the journal Methods.

Coffee stains as inspiration

“I got the idea of using espresso as a staining agent from the circular dried stains in used coffee cups,” says Claudia Mayrhofer, who is responsible for ultramicrotomy at the institute. During preparation, she cuts tissue samples into wafer-thin slices and fixes them onto sample holders. Staining is the last step before examination under the electron microscope. “Initial tests have shown that coffee stains biological samples and enhances contrasts,” says Mayrhofer.

Together with team leader Ilse Letofsky-Papst and graduate student Robert Zandonella, Claudia Mayrhofer investigated how well espresso performs in direct comparison with uranyl acetate. Under identical conditions, they treated ultra-thin sections of mitochondria with various staining agents and assessed the quality of the microscope images using special image analysis software. “Espresso provided comparatively very good contrast values, in some cases they were even better than with uranyl acetate,” explains Claudia Mayrhofer.

Further tests with different tissue types required

Ilse Letofsky-Papst concludes: “Our results show that coffee is a serious alternative to uranyl acetate. However, further investigations on different types of tissues are still required to enable a broad application in life science electron microscopy.”

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Journal

Methods

DOI

10.1016/j.ymeth.2025.08.009 

Method of Research

Experimental study

Subject of Research

Cells

Article Title

Coffee – a ubiquitous substitute for uranyl acetate in staining of biological ultrathin sections for electron microscopy studies

Handheld ‘pocket microscope’ sees molecules directly -- no staining required

Light Publishing Center, Changchun Institute of Optics, Fine Mechanics And Physics, CAS

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Figure | DART enables label-free molecular imaging with quantitative virtual staining. (a) Mesoscale molecular imaging demonstration showing the DART system (inset) acquiring data across a centimeter scale field of view. The topographic height map reveals sample variations from 590 to 635 μm. The recovered nucleic acid mass distribution (0-50.53 femtograms) and protein mass distribution (0-796.90 femtograms) are quantitatively mapped at each pixel without any external labels or stains. (b) Virtual staining based on the quantitative molecular maps produces familiar histological appearances, with nucleic acids (blue) corresponding to cell nuclei and proteins (purple) representing cytoplasm. This physics-based approach provides explainable and reproducible results directly derived from measured molecular content, unlike black-box AI methods.

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Credit: Guoan Zheng et al.

For centuries, biological imaging has relied on dyes and fluorescent labels to visualize cellular structures -- a practice that not only alters samples but can delay critical medical decisions. Traditional staining can take 20-30 minutes during surgery, potentially delaying critical decisions about tumor margins. Even routine pathology requires lengthy preparation protocols that modify the very molecules being studied.

 

In a new paper published in eLight, a team of scientists led by Professor Guoan Zheng from the University of Connecticut, has developed the deep-ultraviolet ptychographic pocket-scope (DART) -- a handheld platform that transforms molecular imaging through intrinsic deep-ultraviolet spectroscopic contrast.

 

DART changes the imaging paradigm by exploiting a fundamental property of biology: DNA and proteins naturally absorb specific wavelengths of deep-ultraviolet light. By imaging at 266 and 280 nanometers -- wavelengths where nucleic acids and proteins have distinct absorption signatures -- DART creates quantitative molecular maps with femtogram precision without any external labels.

 

“DART provides molecular information instantly, and because it’s based on intrinsic molecular properties rather than dye binding, the results are inherently quantitative and reproducible,” explains Zheng.

 

The system achieves remarkable specifications in a handheld form factor: 308-nanometer resolution, centimeter-scale field of view, and millimeter-scale depth of field. This combination, impossible with conventional microscopy, enables comprehensive tissue imaging without mechanical refocusing.

 

A key feature of DART is its ‘virtual staining’ capability. Unlike AI-based approaches that can generate artifacts or hallucinate features, DART's virtual stains derive directly from measured molecular content. The system quantifies nucleic acid and protein masses with femtogram accuracy, then translates these measurements into familiar histological appearances.

 

“We’re essentially giving clinicians molecular X-ray vision,” notes Zheng. “hey can see not just cell structures, but the actual protein and DNA distributions that define cellular identity and pathological states.”

