Researchers at Stanford have created a powerful new imaging system that reveals the inner workings of living cells with remarkable clarity. By combining two advanced microscopy techniques, the instrument achieves a resolution of approximately 120 nanometers, an unprecedented level of detail for an approach that does not require fluorescent labels.

Known as Interferometric Image Scanning Microscopy (iISM), the technology makes it possible to observe multiple cellular structures simultaneously while they move, change, and respond to their surroundings. Scientists can also investigate what happens when cells encounter drugs or disease causing organisms, potentially uncovering biological processes that have been difficult to study.

The breakthrough, described in the Nature portfolio journal Light: Science and Applications, could lead to advances in cancer research, drug development, infectious disease studies, and investigations of how plants interact with microorganisms.

"This new microscope provides a fantastic new view into the cell, where you can see the tiny structures and machines in the cell moving, changing, and interacting without having to add fluorescence to observe them," said senior author W.E. Moerner, the Harry S. Mosher Professor of Chemistry in Stanford's School of Humanities and Sciences. "It's a wonderful look into these complex little cellular boxes that drive our life."

Seeing Inside Living Cells Without Fluorescent Labels

One of the most important advantages of the new microscope is its ability to reveal cellular activity without relying on fluorescent markers.

Many existing microscopy techniques use fluorescent substances to highlight specific structures inside cells. Although these methods have transformed biological research, they come with limitations. Researchers can generally label only a small number of structures at once, making it difficult to observe how different components interact across an entire cell.

Fluorescent signals can also fade during observation through a process known as photobleaching. In addition, introducing the labels can be challenging, and some markers may alter the behavior of the very structures scientists want to study.

The Stanford system avoids these problems by imaging cells without fluorescent labels. This allows researchers to follow numerous cellular components over extended periods while observing their activity in a more natural setting.

Another advantage is that iISM requires substantially less illumination power than comparable label-free methods that produce high-contrast images. Lower light exposure reduces the possibility of photodamage, which occurs when the light used for imaging harms living cells or interferes with their normal functions.

Although other specialized microscopes can achieve finer resolution, the combination of detailed imaging, gentle illumination, and extended observation makes iISM particularly valuable for studying living biological systems.

The researchers emphasize that their technology is intended to complement fluorescence microscopy rather than replace it.

First author Michelle Kueppers, a postdoctoral scholar in Moerner's laboratory, explained that combining the strengths of different imaging approaches could help scientists answer previously inaccessible questions.

"Every method has its advantages and disadvantages, and we believe in a complementary implementation in the future," Kueppers said. "If we use the strengths of fluorescence for molecular specificity and the strength of iISM for label-free context and dynamics, we can really start tackling questions that have been difficult to address before."

How the New Microscope Achieves Unprecedented Detail

The breakthrough comes from bringing together two different microscopy technologies, each with its own advantages.

Moerner received the 2014 Nobel Prize in Chemistry for his pioneering contributions to super-resolution fluorescence microscopy. He specifically recruited Kueppers to Stanford because of her doctoral research involving a technique called "interferometric scattering microscopy."

This method relies on a familiar property of light: scattering.

Light changes direction when it encounters tiny particles. The same basic phenomenon explains why the sky appears blue. As sunlight passes through Earth's atmosphere, molecules, dust, and water droplets scatter its different wavelengths. Shorter blue wavelengths scatter more strongly than longer red wavelengths, producing the blue color we see overhead.

Interferometric scattering microscopy takes advantage of this behavior on a much smaller scale.

A laser illuminates a cell, causing tiny structures inside it to scatter some of the incoming light. Because the scattered signal is extremely weak, a second laser beam is used to amplify it through interference, allowing the microscope to detect structures that would otherwise be difficult to observe.

The Stanford team improved this approach by combining it with technology adapted from advanced confocal microscopes.

Traditional confocal microscopes use a small opening called a pinhole and a single detector to isolate light from a selected area. More advanced versions replace that arrangement with camera-based detector arrays capable of collecting multiple views of the same region.

The researchers incorporated an array detector into their new microscope, allowing it to collect more light than a conventional system built around a pinhole and single detector.

Hundreds of Views Combined Into One Sharper Image

The advantage of collecting multiple perspectives is similar to the way human vision works.

Our two eyes observe the world from slightly different positions, helping the brain distinguish nearby objects from those farther away. The Stanford microscope uses a related principle, but instead of relying on just two perspectives, its detector array gathers tens to hundreds of views.

The researchers developed a computational method that combines these measurements to generate images with improved sharpness, contrast, depth information, and precision.

By merging the strengths of interferometric scattering microscopy and advanced detector arrays, the team created an instrument capable of resolving structures as small as approximately 120 nanometers.

For perspective, a nanometer is one billionth of a meter. At this scale, researchers can examine extremely small cellular structures and follow their movements and interactions.

Crucially, the microscope achieves this resolution without sacrificing imaging speed or requiring intense laser illumination. Scientists can therefore observe living cells for longer periods while minimizing the risk of disturbing or damaging them.

From Cancer Drugs to Malaria: New Possibilities for Research

Moerner and Kueppers are continuing to refine the instrument, with the goal of eventually making it accessible to scientists across many areas of biology.

Three collaborations with other Stanford research groups are already underway or in development, demonstrating the range of questions the technology could help investigate.

One project uses the microscope to observe interactions among plant cells, fungi, and bacteria as they happen. By watching these organisms interact in real time, researchers hope to gain a clearer understanding of the complex relationships between plants and microbes.

Another collaboration focuses on cancer treatment. Scientists are using iISM to investigate how a cancer drug enters a cell, potentially providing valuable information about how medications interact with their targets.

A third project, still in the planning stages, will examine how red blood cells change shape when exposed to malaria infection. Observing these physical changes could offer new insights into how the disease affects blood cells.

These applications illustrate why the researchers believe their microscope could become a useful tool across numerous scientific disciplines rather than serving only a narrow area of research.

"This is not a niche technique," Kueppers said. "It has broad applications, and we hope the life science community will be well served by it, leading to many new discoveries."