Seeing The Invisible: Measuring Crowded Molecular Coatings

Modern medical devices such as biosensors, implants and drug-delivery particles are coated with extremely thin layers of biological molecules. These layers determine how a device behaves in the body, but until now they have been almost impossible to measure when they become very dense. PhD candidate Helen Tan developed a way to see and quantify these crowded molecular coatings using a super-resolution technique called DNA-PAINT.

Medical materials are often covered with short DNA strands, proteins or antibodies that give them their biological function. These coatings act like molecular carpets: they can make a surface recognize certain cells, avoid immune reactions or bind specific molecules. For this to work reliably, scientists must know two things: how many molecules are on the surface, and how they are arranged.

When only a few molecules are present, existing microscopes can do this. But in realistic biomedical applications, surfaces are packed with hundreds to thousands of molecules per square micrometre. At these densities, individual molecules become undistinguishable from each other and standard methods fail.

How can you count molecules when everything is crowded?

Tan ’s research focused on turning DNA-PAINT, a blinking-based super-resolution microscopy technique, into a quantitative tool for these crowded surfaces. She built an analysis framework that answers both key questions: how many molecules are there and how are they arranged?

A central part of her work is a new correction method called BiSC, which can recover the true molecular density even when only a fraction of the molecules is detected. This makes it possible to work in a controlled “undersampling” regime, where overcrowding (undistinguishable molecules) no longer prevents accurate measurements.

She showed that this approach works on biofunctionalized flat surfaces and on 3D microparticles, at realistic high densities of roughly 100 to 1000 molecules per square micrometre.

Key findings

• Polymer coatings are dynamic. In widely used PLL-g-PEG/DNA coatings, Tan discovered that the polymer layer rearranges during the chemical coupling process. This changes both how many molecules bind and how evenly they are distributed, meaning that the final coating cannot be predicted from the formulation alone.
• Not all attachment chemistries behave the same. On microparticles, different coupling strategies gave very different results. Click-chemistry-based coupling produced steadily increasing densities as expected. In contrast, the popular streptavidin–biotin system reached much lower densities than theoretically possible, because at high loading many molecules became physically inaccessible due to crowding.
• Simulations reveal when DNA-PAINT still works. Using Monte Carlo simulations and a neural-network model, Tan mapped out when DNA-PAINT can still provide at least 90% accurate molecular densities and meaningful spatial patterns. The most important design levers turned out to be the binding and unbinding rates of the probes and their concentration, leading to general guidelines for future probes, including non-DNA ones.
• Unexpected effects in real coatings. When comparing different polymer architectures and mixing ratios, Tan found surprising behavior, such as lower functional coverage when more “functional” polymer was added, and clear switches between clustered and evenly spread molecular patterns.

Impact on biomedical engineering
By turning DNA-PAINT into a reliable quantitative tool, Helen Tan’s work enables single-molecule-level quality control of dense biological coatings. This is crucial for developing better biosensors, more predictable biomaterials and more effective drug-delivery systems. Her results also show that surface chemistry and polymer design must be evaluated directly on the final coating, rather than inferred from how it was prepared.

Together, her experiments, correction methods and simulations provide a robust route toward routine, high-precision characterization of the molecular layers that underpin many modern medical technologies.

The research was carried out with support from the European Union’s Horizon 2020 Marie Skłodowska-Curie programme.