 

The implications extend beyond terrestrial laboratories. The compact, label-free design makes DART ideal for space missions, where monitoring astronaut health and studying microbial adaptation under microgravity are critical challenges. The device could also enable field diagnostics in resource-limited settings where traditional staining infrastructure is unavailable.

 

The team achieved these capabilities through several technical innovations. They modified commercial image sensors to improve deep ultraviolet sensitivity. A disorder-engineered coded surface converts high-angle scattered light into detectable signals. Most critically, their ‘virtual state’ algorithm eliminates artifacts from LED coherence limitations and optical imperfections.

 

Looking forward, the researchers envision expanding DART’s wavelength range for finer molecular discrimination and developing endoscopic versions for in-vivo imaging. The technology could transform pathology workflows, enable real-time surgical guidance, and open new frontiers in space biology -- all while preserving samples in their natural, unaltered state.

 

The convergence of computational imaging, spectroscopy, and miniaturization embodied in DART represents more than incremental progress. It offers a fundamentally new way to observe biology: directly, quantitatively, and without perturbation.

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Journal

eLight

DOI

10.1186/s43593-025-00103-y 

Article Title

Deep-ultraviolet ptychographic pocket-scope (DART): mesoscale lensless molecular imaging with label-free spectroscopic contrast

 

A new approach to assessing patients with disorders of consciousness

Institut du Cerveau (Paris Brain Institute)

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After a stroke, traumatic brain injury, or cardiac arrest, some patients sustain severe brain damage and lose consciousness for periods ranging from a few days to several weeks or months. They then may fall along a spectrum of intermediate states between wakefulness and complete loss of consciousness, referred to as disorders of consciousness.

These disorders include, for example, unresponsive wakefulness syndrome (formerly known as the vegetative state), characterized by normal sleep-wake cycles: patients open their eyes and breathe independently but appear unable to interact with their environment. The minimally conscious state, by contrast, shows fleeting signs of awareness: tracking an object with the eyes, reacting to a familiar voice, or making a slight gesture in response to a request.

Helping these patients is extremely difficult. Some certainly understand what is being said to them but cannot express it through voluntary movements—this is known as cognitive–motor dissociation.

“There is no clear boundary between normal and abnormal states of consciousness,” explains Dragana Manasova, a former PhD student in the DreamTeam at Paris Brain Institute and now a postdoctoral researcher at the Lewis Lab at the Massachusetts Institute of Technology (MIT). “By convention, doctors consider patients to be recovering when they can communicate and manipulate objects. However, patients’ condition can fluctuate considerably, and the timing of improvement remains difficult to predict.”

Uncertainty about patient outcomes complicates clinical care and is a major source of distress for families, underscoring the need to better characterize patients’ states of consciousness and to use the most reliable indicators.

A Wide Range of Indicators

This is the challenge addressed by a multicenter European study coordinated by Jacobo Sitt (Inserm) at Paris Brain Institute, with which Dragana Manasova was involved during her doctoral work. The researchers propose a novel approach: combining six assessment techniques, each capturing a distinct aspect of brain function: high-density electroencephalography (EEG) at rest and during an auditory task, structural and functional MRI, diffusion MRI, and positron emission tomography (PET).

The resulting data are then processed using machine-learning algorithms to make them interpretable. The goal? To determine what each of these techniques reveals about consciousness and how they can be used to improve diagnosis and prognosis.

As part of the European PerBrain consortium, the team recruited nearly 400 patients across France, Germany, and Italy and compared their clinical outcomes with predictions from multimodal analysis.

A Synthetic and Precise Assessment

Their results indicate that combining data from multiple techniques significantly improves model performance: the more modalities available, the more reliable the predictions.

The researchers also show that the modalities that contribute most to diagnosis are not necessarily the same as those that predict patient outcomes.

Functional measures of brain activity—metabolic measures from PET or electrical measures from EEG—provide useful information about patients’ current state of consciousness but are less informative about their evolution. Conversely, structural measures, such as diffusion MRI, which analyzes the integrity of brain connections, or conventional MRI, which assesses anatomical integrity, are more relevant for prognosis.

Finally, discrepancies between modalities—for example, between EEG and MRI—are particularly common in patients who ultimately improve. In other words, these divergences, far from being problematic, may actually signal the presence of “islands of consciousness” that are not always captured through clinical observation alone.

Harmonizing patient assessment criteria

“The aim of our study was to bring together a wide range of clinical and brain imaging data within a single, coherent analytical framework. By combining these rich and complementary sources of information, we sought to gain a better understanding of complex brain states, as they occur in real-world clinical practice,” explains Dragana Manasova. “This work also provides insight into how computational analyses, including artificial intelligence models, can support medical decision-making and help clinicians make more informed choices.”

“Now that we have demonstrated the power and usefulness of multimodal analysis in assisting clinicians, we would like to see this tool adopted in expert centers,” explains Jacobo Sitt, co-head of the PICNIC Lab at Paris Brain Institute. “Clinical assessment of patients with disorders of consciousness is not conducted in the same way everywhere; it can vary depending on countries, professional cultures, or access to advanced technologies. Our aim is for all clinicians to share the same reference framework and be able to produce comparable data to advance consciousness research.”

The tool developed by the team is a small, easy-to-use device suitable for clinical settings. It provides a probabilistic and integrative assessment of the patient's condition, giving the medical team full latitude to contextualize and interpret the results.

“This tool does not replace human expertise but offers a way to objectify often ambiguous clinical observations and personalize patient care with a view to achieving the best possible recovery,” the researcher concludes. “It also allows us to better understand the link between brain biology and subjective experience.”

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Journal

Brain

DOI

10.1093/brain/awaf412 

Subject of Research

People

Article Title

Multimodal multicentre investigation of diagnostic and prognostic markers in disorders of consciousness

Article Publication Date

7-Jan-2026

 

Believing or thinking that first impressions are fixed may ease social anxiety, Bar-Ilan University study finds

Bar-Ilan University

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A new study from Bar-Ilan University reveals that people with social anxiety, a common condition marked by fear or discomfort in social situations, may actually feel and perform better when they believe that others’ opinions of them are fixed rather than changeable. Adopting this “fixed mindset” made social interactions feel less stressful and more manageable, leading to improved performance and more positive everyday experiences.

“For most people, believing that others’ opinions can change motivates growth,” explains Prof. Liad Uziel from Bar-Ilan University’s Department of Psychology, who led the research. “But for individuals with high social anxiety, that constant possibility for change can feel overwhelming. Viewing others’ impressions as relatively stable may make the social world seem more predictable and less mentally draining.”

Published in Personality and Social Psychology Bulletin, the multi-phase research included a preliminary study and three follow-up experiments. The initial findings showed that people with higher social anxiety felt less burdened by social interactions when they adopted a fixed mindset about impression formation. Subsequent experiments confirmed this pattern.

In the first experiment, participants prepared self-introductions for an anticipated meeting. Those with high social anxiety made a worse impression when they believed impressions were changeable (“growth mindset”), but not when they believed impressions were fixed. In a second, more stressful video-based task, the same effect emerged: participants high in social anxiety performed better under a fixed mindset. Finally, in a three-day field study, participants guided to adopt a fixed mindset reported more satisfying and less stressful real-world social experiences than those encouraged to think impressions can change.

The findings suggest that for people with social anxiety, predictability and stability. not change, may foster calm and confidence. A fixed mindset appears to reduce the pressure of constant self-monitoring, freeing mental energy for genuine connection. “For those who often worry about how they are perceived, believing that others’ impressions are stable can be both calming and empowering,” says Prof. Uziel.

This line of research points to promising, low-cost interventions for individuals struggling with social anxiety. Simple mindset shifts could help make social interactions feel less taxing and more rewarding. Prof. Uziel and his team plan to further explore these effects in clinically diagnosed populations and examine how such mindsets shape other forms of social behavior.

This research was supported by the Israel Science Foundation (ISF Grant No. 133/23).

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Journal

Personality and Social Psychology Bulletin

Article Title

The Soothing Effect of a Stable World: Social Behavior of Individuals Varying on Social Anxiety Under Fixed and Growth Mindsets About Impression Formation